AU2005213900B2

AU2005213900B2 - Solid-fluid composition and uses thereof

Info

Publication number: AU2005213900B2
Application number: AU2005213900A
Authority: AU
Inventors: Eran Gabbai
Original assignee: Do Coop Tech Ltd
Current assignee: DO-COOP TECHNOLOGIES Ltd
Priority date: 2001-12-12
Filing date: 2005-02-17
Publication date: 2010-05-20
Anticipated expiration: 2022-12-12
Also published as: KR20070028330A; EP1776469A2; AU2005213900A1; WO2005079153A2; JP2007527325A; CA2556913A1; AU2010203023A1; WO2005079153A3

Description

WO 2005/079153 PCT/IL2005/000198 SOLID-FLUID COMPOSITION AND USES THEREOF FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a solid-fluid composition and, more 5 particularly, to a nanostructure and liquid composition having the nanostructure and characterized by a plurality of distinguishing physical, chemical and biological characteristics. Nanoscience is the science of small particles of materials and is one of the most important research frontiers in modem science. These small particles are of interest 10 from a fundamental view point since all properties of a material, such as its melting point and its electronic and optical properties, change when the of the particles that make up the material become nanoscopic. With new properties come new opportunities for technological and commercial development, and applications of nanoparticles have, been shown or proposed in areas as diverse as micro- and 15 nanoelectronics, nanofluidics, coatings and paints and biotechnology. For example, much industrial and academic effort is presently directed towards the development of integrated micro devices or systems combining electrical, mechanical and/or optical/electrooptical components, commonly known as Micro Electro Mechanical Systems (MEMS). MEMS are fabricated using integrated circuit 20 batch processing techniques and can range in size from micrometers to millimeters. These systems -can sense, control and actuate on the micro scale, and are able to function individually or -in arrays to generate effects on the macro scale. In the biotechnology area, nanoparticles are frequently used in nanometer-scale equipment for probing the real-space structure and function of biological molecules. 25 Auxiliary nanoparticles, such as calcium alginate nanospheres, have also been used to help improve gene transfection protocols. In metal' nanoparticles, resonant collective oscillations of conduction electrons, also known as particle plasmons, are excited by optical fields. The resonance frequency of a particle plasmon is determined mainly by the dielectric function of the 30 metal, the surrounding medium and by the shape of the particle. Resonance leads to a narrow spectrally selective absorption and an enhancement of the local field confined on and close to the surface of the metal particle. When the laser wavelength is tuned to the plasmon re onance frequency of the particle, the local electric field in proximity to the nanoparticles can be enhanced by several orders of magnitude.

WO 2005/079153 PCT/IL2005/000198 2 Hence, nanoparticles are used for absorbing or refocusing electromagnetic radiation in proximity to a cell or a molecule, e.g., for the purpose of identification of individual molecules in biological tissue samples, in a similar fashion to the traditional fluorescent labeling. 5 The special radiation absorption characteristics of nanoparticles are also exploited-in the area of solar energy conversion, where gallium selenide nanoparticles are used for selectively absorbing electromagnetic radiation in the visible range while reflecting electromagnetic radiation at the red end of the spectrum, thereby significantly increasing the conversion efficiency. 10 An additional area in which nanoscience can play a role is related to heat transfer. Despite considerable previous research and development focusing on industrial heat'transfer requirements, major improvements in cooling capabilities have been held back because of a fundamental limit in the heat transfer properties of conventional fluids. It is well known that materials in solid form have orders-of 15 magnitude larger thermal conductivities than those of fluids. Therefore, fluids containing suspended solid particles are expected to display significantly enhanced thermal conductivities relative to conventional heat transfer fluids. Low thermal conductivity is a primary limitation in the development of energy efficient heat transfer fluids required in many industrial applications. To overcome this 20 limitation, a new. class of.heat transfer fluids called nanofluids has been developed. These nanofluids are typically liquid compositions in which a considerable amount of nanoparticles are suspended in liquids such as water, oil or ethylene glycol. The resulting nanofluids possess extremely high thermal conductivities compared to the liquids without dispersed nanoparticles. 25 Numerous theoretical and experimental studies of the effective thermal conductivity of dispersions containing particles have been conducted since Maxwell's theoretical work was published more than 100 years ago. However, all previous studies of the. thermal conductivity of suspensions have been confined to those containing millimeter- or micron-sized particles. Maxwell's model shows that the 30 effective thermal conductivity of suspensions containing spherical particles increases with the volume fraction of the solid particles. It is also known that the thermal conductivity of suspensions increases with the ratio of the surface area to volume of the particle. Since the surface area to volume ratio is 1000 times larger for particles with a WO 2005/079153 PCT/IL2005/000198 3 10 nm diameter than for particles with a 10 mm diameter, a much more dramatic improvement in effective thermal conductivity is expected as a result of decreasing the particle size in a solution than can obtained by altering the particle shapes of large particles. 5 Traditionally, nanoparticles are synthesized from a molecular level up, by the application of arc discharge, laser evaporation, pyrolysis process, use of plasma, use of sol gel and the like. Widely used nanoparticles are the fullerene carbon nanotubes, which are broadly defined as objects having a diameter below about 1 tm. In a narrower sense of the words, a material having the carbon hexagonal mesh sheet of 10 carbon substantially in parallel with the axis is called a carbon nanotube, and one with amorphous carbon surrounding a carbon nanotube is also included within the category of carbon nanotube. Also known in the art are nanoshells which are nanoparticles having a dielectric core and a conducting shell layer. Similar to carbon nanotubes, nanoshells are also 15 manufactured from a molecular level up, for example, by bonding atoms of metal on a dielectric substrate; Nanoshells are particularly useful in applications in which it is desired to exploit the above mention optical field enhancement phenomenon. Nanoshells, however, are known to be useful only in cases of near infrared wavelengths applications. 20 It is recognized that nanoparticles produced from a molecular level up tends to loose the physical properties of characterizing the bulk, unless further treatment is involved in the production process. As can be understood from the above non exhaustive list of potential applications in which nanoparticles are already in demand, there is a large diversity of physical properties which are to be considered when 25 producing nanoparticles. In particular, nanoparticles retaining physical properties of larger, micro-sized, particles are of utmost importance. Amongspt.the dive rsityof fields in which the present invention finds uses is the field of molecular biology based research and diagnostics. Over the past ten years, as biological and genomic research have 30 revolutionized the understanding of the molecular basis of life, it has become increasingly clear that the temporal and spatial expression of genes is responsible for all of life's processes. Science has progressed from an understanding of how single genetic defects cause the traditionally recognized hereditary disorders to a realization WO 2005/079153 PCT/IL2005/000198 4 of the importance of the interaction of multiple genetic defects along with environmental factors of more complex disorders. This understanding has become possible with the aid of nucleic acid amplification techniques. In particular, polymerase chain reaction (PCR) has found 5 extensive applications in various fields including the diagnosis of genetic disorders, the detection of nucleic acid sequences of pathogenic organisms in clinical samples, the genetic identification of forensic samples, the analysis of mutations in activated oncogenes and other genes, and the like. In addition, PCR amplification is being used to carry out a variety of tasks in molecular cloning and analysis of DNA. These tasks 10 include the generation of specific sequences of DNA for cloning or use as probes, the detection of segments of DNA for genetic mapping, the detection and analysis of expressed sequences by amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing, the analysis of mutations, and for chromosome crawling. It is 15 expected that PCR, as well as other nucleic acid amplification techniques, will find increasing application in many other aspects of molecular biology. As is well-known, a strand of DNA is comprised of four different nucleotides, as determined by their bases: Adenine, Thymine, Cytosine and Guanine, respectively designated as A, T, C, G. Each strand of DNA matches up with a homologous strand 20 in which A pairs with T, and C pairs with G. A specific sequence of bases which codes for a prote in is referred to as a gene. DNA is often segmented into regions which are responsible for protein compositions (exons) and regions which do not directly contribute to protein composition (introns). The PCR, described generally in U.S. Patent No. 4,683,195, allows in vitro 25 amplification of a target DNA fragment lying between two regions of a known sequence. Double stranded target DNA is first melted to separate the DNA strands, and then oligonucleotide are annealed to the template DNA. The primers are chosen in such a way that they are complementary and hence specifically bind to desired, preselected positions at the 5' and 3' boundaries of the desired target fragment. 30 The oligonucleotides serve as primers for the synthesis of new complementary DNA strands. using a DNA polymerase enzyme in a process known as primer extension. The orientation of the primers with respect to one another is such that the 5' to 3' extension product from each primer contains, when extended far enough, the WO 2005/079153 PCT/IL2005/000198 5 sequence which is complementary to the other oligonucleotide. Thus, each newly synthesized DNA strand becomes a template for synthesis of another DNA strand beginning with the other oligonucleotide as its primer. The cycle of (i) melting, (ii) annealing of oligonucleotide primers, and (iii) primer extension, can be repeated a 5 great number of times resulting in an exponential amplification of the target fragment in between the primers. In prior art PCR techniques, the reaction must be carried out in a reaction buffer containing a DNA polymerase cofactor. A DNA polymerase cofactor is a non protein compound on which the enzyme depends for activity. Without the presence of 10 the cofactor the enzyme is catalytically inactive. Known cofactors include compounds containing manganese or. magnesium in such a form that divalent cations are released into an aqueous solution. Typically these cofactors are in a form of manganese or magnesium salts, such as chlorides, sulfates, acetates and fatty acid salts. The use ofa buffer with a low concentration of cofactors results in mispriming 15 and amplification of non-target sequences. Conversely, too high a concentration reduces primer annealing and results in inefficient DNA amplification. In addition, thermostable DNA polymerases, such as Thermus aquaticus (Taq) DNA polymerase, are magnesium-dependent. Therefore, a precise concentration of magnesium ions is necessary to both maximize the efficiency of the polymerase and the specificity of the 20 reaction. Over the years, many attempts have been made to optimize the PCR, inter alia, by a proper selection of the primer length and sequence, annealing temperature, length of amplifidate, concentration of buffers reaction supplements and the like. As the number of variants which are responsible to the efficiency of.the PCR is extremely 25 large, it is extremely difficult to find an optimal set of parameters for all the components participating in the process. As further detailed in the following sections, the efficiency of nucleic acid amplification techniques can be significantly improved with the aid of a liquid composition incorporating nanostructures therein. 30 SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a nanostructure comprising a core material of a nanometric size surrounded by an WO 2005/079153 PCT/IL2005/000198 6 envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to another aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, each of the nanostructures 5 comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the. core material and the envelope of ordered fluid molecules being in a steady physical state; the nanostructures are designed such that when the liquid composition is first contacted -with a surface and then washed by a predetermined wash protocol, an electrochemical signature of the composition is preserved on the 10 surface. According to yet another aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition facilitates increrment of bacterial colony expansion rate, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an 15 envelope of ordered: fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to still another aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition facilitates increment of phage-bacteria or virus-cell interaction, whereby each of the 20 nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to an additional aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is 25 characterized by a zeta potential which is substantial larger than a zeta potential of the liquid per se, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material.and the envelope of ordered fluid molecules being in a steady physical state. According to.yet an additional aspect of the present invention there is provided 30 a liquid composition comprising a liquid and nanostructures, each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of. ordered, fluid molecules, the core material and the envelope of ordered WO 2005/079153 PCT/IL2005/000198 7 fluid molecules being in a steady physical state, and each of the nanostructures having a specific gravity lower than or equal to a specific gravity of the liquid. According to still further features in the described preferred embodiments the nanostructures are designed such that when the liquid composition is mixed with a 5 dyed solution; spectral properties of the dyed solution are substantially changed. According to still an additional aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered 10 fluid molecules being in a steady physical state; the nanostructures are designed such that when the liquid composition is mixed with a dyed solution, spectral properties of the dyed solution are substantially changed. According to yet a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures., the liquid composition 15 enhances macromolecule- binding to solid phase matrix, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to further features in preferred embodiments of the invention 20 described below, the composition wherein the solid phase matrix is hydrophilic. According to still further features in the described preferred embodiments the solid phase matrix is hydrophobic. According to still further features in the described preferred embodiments the solid phase matrix comprises hydrophobic regions and hydrophilic regions. 25 According to still further features in the described preferred embodiments the macromolecule is an antibody. According to still further features in the described preferred embodiments the antibody is a polyclonal antibody. According to still further features in the described preferred embodiments the 30 macromolecule comprises at least one carbohydrate hydrophilic region. Accordingto still further features in the described preferred embodiments the macromolecule coinprises at least one carbohydrate hydrophobic region.

WO 2005/079153 PCT/IL2005/000198 8 According to still further features in the described preferred embodiments the macromolecule is a lectin. According to still further features in the described preferred embodiments the macromolecule is a DNA molecule. 5 According to still further features in the described preferred embodiments the macromolecule is an RNA molecule. According to still a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is capable of at least partially de-folding DNA molecules, whereby each of the 10 nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to, still .a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is 15 capable of altering bacterial adherence to biomaterial, whereby each nanostructure comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to further features in the described preferred embodiments the 20 composition of the present invention decreases its adherence to biomaterial. According to still further features in the described preferred embodiments the biomaterial is selected from the group consisting of plastic, polyester and cement. According to still further features in the described preferred embodiments, the biomaterial is suitable for being surgically implanted in a subject. 25 According to still further features in the described preferred embodiments, the bacterial adherence is Staphylococcus epidermidis adherence. According to still further features in the described preferred embodiments the StaphylococcusI epidermidis adherence is selected from the group consisting of Staphylococcus epidermidis RP 62 A adherence , Staphylococcus epidermidis M7 30 adherence and Staphylocoecus epidermidis (API-6706112) adherence. According to still a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is capable of stabilizing enzyme activity, whereby each of the nanostructures comprises a WO 2005/079153 PCT/IL2005/000198 9 core material. of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to further features in preferred embodiments of the invention 5 described below, the enz yme activity is of an unbound enzyme. According to still further features in the described preferred embodiments the enzyme activity is of a bound enzyme. According to still further features in the described preferred embodiments the enzyme activity is of an enzyme selected from the group consisting of Alkaline 10 Phosphatase, and 3-Galactosidase. According to still a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is capable of improving affinity binding of nucleic acids to a resin and improving gel electrophoresis separation, whereby each of the nanostructures comprises a core 15 material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material nd the envelope of ordered fluid molecules being in a steady physical state. According. to still a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition is 20 capable of. increasing a capacity of a column, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to still a further aspect of the present invention there is provided a 25 liquid composition comprising a liquid and nanostructures, the liquid composition is capable of improving efficiency of nucleic acid amplification process, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered-fluid molecules, the core material and the envelope of ordered fluid moleules being in a steady physical state. 30 According to further features in preferred embodiments of the invention described below, the nucleic acid amplification process is a polymerase chain reaction.

WO 2005/079153 PCT/IL2005/000198 10 According to still further features in the described preferred embodiments the composition is capable of enhancing catalytic activity of a DNA polymerase of said polymerase chain reaction. According to still further features in the described preferred embodiments the 5 polymerase chain reaction is magnesium free. According to. still further features in the described preferred embodiments the polymerase chain reaction is manganese free. According to still a further aspect of the present invention there is provided a kit for polymerase chain reaction, comprising, in separate packaging (a) a thermostable 10 DNA polymerase and (b) a liquid composition having a liquid and nanostructures, each of said nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, said core material and said envelope of ordered fluid molecules being in a steady physical state. According to further features in preferred embodiments of the invention 15 described below, the kit further comprises at least one dNTP. According to still further features in the described preferred embodiments the kit further combrises at least one control template DNA. According to still further features in the described preferred embodiments the kit further comprises at least one control primer. 20 According to still a further aspect of the present invention there is provided a method of amplifying a DNA sequence, the method comprising (a) providing a liquid composition having a liquid and nanostructures, each of the nanostructures comprising a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a 25 steady physical state; and (b) in the presence of the liquid composition, executing a plurality of polymerase chain reaction cycles on the DNA sequence, thereby amplifying the DNA sequence. According to still a further aspect of the present invention there is provided a liquid composition comprising a liquid and nanostructures, the liquid composition 30 being capable of allowing the manipulation of at least one macromolecule in the presence of :a solid support, whereby each of the nanostructures comprises a core material of a nanometric size surrounded by an envelope of ordered fluid molecules, WO 2005/079153 PCT/IL2005/000198 11 the core material and the envelope of ordered fluid molecules being in a steady physical state. According to further features in the described preferred embodiments, the macromolecule is a polynucleotide. 5 According to .still further features in the described preferred embodiments, the polynucleotide is selected from the group consisting of DNA and RNA. According to further features in the described preferred embodiments, the solid support comprises glass' beads. According to further features in the described preferred embodiments, the glass 10 beads are between about 80 and 150 microns in diameter. According to further features in the described preferred embodiments, the manipulation is effected by a chemical reaction. According to still further features in the described preferred embodiments, the chemical reaction is selected from the group consisting of an amplification reaction, a 15 ligation reaction, a transformation reaction, transcription reaction, reverse transcription reaction, restriction digestion and transfection reaction. According to yet. another aspect of the present invention, there is provided a liquid composition comprising a liquid, beads and nanostructures, the liquid composition being capable of allowing the manipulation of at least one macromolecule 20 in the presence of the beads, whereby each nanostructure comprises a core material of a nanometric 'size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. According to- further: features in preferred embodiments of the invention described below, at least a portion of the fluid molecules are in a gaseous state. 25 According to: still further features in the described preferred embodiments the nanostructures are capable of clustering with at least one additional nanostructure. According to' still further features in the described preferred embodiments the nanostructures are capable of maintaining long range interaction with at least one additional nanostructure. 30 According to still further features in the described preferred embodiments at least a portion of the fluid molecules are identical to molecule of the liquid.

WO 2005/079153 PCT/IL2005/000198 12 According to still further features in the described preferred embodiments a concentration of the nanostructures is lower than 102 nanostructures per liter, more preferably lower than 1015 nanostructures per liter. According to still further features in the described preferred embodiments the 5 nanostructures are capable of maintaining long range interaction thereamongst. According to still further features in the described preferred embodiments the core material is selected from the group consisting of a ferroelectric core material, a ferromagnetic cor& material and a piezoelectric core material. According to still further features in the described preferred embodiments the 10 core material is a crystalline core material. According to still further features in the described preferred embodiments the liquid is water. According to still further features in the described preferred embodiments the nanostructures are designed such that a contact angle between the composition and a 15 solid surface is smaller than a contact angle between the liquid and the solid surface. According to a further aspect of the present invention there is provided a method of producing a liquid composition from a solid powder, the method comprising: (a) heating the solid powder, thereby providing a heated solid powder; (b) immersing the heated solid powder in a cold liquid; and (c) substantially 20 contemporaneously with the step (b), irradiating the cold liquid and the heated solid powder by electromagnetic radiation, the electromagnetic radiation being characterized by .a frequency selected such that nanostructures are formed from particles of the solid powder. According to further features in preferred embodiments of the invention 25 described below, the solid powder comprises micro-sized particles. According to still further features in the.described preferred embodiments the micro-sized particles are crystalline particles. According to still further features in the described preferred embodiments the nanostructures are crystalline nanostructures. 30 According to still further features in the described preferred embodiments the solid powder is selected from the group consisting of a ferroelectric material and a ferromagnetic material.

13 According to still further features in the described preferred embodiments the solid powder is selected from the group consisting of BaTiO 3 , W0 3 and Ba 2

F

9 0 12 . According to still further features in the described preferred embodiments the solid powder comprises a material selected from the group consisting of a mineral, a 5 ceramic material, glass metal and synthetic polymer. According to still further features in the described preferred embodiments the electromagnetic radiation is in the radiofrequency range. According to still further features in the described preferred embodiments the electromagnetic radiation is continues wave electromagnetic radiation. 10 According to still further features in the described preferred embodiments the electromagnetic radiation is modulated electromagnetic radiation. The present invention successfully addresses the shortcomings of the presently known configurations by providing a nanostructure and liquid composition having the nanostructure, which is characterized by numerous distinguishing physical, chemical 15 and biological characteristics. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable 20 methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Definitions of the specific embodiments of the invention as claimed herein follow. 25 According to a first embodiment of the invention, there is provided a method of making nanoparticles, comprising the steps of: (a) heating a solid powder; (b) immersing said solid in a liquid that is colder than said hot powder; and (c) during said immersing, irradiating said liquid with radiofrequency ('RF') 30 radiation, wherein said immersing and said irradiating serve to break up said powder into the nanoparticles. According to a second embodiment, there is provided a method of producing a liquid composition from a solid powder, the method comprising: 13a (a) heating the solid powder, thereby providing a heated solid powder; (b) immersing said heated solid powder in a liquid that is colder than said heated solid powder; and (c) substantially contemporaneously with said step (b), irradiating said liquid 5 and said heated solid powder by electromagnetic radiation, said electromagnetic radiation being characterized by a frequency selected such that nanostructures are formed from particles of the solid powder. According to a third embodiment, there is provided, nanoparticles produced by the method according to the first or second embodiments. 10 According to a fourth embodiment, there is provided ferroelectric crystalline nanoparticles produced by the method according to the first or second embodiments. According to a fifth embodiment, there is provided ferromagnetic crystalline nanoparticles produced by the method according to the first or second embodiments. According to a sixth embodiment, there is provided piezoelectric crystalline 15 nanoparticles produced by the method according to the first or second embodiments. According to the seventh embodiment, there is provided an apparatus for converting a solid powder into nanoparticles, comprising: (a) a furnace for heating the powder; (b) a container of a liquid into which said hot powder is dropped to cool and 20 break up the hot powder; and (c) a mechanism for irradiating said liquid with radiofrequency ('RF') radiation while said hot powder is dropped into said liquid. BRIEF DECRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the 25 accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. 30 In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the [Text continues page 14.1 WO 2005/079153 PCT/IL2005/000198 14 description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. In the drawings: FIG. 1 is a schematic illustration of a nanostructure, according to a preferred 5 embodiment of the present invention; FIG. 2a is a flowchart diagram of a method of producing a liquid composition, according to a preferred embodiment of the present invention; FIG. 2b is. a flowchart diagram of a method of amplifying a DNA sequence, according to a preferred embodiment of the present invention; 10 FIGs. 3a-e are TEM images of the nanostructures of the present invention; FIG. 4 shows the effect of. dye on the liquid composition of the present invention; FIGs. 5a-b show the effect of high g centrifugation on the liquid composition, where Figure 5a shows signals recorded of a lower portion of a tube and Figure 5b 15 shows signals recorded of an upper portion of the tube; FIGs. 6a-c show results of pH tests, performed on the liquid composition of the present invention; FIG. 7 shows the absorption spectrum of the liquid composition of the present invention; 20 FIG. 8 shows results of ( potential measurements of the liquid composition of the present invention; FIGs. 9a-b show a bacteriophage reaction in the presence of the liquid composition of the present invention (left) and in the presence of a control medium (right); 25 FIG. 10 shows a comparison between bacteriolysis surface areas of a control liquid and the liquid composition of the present invention; FIG. 11 shows phage typing concentration at 100 routine test dilution, .in the presence of the liquid composition of the present invention (left) and in the presence of a control medium-(right); 30 FIG. 12 shows optic density, as a function of time, of the liquid composition of the present invention and a control medium; FIGs. 13a-c show optic density in slime-producing Staphylococcus epidermidis in an experiment directed to investigate the effect of the liquid composition of the WO 2005/079153 PCT/IL2005/000198 15 -present invention on the adherence of coagulase-negative staphylococci to microtiter plates; FIG. 14 is a histogram representing 15 repeated experiments of slime adherence to different micro titer plates; 5 FIG. 15 shows differences in slime adherence to the liquid composition of the present invention and the control on the same micro titer plate; FIGs. 16a-c show an electrochemical deposition experimental setup; FIGs. 17a-b show electrochemical deposition of the liquid composition of the present invention (Figure 17a) and the control (Figure 17b); 10 FIG. 18 shows electrochemical deposition of reverse osmosis (RO) water in a cell which was in contact with the liquid composition of the present invention for a period of 30,minutes; FIGs. 19a-b show results of Bacillus subtilis colony growth for the liquid composition of the present invention (Figure 19a) and a control medium (Figure 19b); 15 FIGs. 20a-c show results of Bacillus subtilis colony growth, for the water with a raw powder (Figure 20a), reverse osmosis water (Figure 20b) and the liquid composition of the present invention (Figure 20c); FIGs. 2la-d show bindings of labeled and non-labeled antibodies to medium costar microtitration plate (Figure 21a), non-sorp microtitration plate (Figure 21b), 20 maxisorp microtitration plate (Figure 21c) and polysorp microtitration plate (Figure 21d), using the liquid composition of the present invention or control buffer; FIGs. 22a-d show bindings of labeled antibodies to medium costar microtitration plate (Figure 22a), non-sorp microtitration plate (Figure 22b), maxisorp microtitration plate (Figure 22c) and polysorp microtitration plate (Figure 22d), using 25 the liquid composition Qfthe present invention or control buffer; FIGs. 23a-d show bindings of labeled antibodies after overnight incubation at 4 0 C, to non-sorp microtirati6n plate (Figure 23a), medium costar microtitration plate (Figure 23b), polysorp microtitration plate (Figure 23c) and maxisorp microtitration plate (Figure 23d), using the liquid composition of the present invention and using 30 buffer; FIGs. 24a-d show bindings of labeled antibodies 2 hours post incubation at 37 'C, to nonssorp microtitration plate (Figure 24a), medium costar microtitration plate (Figure 24b), polysorp microtitration plate (Figure 24c) and maxisorp WO 2005/079153 PCT/IL2005/000198 16 microtitration plate (Figure 24d), using the liquid composition of the present invention or control buffer; FIGs. 25a-d show binding of labeled and non-labeled antibodies after overnight incubation at 4 ' to medium costar microtitration plate (Figure 25a), polysorp 5 microtitration plate (Figure 25b), maxisorp microtitration plate (Figure 25c) and non sorp microtitration plate (Figure 25d), using the liquid composition of the present invention or control buffer; FIGs. 26a-d show binding of labeled and non-labeled antibodies after overnight incubation at room temperature, to medium costar microtitration plate (Figure 25a), 10 polysorp micrOtitration plate (Figure 25b), maxisorp microtitration plate (Figure 25c) and non-sorp microtitration plate (Figure 25d), using the liquid composition of the present invention or control buffer; FIGs. 27a-b show binding results of labeled and non-labeled antibodies (Figure 27a) and only labeled antibodies (Figure 27b) using phosphate washing buffer, for the 15 liquid composition of the present invention or control buffer; FIGs. 27c-d show binding results of labeled and non-labeled antibodies (Figure 27a) and only labeled antibodies (Figure 27b) using PBS washing buffer, for the liquid composition of the present invention or control buffer; FIGs. 2Sa-b show binding of labeled and non-labeled antibodies (Figure 28a) 20 and only labeled antibodies (Figure 2Sa), after overnight incubation at 4 'C, to medium costar microtitration plate, using the liquid composition of the present invention or control buffer; FIG.. 29a-c show binding of labeled lectin to non-sorp microtitration plate for acetate (Figure 29a), carbonate (Figure 29b) and phosphate (Figure 29c) buffers, using 25 the liquid composition of the present invention or control buffer; Figures 3Ca-d show binding of labeled lectin to maxisorp microtitration plate for carbonate (Figure- 30a-b), acetate (Figure 30c) and phosphate (Figure 30d) buffers, using the liquid composition of the present invention or control buffer, where the graph shown in Figure 30b is a linear portion of the graph shown in Figure 30a; 30 FIGs. 31 a-b show an average binding enhancement capability of the liquid composition of thd present invention for nucleic acid; FIGs.::32-35b are images of PCR product samples before and after purifications for different-buffer combinations and different elution steps; WO 2005/079153 PCT/IL2005/000198 17 FIGs. 36-37 are an image (Figure 36) and quantitative analysis (Figure 37) of PCR products having been passed through columns in varying amounts, concentrations and elution steps; FIGs. 38a-c are images of PCR products columns having been passed through 5 columns 5-17 shown in Figure 36, in three elution steps; FIG. 39a shows the area of control buffer (designated CO) and the liquid composition of.the. present invention (designated LC) as a function of the loading volume for each of the three elution steps of Figures 38a-c; FIG. 39b shows the ratio LC/CO as a function of the loading volume for each 10 of the three elution steps of Figures 38a-c; FIGs. 40a-42b are lane images comparing the migration speed of DNA in gel electrophoresis experiments in the presence of RO water (Figures 40a, 41a and 42a) and in the presence of the liquid composition of the present invention (Figures 40b, 41b and 42b); 15 FIGs. 43a-45d are lane images captured in gel electrophoresis experiments in which the effect of the liquid composition of the present invention on running buffer was investigated; FIGs. 46a-48d are lane images captured in gel electrophoresis experiments in which the effect of the liquid composition of the present invention on the gel buffer 20 was investigated; Figure 49 shows values of a stability enhancement parameter, Se, as a function of the dilution, in an experiment in which the effect of the liquid composition of the present invention on the activity and stability of unbound form of alkaline phosphatase was investigated; 25 FIG. 50 shows enzyme activity of alkaline phosphatase bound to Strept-Avidin, diluted in RO water and the liquid composition of the present invention as a function of the dilution,' in afi. experiment -in which the effect of the liquid composition of the present invention on -the activity and stability of the bound form of alkaline phosphatase was investigated; 30 FIG. 5.a-d show stability of p-Galactosidase after 24 hours (Figure 51a), 48 hours (Figure, 51b), 72 hours (Figure 51c) and 120 hours (Figure 51d), in an experiment in which the effect of the liquid composition of the present invention on the activity and, stability of P-Galactosidase was investigated; WO 2005/079153 PCT/IL2005/000198 18 FIG. 52a-d shows values of a stability enhancement parameter, Se, after 24 hours (Figure 52a), 48 hours (Figure 52b), 72 hours (Figure 52c) and 120 hours (Figure 52d), in an experiment -in which the effect of the liquid composition of the present invention on the activity and stability of P-Galactosidase was investigated; 5 FIG. 53a shows remaining activity of alkaline phosphatase after drying and heat treatment; FIG. 53b show values of the stability enhancement parameter, Se, of alkaline phosphatase after drying and heat treatment; and FIG. 54 shows lane images captured in gel electrophoresis experiments in 10 which the effect of the liquid- composition of the present invention on the ability of glass beads to affect DNA during a PCR reaction was investigated. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a nanostructure and liquid composition having the 15 nanostructure and characterized by a plurality of distinguishing physical, chemical and biological characteristics. The liquid composition of the present invention can be used for many biological and chemical application such as, but not limited to, bacterial colony growth, electrochemical deposition and the like. The principles of a nanostructure and liquid composition according to the 20 present invention .may be better understood with reference to the drawings and accompanying descriptions. Before, explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following 25 description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Referring 'now -to the drawings, Figure 1 illustrates a nanostructure 10 30 comprising a core material 12 of a nanometric size, surrounded by an envelope 14 of ordered fluid .'molecules. Core material 12 and envelope 14 are in a steady physical state.

WO 2005/079153 PCT/IL2005/000198 19 As used herein the phrase "steady physical state" is referred to a situation in which objects or molecules are bound by any potential having at least a local minimum. Representative examples, for such a potential include, without limitation, Van der Waals potential, Yukawa potential, Lenard-Jones potential and the like. Other 5 forms of potentials are also contemplated. As used herein the phrase "ordered fluid molecules" is referred to an organized arrangement of fluid molecules having correlations thereamongst. As used herein the term "about" refers to ± 10 %. According to a preferred embodiment of the present invention, the fluid 10 molecules of envelope 14 may be either in a liquid state or in a gaseous state. As further demonstrated in the Example section that follows (see Example 3), when envelope 14 comprises gaseous material, the nanostructure is capable of floating when subjected to sufficient g-forces. Core material 12 is not limited to a certain type or family of materials, and can 15 be selected in accordance with the application for which the nanostructure is designed. Representative examples include, without limitation, ferroelectric material, a ferromagnetic material and a piezoelectric material. As demonstrated in the Examples section that follows (see. Example 1) core material 12 may also have a crystalline structure. 20 A ferroelectric material is a material that maintains, over some temperature range, a permanent electric polarization that can be reversed or reoriented by the application of anelectric field. A ferromagnetic material is a material that maintains permanent magnetization, which is reversible by applying a magnetic field. According to a preferred embodiment of the present invention, when core material 12 is 25 ferroelectric or ferromagnetic, nanostructure 10 retains its ferroelectric or ferromagnetic properties. Hence, nanostructure 10 has a particular feature in which macro scale physical properties are brought into a nanoscale environment. According to a preferred embodiment of the present invention nanostructure 10 is capable of clustering with at -least one additional nanostructure. More specifically, 30 when a certain concentration of nanostructure 10 is mixed in a liquid (e.g., water), attractive electrostatic forces between several nanostructures may cause adherence thereamongst so as to form a cluster of nanostructures. Preferably, even when the distance between the nanostructures prevents cluster formation, nanostructure 10 is WO 2005/079153 PCT/IL2005/000198 20 capable of niaintaining long range interaction (about 0.5-10 tm), with the other nanostructures. Long range interactions between nanostructures present in a liquid, induce unique characteristics on the liquid, which can be exploited in many applications, such as, but not limited to, biological and chemical assays. 5 The unique properties of nanostructure 10 may be accomplished, for example, by producing nanostructure 10 using a "top-down" process. More specifically, nanostructure 10 can be produced from a raw powder of micro-sized particles, say, above 1 tm or above 10 jim in diameter, which are broken in a controlled manner, to provide nanometer-sized particles. Typically, such a process is performed in a cold 10 liquid (preferably, but not obligatorily, water) into which high-temperature raw powder is inserted, under condition of electromagnetic radiofrequency (RF) radiation. A more detailed description of the production process, is preceded by the following review of the physical properties of water, which, as stated, is the preferred liquid. 15 Hence, water is one of a remarkable substance, which has been very well studied. Although it appears to be a very simple molecule consisting of two hydrogen atoms, attached to an oxygen atom, it has complex properties. Water has numerous special properties due to, hydrogen bonding, such as high surface tension, high viscosity, and the capability of forming ordered hexagonal, pentagonal of 20 dodecahedral water arrays by themselves of around other substances. The melting point of water is over 100 K higher than expected when considering other molecules with similar molecular weight. In the hexagonal ice phase of.the water (the normal form of ice and snow), all water molecules participate in four hydrogen bonds (two as donor and two as acceptor) and are held relatively 25 static. In liquid Water, some hydrogen bonds must be broken to allow the molecules move around. The large energy required for breaking these bonds must be supplied during the melting process and only a relatively minor amount of energy is reclaimed from the change in volume. The free energy change must be zero at the melting point. As temperature increases, the amount of hydrogen bonding in liquid water decreases 30 and its entropy increases. Melting will only occur when there is a sufficient entropy change to provide the energy required for the bond breaking. The low entropy (high organization) of liquid Water causes this melting point to be high.

WO 2005/079153 PCT/IL2005/000198 21 Most of the 'Water properties are attributed to the above mentioned hydrogen bonding occurring when an atom of hydrogen is attracted by rather strong forces to two oxygen atoms (as opposed to one), so that it can be considered to be acting as a bind between the two atoms. 5 Water has high density, which increases with the temperature, up to a local maximum occurring at a temperature of 3.984 IC. This. phenomenon is known as the density anomaly 'of water. The high density of liquid water is mainly due to the cohesive nature of the hydrogen-bonded network. This reduces the free volume and ensures a relatively high-density, compensating for the partial open nature of the 10 hydrogen-bonded network. The anomalous temperature-density behavior of water can be explained utilizing the range of environments within whole or partially formed clusters with differing degrees of dodecahedral puckering. The density maxinum (and molar volume minimum) is brought about by the opposing effects of increasing temperature, causing both structural collapse that 15 increases density and thermal expansion that lowers density. At lower temperatures, there is a higher concentration of expanded structures whereas at higher temperatures there is a higher concentration of collapsed structures and fragments, but the volume they occupy expands with temperature. The change from expanded structures to collapsed structures as the temperature rises is accompanied by positive changes in 20 entropy and enthalpy due to, the less ordered structure and greater hydrogen bond bending, respectively. Generally, the hydrogen bonds of water create extensive networks, that can form numerous hexagonal, pentagonal of dodecahedral water arrays. The hydrogen bonded network possesses a large extent of order. Additionally, there is temperature 25 dependent compe tuition between the ordering effects of hydrogen bonding and the disordering kinetic effects. As known, water molecules can form ordered structures and superstructures. For example, shells of ordered water form around various biomolecules such as proteins and carbohydrates. The ordered water environment around these 30 biomolecules are strongly involved in biological function with regards to intracellular function including, for example, signal transduction from receptors to cell nuclei. Additionally thes& water structures are stable and can protect the surface of the molecule.

WO 2005/079153 PCT/IL2005/000198 22 Most of the ordered structure of liquefied water is on a short-range scale, typically about 1 nm. Although long-range order may, in principle exists, when the water is in its liquid phase, such long-range order has extremely low probability to occur spontaneously, because molecules in a liquid state are in constant thermal 5 motion. Due to hydrogen bonding and non-bonding interactions, water molecules can form an infinite hydrogen-bonded network with specific and structured clustering. Thus, small clusters of water molecules can form water octamers that can further cluster with other smaller clusters to form icosahedral water clusters consisting of hundreds of water molecules. Therefore, water molecules can form ordered structures. 10 Other properties of water include a high boiling point, a high critical point, reduction of melting point with pressure (the pressure anomaly), compressibility which decreases with increasing temperature up to a minimum at about 46 'C, and the like. The unique .properties of water have been exploited by the Inventor of the 15 present invention for the purpose of producing nanostructure 10. Thus, according to another aspect of the present invention there is provided a method of producing a liquid composition. Reference is now made to Figure 2a which is a flowchart diagram of the method, according to a preferred embodiment of the present invention. The method 20 comprises the following method steps, in which in a first step, a solid powder (e.g., a mineral, a ceramic powder, a glass powder, a metal powder, a synthetic polymer, etc.) is heated, to 'a sufficiently high temperature, preferably more than about 700 'C. Representative examples of solid powders which are contemplated include, without limitation, :'BaTiO 3 , W0 3 and Ba 2

F

9 0 1 2 . In a second step, the heated powder is 25 inmersed in a cold liquid, preferably water, below its density anomaly temperature, e.g., 3 'C or 2 'C. In a third step of the method, which is preferably executed substantially contemporaneously with the second step, the cold liquid and the powder are irradiated by electronmagnetic RF radiation, preferably above 500 MHz, which may be either continuous wave RF radiation or modulated RF radiation. 30 The formation of the nanostructures in the liquid may be explained as follows. The combination of cold liquid, and RF radiation (i.e., highly oscillating electromagnetic field) influences the interface between the particles and the liquid, thereby breaking the liquid molecules and the particles. The broken liquid molecules WO 2005/079153 PCT/IL2005/000198 23 are in the form of free radicals, which envelope the (nano-sized) debris of the particles. Being at a small -temperature, the free radicals and the debris enter a steady physical state. The attraction of the free radicals to the nanostructures can be understood from the relatively small size of the nanostructures, compared to the correlation length of 5 the liquid molecules. It has been argued [D. Bartolo, et al., Europhys. Lett., 2000, 49(6):729-734], that a small size perturbation may contribute to a pure Casimir effect, which is manifested by long-range interactions. Performing' the. above method according to present invention successfully produces the nanostructure of the present invention. In particular, the above method 10 allows the formation of envelope 14 as further detailed hereinabove. Thus, according to another aspect of the present invention, there is provided a liquid composition having a liquid and nanostructures 10. When the liquid composition is manufactured by the above method, with no additional steps, envelope 14 of nanostructure 10 is preferably made of molecules which are identical to the molecule of the liquid. 15 Alternatively, the nahostructure may be further mixed (with or without RF irradiation) with a different liquid, so that in the final composition, at least a portion of envelope 14 is made of.molecules which are different than the molecules of the liquid. Due to the formation of envelope 14 the nanostructures preferably have a specific gravity which is lower than'or equal to a specific gravity of liquid. 20 The concentration of the nanostructures is not limited. A preferred concentration is. below 1020 nanostructures per liter, more preferably below 1015 nanostructures per litter. One ordinarily skilled in the art would appreciate that with such concentrations, the average distance between the nanostructures in the composition is rather large, of the order of microns. As further detailed hereinunder 25 and demonstrated in the Example section that follows, the liquid composition of the present invention 'has many unique characteristics. These characteristics may be facilitated, for example, by long range interactions between the nanostructures. In particular, long range interactions allow that employment of the above relatively low concentrations. 30 Interactions between the nanostructures (both long range and short range interactions) facilitate self organization capability of the liquid composition, similar to a self organization of bacterial colonies. When a bacterial colony grows, self organization allows 'it to cope with adverse external conditions and to "collectively WO 2005/079153 PCT/IL2005/000198 24 learn" from the environment.for improving the growth rate. Similarly, the long range interaction and thereby the long range order of the liquid composition allows the liquid composition to perform self-organization, so as to adjust to different environmental conditions, such as, but not limited to, different temperatures, electrical currents, 5 radiation and the like. The long range order of the liquid composition of the present invention is best seen when the. liquid composition is subjected to an electrochemical deposition (ECD) experiment (see also Example 9 in the Examples section that follows). ECD is a.process in which a substance is subjected to a potential difference 10 (for example using two electrodes), so that an electrochemical process is initiated. A particular property of the ECD process is the material distribution obtained thereby. During the electrochemical process, the potential measured between the electrodes at a given current, is the sum of several types of over-voltage and the Ohmic drop in the substrate. The size of the Ohmic drop depends on the conductivity of the substrate and 15 the distance between the electrodes. The current density of a specific local area of an electrode is a function of the distance to the opposite electrode. This effect is called the primary current distribution, and depends on the geometry of the electrodes and the conductivity of the substrate. When the potential difference between the electrodes is large, compared to the 20 equilibrium voltage, the substrates experience a transition to a non-equilibrium state, and as a result, structures of different morphologies are formed. It has been found [E. Ben-Jacob, "From snowflake formation to growth of bacterial colonies," Cont. Phys., 1993, 34(5)] that systems in non-equilibrium states may select a morphology and/or experience transitions between two morphologies: dense branching morphology and a 25 dendritic morphology. According to a preferred embodiment of the present invention when the liquid composition of the present invention is placed in an electrochemical deposition cell, a predetermined morphology (e.g., dense branching and/or dendritic) is formed. Preferably, the liquid composition of the present invention is capable of preserving an 30 electrochemical signature on the surface of the cell even when replaced by a different liquid (e.g., water). More specifically, according to a preferred embodiment of the present invention, when the liquid composition is first contacted with the surface of the electrochemical deposition cell and then washed by a predetermined wash WO 2005/079153 PCT/IL2005/000198 25 protocol, an electrochemical signature of the composition is preserved on the surface of the cell. An additional characteristic of the present invention is a small contact angle between the liquid composition and solid surface. Preferably, the contact angle 5 between the liquid composition and the surface is smaller than a contact angle between liquid (without the nanostructures) and the surface. One ordinarily skilled in the art would appreciate that small contact angle allows the liquid composition to "wet" the surface in larger extent. It is to be understood that this feature of the present invention is not limited to large concentrations of the nanostructures in the liquid, but rather also 10 to low concentrations, with the aid of the above-mentioned long range interactions between the nanostructures. While reducing the present invention to practice, it has been unexpectedly realized (see ExamIples 6, 7 and 10 in the Examples section that follows) that the liquid composition of the present invention is capable of facilitating the increment of 15 bacterial colony expansion rate and phage-bacteria or virus-cell interaction, even when the solid powder used for preparing the liquid composition is toxic to the bacteria. The unique process by which the liquid composition is produced, which, as stated, allows the formation of envelope 14 surrounding core material 12, significantly suppresses any toxic influence of the liquid composition on the bacteria or phages. 20 An additional characteristic of the liquid composition of the present invention is related to the so called zeta (C) potential. ( potential is related to physical phenomena called electrophoresis and dielectrophoresis in which particles can move in a liquid under the influence of electric fields present therein. The ( potential is the electric potential at a shear plane, defined at the boundary between two regions of the 25 liquid having different behaviors. The electrophoretic mobility of particles (the ratio of the velocity of particles to the field strength) is proportional to the ( potential. Being surfacee related quantity, the ( potential is particularly important in systems with small particle size, where the total surface area of the particles is large relative to; their total volume, so that surface related phenomena determine their 30 behavior. According to a preferred embodiment of the present invention, the liquid composition is characterized by a ( potential which is substantially larger than the ( potential of the liquid per se. Large ( potential corresponds to enhanced mobility of WO 2005/079153 PCT/IL2005/000198 26 the nanostructures in the liquid, hence, it may contribute, for example, to the formation of special morphologies in the electrochemical deposition process. There are many methods of measuring the ( potential of the liquid composition, including, without limitation, microelectrophoresis, light scattering, light 5 diffraction, acoustics, electroacoustics etc. For example, one method of measuring ( potential is disclosed in U.S. Patent No, 6,449,563, the contents of which are hereby incorporated by reference. As stated in the Background section hereinabove, the present invention also relates to the field of molecular biology research and diagnosis, particularly to nucleic 10 acid amplification techniques, such as, but not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and self sustained sequence replication (SS SR). It has been found by the inventor of the present invention, that the liquid composition of the present invention is capable of improving the efficiency of a 15 nucleic acid amplification process, e.g., by enhancing the catalytic activity of a DNA polymerase in PCR procedures. The enhancement of catalytic activity is preferably achieved without the use of additional cofactors such as, but not limited to, magnesium or manganese. As will be appreciated by one of ordinary skill in the art, the ability to employ a magnesium-free or manganese-free PCR is highly 20 advantageous. This is because the efficiency of a PCR procedure is known to be very sensitive to the concentration of the cofactors present in the reaction. An expert scientist is often required to calculate in advance the concentration of cofactors or to perform many tests, with varying concentrations of cofactors, before achieving the desired amplification efficiency. 25 The use of the liquid composition of the present invention thus allows the user to execute a simple and highly efficient multi-cycle PCR procedure without having to calculate or vary the concentration of cofactors in the PCR mix. Additionally, it has been found by the present inventor that polymerase chain reaction can take place devoid of any additional buffers or liquids. One of the major 30 problems associated with the application of PCR to clinical diagnostics is the susceptibility of PCR to carryover contamination. These are false positives due to the contamination of the sample with molecules amplified in a previous PCR. The use of the liquid composition of the present invention as a sole PCR mix significantly WO 2005/079153 PCT/IL2005/000198 27 reduces the'probability of carryover contamination, because the entire procedure can be carried out without the need for any additional buffers or liquids, hence avoiding the risk of contamination. Thus, according -to a preferred embodiment of the present invention there is 5 provided a kit for polymerase chain reaction. The PCR kit of the present invention may, if desired; be presented. in a pack which may contain one or more units of the kit of the present invention. The pack may be accompanied by instructions for using the kit. The pack may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of 10 laboratory supplements, which notice is reflective of approval by the agency of the form of the compositions: The kit comprises, preferably in separate packaging, a thermostable DNA polymerase, such as, but not limited to, Taq polymerase and the liquid composition of the present invention. Additionally, the kit may comprise at least one dNTP, such as, 15 but not limited to, dATP, dCTP, dGTP, dTTP. Analogues such as dITP and 7-deaza dGTP are also, contemplated. According to a preferred embodiment of the present invention the kit may further comprise at least one control template DNA and/or at least one at least one control primer to allow the user to perform at least one control test to ensure the PCR 20 performance. According to an additional aspect of the present invention there is provided a method of amplifying a DNA sequence, the method comprises the following method steps illustrated in the flowchart of Figure 2b. In a first step of the method, the liquid composition of the present invention is provided, and in a second step, a plurality of 25 PCR cycles is executed on 'the DNA sequence in the presence of the liquid composition, The PCR cycles can be performed in any way known in the art, such as, but not limited to, the PCR cycles, disclosed in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965 188, 5 512,462,- 6,007,231, 6,150,094, 6,214,557, 6,231,812, 30 6,391,559, 6,740,510 and International Patent application No. WO/9911823. Preferably, in each PCR cycle, the DNA sequence is first treated to form single-stranded complementary strands. Subsequently, pair of oligonucleotide primers which are specific to the DNA sequence are added to the liquid composition. The WO 2005/079153 PCT/IL2005/000198 28 primer pair is then annealed to the complementary sequences on the single-stranded complementary strands. Under proper conditions, the annealed primers extend to synthesize extension products which are respectively complementary to each of the single-strands. 5 Anchoring polynucleotide to a solid support such as glass beads can be of utmost benefit in the field of molecular biology research and medicine. As used herein "polynucleotides" are defined as DNA or RNA molecules linked to form a chain of any size. Polynucleotides may be manipulated in many ways during the course of 10 research and. medical applications, including, but not limited to amplification, transcription, reverse transcription, ligation, restriction digestion, transfection and transformation. As used herein, "ligation" is defined as the joining of the 3' end of one nucleic acid strand with the 5'end of another, forming a continuous strand. "Transcription" is 15 defined as the synthesis of messenger RNA from DNA. "Reverse transcription" is defined as the synthesis of DNA from RNA. "Restriction digestion" is defined as the process of cutting DNA molecules- into smaller pieces with special enzymes called Restriction Endonucleases. "Transformation" is the process by which bacterial cells take up naked DNA molecules "Transfection" is the process by which cells take up 20 DNA molecules. Typically, DNA manipulations comprise a sequence of reactions, one following the other. Thus, as a typical example DNA can be initially restriction digested, amplified and then transformed into bacteria. Each reaction is preferably performed under its, own suitable reaction conditions requiring its own specific buffer. 25 Typically, in between each reaction, the DNA or RNA sample must be precipitated and then' reconstituted in its new appropriate buffer. Repeated precipitations and reconstitutions takes time and more importantly leads to loss of starting material, which can be of utmost relevance when this material is rare. By anchoring the polynucleotides to a solid support, this is avoided. 30 Thus, according to an additional aspect of the present invention, there is provided a liquid, composition comprising a liquid and nanostructures, the liquid composition is capable of allowing the manipulation of at least one macromolecule in the presence of a solid support, whereby each of the nanostructures comprise a core WO 2005/079153 PCT/IL2005/000198 29 material of a nanometric size surrounded by an envelope of ordered fluid molecules, the core material and the envelope of ordered fluid molecules being in a steady physical state. The solid support can. be any solid support capable of binding DNA and RNA 5 while allowing access of other molecules to bind and interact with the DNA and RNA in subsequent reactions as discussed above. The inventor of the present invention found that glass beads, which are capable of anchoring polynucleotides, require the liquid composition of the present invention in order for the polynucleotides to remain intact. Thus, as described in example 16, 10 DNA undergoing PCR amplification in the presence of glass beads requires the presence of the liquid composition of the present invention for the PCR product to be visualized. Beside nucleic acid amplification, the liquid composition of the present invention can be used as a buffer or an add-on to an existing buffer, for improving 15 many chemical and biological assays and reactions. Hence, in one embodiment the liquid composition of the present invention can be used to at least partially de-fold DNA molecules. In another embodiment, the liquid composition of the present invention can be used to facilitate isolation and purification of DNA. 20 In an additional embodiment, the liquid composition of the present invention can be used for stabilizing enzyme activity of many enzymes, either bound or unbound enzymes, such as, but not limited to, Alkaline Phosphatase or #-Galactosidase. In still another embodiment, the liquid composition of the present invention can also be used for enhancing binding of macromolecule to a solid phase matrix. As 25 further demonstrated in the Examples section that follows (see Example 11), the liquid composition of the. present invention can enhance binding to both hydrophilic and hydrophobic substances. In addition, the liquid composition of the present invention can enhance binding-to substances having hydrophobic regions and hydrophilic regions. The binding of many macromolecules to the above substances can be 30 enhanced, including, without limitation macromolecule having one or more carbohydrate hydrophilic or carbohydrate hydrophobic regions, antibodies, polyclonal antibodies, lectin, DNA molecules, RNA moleculs and the like.

WO 2005/079153 PCT/IL2005/000198 30 Additionally, as demonstrated in the Examples section that follows (see Examples 12-14), it has been found by the present inventor that the liquid composition of the present invention can be used for increasing a capacity of a column, binding of nucleic acids to a rdsin and improving gel electrophoresis separation. 5 Additional objects, advantages and novel features of the present invention will become apparent'to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as 10 claimed in the claims section below finds experimental support in the following examples. EXAMPLES Reference is now made to the following examples, which, together with the above descriptions, illustrate the invention in a non limiting fashion. 15 The examples below are directed at various characterization experiments, which have been performed using the nanostructure and the liquid composition of the present invention. The nanostructure and the liquid composition used in the following experiments were manufactured in accordance with the present invention as further detailed hereinabove> More specifically, in the production method which was 20 employed, to provide the nanostructure and the liquid composition, the following protocol was used: First, a .powder of micro-sized BaTiO 3 was heated, to a temperature of 880 'C. Second, under, condition of continues wave RF radiation at a frequency of 915 MHz, the heated powder was immersed in water at a temperature of 2 'C. The radiation and 25 sudden cooling causes the micro-sized particles of the powder to break into nanostructures. Subsequently, the liquid composition (nanostructure and water) was allowed to heat to room temperature. In the following examples, various liquid compositions, manufactured according to a preferred embodiment of the present invention, are referred to as LCl, 30 LC2, LC3, LC4, LC5, LC6, LC7, LC8 and LC9.

WO 2005/079153 PCT/IL2005/000198 31 EXAMPLE 1 Solid-Fluid Coupling and Clustering of the Nanostructure In this Example, the coupling of the surrounding fluid molecules to the core material was investigated by Cryogenic-temperature transmission electron microscopy 5 (cryo-TEM), which is a modern technique of structural fluid systems. The analysis involved the following steps in which in a first step, the liquid composition of the present invention (LC1) was cooled ultra-rapidly, so that vitreous sample was provided, and in a second, step the vitreous sample was examined in via TEM at cryogenic temperatures. 10 Figures 3a-e. show TEM images of the nanostructures of the present invention. Figure 3a is an image of a region, about 200 nm long and about 150 nm wide, occupied' by four nanostructures. As shown in Figure 3a, the nanostructures form a cluster via intermediate regions of fluid molecules; one such region is marked by a black arrow. Striations surrounding the nanostructures, marked by a white arrow in 15 Figure 3a, suggest a crystalline structure thereof. Figure 3b is an image of a single nanostructure, about 20 nm in diameter. A bright corona, niarked by a white arrow, may be a consequence of an optical interference. effect,- commonly known as the Fresnel effect. An additional, darker, corona (marked by a black arrow in Figure 3b) was observed at a further distance from 20 the center of the nanostrugture, as compared to the bright corona. The dark corona indicate an ordered. structure of fluid molecules surrounding the core, so that the entire nanostructure is in a steady physical state. Figures 3c -e are of equal magnification, which is illustrated by a scale-bar shown in Figure 3c. Figure 3c further demonstrates, in a larger magnification than in 25 Figure 3a, the ability of the nanostructures of the present invention to cluster. Figure 3d shows a single nanostructure characterized by crystalline facets and Figure 3e shows a. cluster of two nanostructures in which one is characterized by crystalline facets and the other has a well defined dark area which is also attributed to its crystalline structure.

WO 2005/079153 PCT/IL2005/000198 32 EXAMPLE 2 Effect of dye on the Liquid Composition The interaction of the liquid composition of the present invention with dye was investigated. A liquid composition, manufactured as further detailed above, was dyed 5 with a Ru based dye (N3) dissolved in ethanol. One cuvette containing the liquid composition of the present invention (LC1) was exposed to the dye solution for 24 hours. A second cuvette containing the liquid composition was exposed to the following protocol: (i) stirring, (ii) drying with air stream, and (iii) dying. Two additional cuvettes, containing pure water were subjected 10 to the above tests as control groups. Figure 4 shows the results of the four tests. As shown in Figure 4 the addition of the dye results in the disappearance of the dye color (see the lower curves in Figure 4), in contrast to the case of pure water (see the lower curves in Figure 4) where the color was maintained. Hence, the interaction with the nanostructures affects the dye 15 spectrum by either changing the electronic structure or by dye oxidation. The color disappearance is best evident in the picture of the cuvette. All samples presented in Figure .4 containing the liquid composition of the present invention were stirred. The sample designated "dry S-R" was kept dry for 24 hours; the sample designated wet "S-R" was maintained with ethanol; the sample designated 20 "dye S-R" was dyed (dye in ethanol) and the sample designated "dye S-dry R" was dried and remeasured. EXAMPLE 3 Effect of High g Centrifugation on the Liquid Composition 25 Tubes containing the liquid composition of the present invention were centrifuged at high g values (about 3 Og) . Figures 5a-b Show results of five integrated light scattering (ILS) measurements of the liquid composition of the present invention (LCl) after centrifugation. Figure 5a shows signals recorded at the lower portion of the tubes. As shown, no signal from 30 structures less that 1 p'tm was recorded from the lower portion. Figure 5b shows signals recorded at the upper portion of the tubes. A clear presence of structures less than 1 pm is shown. I all the measurements, the location of the peaks are consistent with nanostructures of about 200-300 nm.

WO 2005/079153 PCT/IL2005/000198 33 This experiment demonstrated that the nanostructures have a specific gravity which is lower than the specific gravity of the host liquid (water). EXAMPLE 4 5 pH Tests The liquid composition of the present invention was subjected to two pH tests. In a first test, caraminic indicator was added to the liquid composition of the present invention (LC1) so as to provide an indication of affective pH. Figure I6a shows the spectral change of the caraminic indicator during titration. 10 These spectra are used to examine the pH of the liquid composition. Figure 6b shows that the liquid composition spectrum is close to the spectrum of water at pH 7.5. Figure 6c shows that unlike the original water used in the process several liquid composition samples have pH 75spectra. The results of the first test indicate that the liquid composition has a pH of 7.5, 15 which is more than the pH value of pure water. In a second, test, Bromo Thymol Blue (BTB) was added to the liquid composition of the present invention (LCl). This indicator does not affect the pH itself but changes colors in the pH range of interest. The absorption spectrum for samples No. 1 and 4 is shown in Figure 7, where 20 "HW" represents the spectrum of the liquid composition; '+' represents positive quality result and "-." represents negative quality result. Two absorption peaks of BTB are shown in Figure 7. These are peaks result in a yellow color for the more acidic case and green-blue when movie basic. When added to liquid composition, a correlation between the color and the quality of the liquid composition was found. The green color 25 (basic) of the liquid composition indicates higher quality. EXAMPLE 5 Zeta Potential Measurement Zeta (C) potential measurements were performed on the liquid composition of 30 the present invention. Figure 8 shows Q potential of 6 samples: extra pure water, extra pure water shifted .to pH 8, extra pure water shifted to pH 10, two samples of the liquid composition with sitive quality and one sample of the liquid composition with WO 2005/079153 PCT/IL2005/000198 34 negative quality. The measurement of the ( potential was performed using a Zeta Sizer. As shown, the ( potential of the liquid composition of the present invention is significantly higher, indicating a high mobility of the nanostructures in the liquid. 5 EXAMPLE 6 Bacteriophage Reaction The effect of the liquid composition of the present invention (LC9) on bacteriophage typing was investigated. 10 Materials and methods 1) Bacteriophages No. 6 and 83A of a standard international kit for phage typing of staphylococcus aureus (SA), obtained from Public Health Laboratory In Colindale, UK, The International Reference Laboratory (URL: www.phls.co.uk), were examined. 15 2) Media for agar plates: Nutrient agar Oxoid No2 (catalog number CM 67 Oxoid Ltd.) + CaCl 2 . After autoclave sterilization 20 ml of CaCl 2 was added for each liter of medium. 3) Media for liquid cultures: Nutrient Broth No2 Oxoid: 28 gr/lliter. 4) Phage typing concentration: each bacteriophage was tested at 1 and 100 20 RTD (Routine Test Dilution). 5) -Propagation ofphage: each phage was propagated in parallel in control d nd -in tested media based on the liquid composition of the present invention.. 6) ' The. bacteriolysis surface area was measured using computerizes 25 !'Sketch" software for surface area measurements. 7) statistical analysis: analysis-of-variance (ANOVA) with repeated measures was used for optic density analysis, and 2 ways ANOVA for lysis surface area measurements using SPSSTM Software for Microsoft WindoWSTM. 30 Results Acceler'atio of bacteriophage reaction. Figures 9a-b illustrate'. the bacteriophage reaction in the tested media, as follows: Figure . 9a 'Shows Bacteriophages No. 6 in a control medium (right hand side) WO 2005/079153 PCT/IL2005/000198 35 and in the liquid composition of the present invention (left hand side); Figure 9b shows Bacteriophages No. 83A in a control medium (right hand side) and in the liquid composition of the present invention. The bacteriophage reaction in the liquid composition of the present invention demonstrated an accelerated lysis of bacteria 5 (within 1 hour in the liquid composition and 3 hours in the control media). Superior lysis areas on the tested plates were observed immediately and remained larger. at, 24 hours of -incubation. Vivid differences between the control and tested plates were demonstrated by measuring RTD concentrations. Area measurements 10 Figure 10 is a histogram showing a comparison between the bacteriolysis surface areas of the control and liquid composition. Statistic significance was determined using 2 ways ANOVA for phage typing. The corresponding numbers are given in Tables 2 and 3, below. Table 1 Phage Control Composition No.6 2.488 6.084 2.238 -2.441 3.246 5.121 Average 2.657333 4.548667 STD 0.524901 1.887733 No.83 2.898 7.369 2.61 4.748 4.692 8.261 -Average 3.4 6.792667 STD 1.128133 1.826037 15 Table 2 .2 way ANOVA - dependent variable: Area Factor SS d.f. MS F Significance Phage 6.69 1 6.69 3.168 0.113 RO Water 20.94 1 20.94 9.917 0.014 Phage-Water 1.691 1 1.691 0.801 0.397 A significant increase in phage reaction area was found with the liquid composition (p=0.014). There was no significant difference between the phages (p=0.113) and. media interactions (p=0.397), which demonstrate that the liquid WO 2005/079153 PCT/IL2005/000198 36 composition of the present invention has identical trends of effect on both tested phages. R TD determination Figure 11 shows increased dilution by 10 times in each increment. Increased 5 concentration of* phages in the liquid composition of the present invention was observed in well 3 in-which dilution was 100 times more than well 1. Bacteriolysis- optic density reading Figure 12 is a graph of.the optical density (OD) in phage No. 6, as a function of time. The corresponding numbers for mean change from start and the OD of phage 10 reaction are given in Tables 3 and 4, respectively. The ANOVA for repeated measures is presented in Table 5. Table 3 -_ phage No. 6 phage No. 83A Time control composition control composition 15' 1.079109 1.052213 1.035938 1.038375 36'. .1.142857 1.102157 1.139063 1.128668 67' 1.207373 1.205448 1.221875 1.180587 150' 1.407066 1.321226 1.366406 1.345372 275' 1.515361 1.434733 1.810938 1.3386 311' 1.483871 1.449489 1.686719 1.327314 22h 1.616743. 1.094211 2.735938 0.87246 Table 4 phage No. 6 phage No. 83A Time - control composition control composition 0 '0.668. 0.446 0.642 0.428 0 0.634 0.435 0.638 0.458 Average' 0.651 .0.4405 0.64 0.443 STD 0.024042 0.007778 0.002828 0.021213 15 0.733- 0.471 0.642 0.458 15 0.672 0.456 0.684 0.462 Average . 0.7025 0.4635 0.687 0.46 STD 0.043134 0.010607 0.029698 0.002828 36 0.764 0.485 0.728 0.486 36 0.724 0.486 0.73 0.514 Average 0.744 0.4855 0.729 0.5 STD 0.028284 0.000707 0.001414 0.019799 67 0.799 0.537 0.777 0.523 67 0.773 0.525 0.787 0.523 Average 0.786 '0.531 0.687 0.523 WO 2005/079153 PCT/IL2005/000198 37 STD 0.018385 0.008485 ]0.007071 0 150 0.966 0.571 0.87 0.596 150 0.866 0.593 0.879 0.596 Average 0.916 0.582 0.8745 0.596 STD 0.070711 0.015556 0.006364 0 275 0.978 0.639 1.132 0.602 275 0.995 . 0.625 1.186 0.584 Average 0.9865 0.632 0.687 0.593 STD 0.012021 0.009899 0.038184 0.012728 311 0.964 0.644 1.081 0.602 311 0.968 0.633 1.078 0.574 Average 0.966 0.6385 1.0795 0.588 STD 0.002828 0.007778 0.002121 0.019799 22h 1.003 0.463 1.691 0.388 22h 1.102 0.501 1.811 0.385 Average 1.0525 0.482 0.687 0.3865 STD 0.070004 0.02687 0.084853 0.002121 Table 5 Phage Factor SS d.f. MS F Significance No.83 time 17804.37 6 2967.396 164.069 0.001 time-water 27350 6 4558.334 252.033 0.001 control-LC 10851.38 1 10851.38 55.805 0.017 No.6 time 6449.544 6 10.74.924 32.31 0.001 time-water '2024.998 6 337.5 10.145 0.001 control- LC 904.547 1 904.547 15.385 0.059 As demonstrated in Figure 12 and Tables 3-5, there is a significant correlation between the medium and the time. More specifically, there is a significant different 5 trends in time between the control and the liquid composition of the present invention (p=0.001) both in pliage No. 6 and in phage No. 83A. The phage reaction in the liquid composition of. the present invention has significantly different trend with opposite direction. At 22 hour an addition "kick" of lysis was observed which may be due to 10 increased-potency of the phage. All the controls OD (media alone, phage alone, bacteria alone, in control and composition with different phages) demonstrated no difference between themselves and were significant different from tested reaction.

WO 2005/079153 PCT/IL2005/000198 38 Conclusions The liquid composition of the present invention accelerates the phage reaction time (x3); and- increases the bacteriolysis surface area; increases the RTD (x100 or more) 5 The bacteriophage reactions in the liquid composition of the present invention demonstrate opposite trends compare to control in OD measurements, and increased potency with time. Discussion The kinetics of phage-host interaction has been enhanced in media containing 10 the liquid composition. This was observed in repeated experiments and in measured "growth curve kinetics." The parameters influencing the kinetics are independent of measured factors (e.g., pH, temperature, etc.) Not only does phage concentration increase but also its potency, as was observed after 22 hours of reaction. Phages in control media are non effective at a time when phages in the liquid composition of the 15 present invention are still effective. In addition, the propagating strains pre-treated with the liquid composition are much more effective. EXAMPLE 7 Effect of the Liquid Composition on Phage-Bacteria Interaction 20 The effect of the liquid composition of the present invention on Lambda (k) phage was investigated. % phage is used in molecular biology for representing the genome DNA of. organisms. The following experiment relies on standard % phage interaction applications. In all the experiments the materials in the test groups were prepared with the liquid composition as a solvent. The materials in control groups 25 were prepared as described hereinbelow. The pH of the control groups was adjusted to the pH of the liquid, composition solutions, which was between 7.2 and 7.4. Materials and Methods 1) LB mediuin 10 g. of Bacto .Tryptone, 5 g of Yeast extract, 10 g of NaCl dissolved in 30 1000 ml of distilled water, and then sterilized by autoclave (121 'C, 1.5 atm for 45 minutes).

WO 2005/079153 PCT/IL2005/000198 39 2) LB plates 15g of Bacto Agar were added to 1000 ml of LB medium, mixed and autoclaved as described above. After cooling to 50'C, the medium was poured into sterile plastic plates. The plates were pre-incubated for two 5 days before use. 3) Top Agarose 0.7 % 100 ml of LB medium were mixed with 0.7 g of chemically pure, electrophoresis grade agarose (from Difco or other supplier), and then sterilized by autoclave (121 'C, 1.5 atm during 45 minutes). 10 4) MgSO 4 -10 mM 1.2 g of MgSO 4 were dissolved in 1000 ml distilled water and sterilized by autoclaving. 5) Maltose 20 % (w/v) 200 g of maltose were dissolved in 1000 ml distilled water, and 15 sterilized by filtration through a 20 ptm filter. 6) MgSO 4 -1 M 120.37 g of MgSO 4 were dissolved in 1000 ml distilled water and sterilized by autoclaving. 7) LB with 10 mM of MgSO 4 and 0.2 % of maltose 20 100 ptl of MgSO 4 1M and 100 pl of maltose 20% were added to 99.8 ml of LB medium. 8) SM buffer (phage storage buffer) 5.8 g of NaCl, 2 g of MgSO 4 , 50 ml of 1M Tris HCl (pH 7.5), 5 ml of 2 % (w/v) gelatin were dissolved in distilled water, to a final volume of 25 1000 ml, and then, sterilized by autoclaving. 9) Bacterial strain (Host) E. coli XL1 Blue MRA (Stratagene). 10) Phage: k GEM 11 (Promega). 30 1) Bacterial cultivation on LB plates X 1 cells w ere dispersed on the LB plate with a bacteriological loop according to a common procedure of bacterial inoculation. The plates were incubated at 37 'C for 16 hours.

WO 2005/079153 PCT/IL2005/000198 40 12) Bacterial cultivation in LB liquid medium A single colony of XL1 cells was picked from an LB plate and inoculated in LB liquid medium with subsequent incubation at 37 0 C for 16 hours (overnight), with shaking at 200 rpm. 5 13) Infection of the host bacterial strain by the phage XL1 cells were inoculated into the LB medium supplemented with 10 mM of MgSO4 and 0.2% of maltose. Incubation at 37 IC with shaking at 200 rpm continued, until turbidity of 0.6 at a wavelength of 600 nm was achieved (4-5 hours). The grown culture was centrifuged at 4000 10 rpni for 5 minutes. Supernatant was discarded, and the bacteria were re-suspended into the 10 mM of MgSO 4 , until turbidity of 0.6 at wavelength of 600 nm was achieved. A required volume of SM buffer containing the phages was added to 200 ml of the re-suspended bacteria. After incubation at 37 'C for 15 minutes two alternative 15 procedures wericarried out: (i) For lysate preparation an appropriate volume of LB medium was added to the host-phage mixture, and incubated at 37 'C for 16 hours (bvernight),-with shaking at 200 rpm. (ii) For phage appearance on solid medium (plaques), a molten Top 20 Agarose (50 C) was poured on the host-phage mixture and quickly mixed and spread on the pre-warmed LB plate. After agarose solidification, incubation was performed at 37 'C for 16 hours (overnight). 14) Extraction of the phage DNA 25 Bacterial lysates were centrifuged at 6000 rpm for 5-10 minutes for sedimentation of the bacterial debris. Supernatant was collected and centrifuged at -14000 rpm for 30 minutes for sedimentation of the phage particles. Supernatant was discarded and the phage pellet was re suspended in SM buffer without gelatin. A mixture of nucleases 30 (RNase and DNase from any supplier) was added to the re-suspended hage for a final concentration of 5 - 10 Weiss units per 1 tl of the phage suspension. After an incubation of 30 minutes at 37 'C, as WO 2005/079153 PCT/IL2005/000198 41 required for complete digestion of any residual bacterial nucleic acids, the DNA of the phage was extracted by the following procedure: (i) extraction with phenol: chloroform: iso-amil-alcohol (25:24:1 v/v); (ii) removing of phenol contamination by chloroform; 5 (iii) precipitation to final concentration of 0.3 M Potassium Acetate and one volume of iso-propanol; (iii) washing with 70% ethanol; and (iv) drying and re-suspension in distilled water for further analysis. Results 10 Plaque Forming Unit (PFU) titer experiment Phage suspensions were prepared from phage stock in SM buffer in series of 1/10 dilutions: one in SM buffer based on liquid composition of the present invention and one in SM buffer based on ddH 2 0. 1 pl of each dilution was incubated with 200 pl of competent bacterial host (see 15 methods, item 13). The suspension was incubated at 37 IC for 15 minutes to allow the bacteriophage to inject its DNA into the host bacteria. After incubation a hot (45 50 'C) top agarose -was added and dispersed on the LB plate. Nine replications of each dilution and treatment were prepared. Table 6 below presents the PFU levels which were counted after overnight 20 incubation. Table 6 Phage Dilution Control Composition 506 724 684 845 761 704 651 879 101 618 617 683 612 932 860 697 652 746 891 Average 697.5556 753.7778 S.D. 115.6083 115.4597 WO 2005/079153 PCT/IL2005/000198 42 70 119 11 129 77 90 32 111 10i 96 91 53 106 56 120 71 100 25 183 Average 54.55556 116.5556 S.D. 27.41857 28.20067 The numbers were modified by square root transformation to normalize the data as required for performing parametrical tests. Table 7 below shows results of data 5 analysis by factorial ANOVA. Table 7 Factors SS d.f. MS Significance Treatment 48.9147 1 48.9147 P = 0.01 Concentration 2893.0255 1 2893.0255 P = 0.01 Interaction 147506 1 14.7506 P = 0.01 Error. .. 239.8006 32 7.4938 1 Significance levels: P 0.05 (d.f. 1; 32)= 4.14909, P 0.01 (d.f. 1; 32) = 7.49924. A significant effect in the PFU titer was detected between concentrations (0.001 10 against 0.0001), treatment (test against control) and interactions (any combination of treatment and concentration). Significant differences between concentrations were expected as a.ensequence of experiment structure. However, a significant increase in the PFU titer, as caused by the liquid composition of the present invention treatment requires special explanation, which is presented in the discussion section of this 15 example, hereinbelow. E. coli Strain XLI-Blue Bacterial growth in LB. 2l of a bacterial suspension were inoculated on each 1/8 sector of two LB plates (16 inoculation totally), both in control and liquid composition of the present invention based media. After incubation at 37 IC for 3 days, colony shapes and sizes 20 were observed. No significant differences were observed, between control and the liquid composition treatments..

WO 2005/079153 PCT/IL2005/000198 43 Phage growth on LB bacterial culture (lysate) Lysates were prepared as described in methods (item 13), centrifuged at 6000 rpm for 5-10 minutes to sediment bacterial debris and turbidity was measured at 600 nn. DNA was then extracted from lysates as described hereinabove in the methods 5 (item 14). No significant differences were observed between control and the liquid composition treatments both in turbidity and extracted DNA concentration (0.726 pg/pl in control; 0.718 p g/l in the liquid composition). Discussion In two independent tests out of three, a significant increase in PFU at low phage 10 dilutions (103 ,and 104) was observed, when the liquid composition of the present invention was used.compared to the control. The probable explanation of the above observation lies in the fact that plaque formation depends on two separate processes: the phage's ability to infect their hosts (infectivity) and the host compatibility to the phage. 15 The host compatibility depends on the ability of the phage to adopt bacterial mechanisms for phage reproduction. No correlation between the liquid composition of the present invention to the host compatibility was found. Increased compatibility can be established by the observation of either larger plaques than those of control (a greater distance from the initial infection site), or a greater number of phage particles 20 than that of the control. The fact that the liquid composition of the present invention did not affect DNA phage level supports the previous finding. The infectivity depends on essential phage particles and/or on the bacterial cell's capability to be infected by the phage. The significant increase in PFU when the 25 liquid composition of the present invention was used (about 2-fold greater than the control) indicates that the liquid composition of the present invention affects the infectivity. Pre-infection treatments (see methods, item 13), are required for increasing probability of infection by preparing competent bacteria, which are easier infected by phage than non-treated bacteria. 30 At low phage dilutions the limiting factor of the PFU formation is the host cell's ability to be infected by the phage. It seems that bacteria treated and grown with the liquid composition of the present invention had an increased capability of infection by the phage.. It is therefore WO 2005/079153 PCT/IL2005/000198 44 assumed that the liquid composition increases the affinity between bacterial receptors and phage particles. EXAMPLE 8 5 Effect of the Liquid Composition on the Adherence of Coagulase-Negative Staphylococci to Microtiter Plate Production of slime polysaccharide, is crucial to biofilm generation and maintenance, and plays a major part as a virulence factor in bacteria [Gotz F., "Staphylococcus and biofilms," Mol Microbiol 2002, 43(6):1367-78]. The slime 10 facilitates adherence of bacteria to a surface and their accumulation to form multi layered clusters. Slime also protects against the host's immune defense and antibiotic treatment [Kolari M. et al., "Colored moderately thennophilic bacteria in paper machine biofilms," to apear in J Ind Microbiol Biotechnol 2003]. Biofilm produced by bacteria can cause problems also in industry. 15 Most of current concepts for the prevention of slime are associated with search for new anti-infective active in biofilm and new biocompatible materials that complicate biofilm. It has been demonstrated [Besnier JM et al., "Effect of subinhibitory concentrations of antimicrobial agents on adherence to silicone and hydrophobicity of 20 coagulase-negative staphylococci," Clin Microbiol Infect 1996, 1(4):244-248] that the adherence of coagulase-negative staphylococci onto silicone can be modified by sub MICs of antimicrobial agents. This effect was different in the slime-producing and non-slime-prbducinj strains, and was not correlated with the mechanism of the inhibitory effect of these antimicrobial agents, or the modification of hydrophobicity 25 suggesting that some surface components, not involved in hydrophobicity, could play a role in vitro adherence. The bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms [Konig 30 DP et al., "In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements," Langenbecks Arch Surg 2001, 386(5):328-32]. In vitro studies of different bone cements containing antibiotics, developd for the prevention of biomaterial-associated infection, could not WO 2005/079153 PCT/IL2005/000198 45 always demonstrate complete eradication of biomaterial-adherent bacteria. Further efforts are done to find better protection from slime adherence. In addition, surface interaction can modify slime adherence. For example, Farooq et al. [Farooq M et al., "Gelatin-sealed polyester resists Staphylococcus 5 epidermidis biofilm infection," J Surg Res 1999, 87(l):57-61] demonstrated that gelatin-impregnated polyester grafts inhibit Staphylococcus epidermidis biofilm infection in a canine model of aortic graft interposition. Gelatin-impregnated polyester grafts demonstrated in vivo resistance to coagulase-negative staphylococcal biofilm infection. 10 The objectives of the experiments in this example were to investigate the effect of the liquid composition of the present invention on the adherence to plastic of a slime-producing Staphylococcus epidermidis (API-6706112) Methods The bacteria used were identified using Bio Merieux sa Marcy 1' Eoile, France 15 (API) with 98.4 %. confidence for Staphylococcus epidermidis 6706112. Table 8, below summarizes the three bacterial strains which were used. Table 8 Bacterial strain API No. Confidence 24 6706112 98.4% 44 6706112 98.4% 56 6706112 98.4% Slime adherence was quantitatively examined with a spectrophotometer optical density (OD) technique, as 'follows. Overnight cultures in TSB with the liquid composition of the present invention and with regular water were diluted 1:2.5 with 20 corresponding media and placed in sterile micro titer tissue culture plates (Cellstar, Greniner labortechnik, Tissue culture plate, 96W Flat bottom, with LID, sterile No. 655180) in a total volume of 250 il each and incubated at 37 'C. The plates were rinsed 3 times with tap water, stained with crystal violet, and rinsed 3 more times with tap water. After drying, the OD of the stained adherent bacterial films was measured 25 with a MicroElisa Auto reader (M5000; Dynatech Laboratories, Alexandria VA.) by using wavelength of 550nm. OD of bacterial culture was measured before each staining using dual filter of 450nm and 630nm. The test of each bacterial strain was performed in quadruplicates.

WO 2005/079153 PCT/IL2005/000198 46 The experiment was designed to evaluate slime adherence at intervals. The time table for the kinetics assessment was 18, 20, 22, 24 and 43 hours. All three (3) strains were evaluated on the same plate. The liquid composition was used for standard media preparation and underwent standard autoclave sterilization. 5 Adherence values were compared using ANOVA with repeated measurements for the same plate examination; grouping factors were plate and strain. A three-way ANOVA was used for the different plate examination using SPSSTM 11.0 for Microsoft WindowsTM. Results 10 Figures 13a-c show the OD in all the slime-producing Staphylococcus epidermidis (see Table above). Adherence was significantly different (p < 0.001) in the liquid composition of the present invention. The kinetics of Strains 24 and 44 demonstrated increased slime adherence (Figures 13a-b, respectively) and strain 56 demonstrated decreased adherences (Figure 15 13c). Time was found to be a significant factor in decreasing adherence where in the last hour the lowest adherences were observed. Significant differences were found between the stains (p<0.001), each strain having its own adherence characteristics. A significant interaction was found between the different strains and time (p<0.001), the differences between the strains being time dependent. Regression analysis found no 20 interaction between time and type of water used (p=0.787). The differences between the adherence in the liquid composition and in the control was maintained at all times, beginning at the 18th hour and peaking at the 43rd hour. A significant interaction between the strains and water (p<0.001) was found. The differences between the. liquid composition and the control water were strain 25 dependent. Each strain had its own adherence characteristics. No interaction was found betweenstrains, time and water (p=0.539). Table 9, below suimarizes the results of Slime adherence kinetics (Three-way ANOVA). -_ Table 9 Factor SS. df MS F Significance Time 1.356 4 0.339 8.624 0.001 Strain 28.285 2 14.143 359.743 0.001 Water 0.731 . 1 0.731 18.599 0.001 WO 2005/079153 PCT/IL2005/000198 47 Time-Strain - 1.072. 8 0.134 3.41 0.002 Time-water 6.75E-02 4 1.69E-02 0.429 0.787 Strain-Water 1.052 2 0.526 13.374 0.001 Time-Strain-water 0.276 8 3.45E-02 0.877 0.539 Repeat slime adherence experiments were performed at 24 hours post incubation on different plates of the same type, where each strain was incubated on a separate micro titer plate. 5 Figure 14 is a histogram representing 15 repeat experiments of slime adherence on different micro titer plates. As shown, the adherence in the presence of the liquid composition is higher than the adherence in the control. Significant adherence differences in the liquid composition and control, between the' micro titer plates, and, among the strains were found (p<0.001). 10 Significant interactions were found between plates, strain and the type of water used. The extent of adherence is dependent on the strain, on the plate, and, on the water used. Table 10, below summarizes the results of slime adherence on separate micro titer plates (Three-way ANOVA). Table 10 Factor SS df MS F Significance Plate 0.572 2 0.286 29.798 0.001 Strain 9.484 2 4.742 494.346 0.001 Water 1.288 1 1.288 134.276 0.001 Plate-Strain 1.265 4 0.316 32.976 0.001 Plate-water 215E-01 2 1.07E-01 11.183 0.001 Strain-Water 0.288 2 0.144 15.021 0.001 Plate-Strain-wate 0.259 4 6.47E-02 6.744 0.001 15 To examine he possibility of plate to plate variation, multiple analyses were performed on the same plate (all strains). Figure 15s hows slime adherence differences in the liquid composition of the present invention and the control on the same micro titer plate. Tables 11-12, below 20 summarizes the results of slime adherence on the same micro titer plat (ANOVA with repeated measurements). As shown in Tables 11-12, a significant difference between slime adherence with the liquid composition and Control was once more confirmed. However, new significant interactions between plate (p<0.001), strain (p<0.001), and water (p<0.001) WO 2005/079153 PCT/IL2005/000198 48 were also found, confirming that the- adherence differences in the liquid composition depend also on the plate, strain and interactions therebetween. A significance difference in adherence between the strains and the plate points out the possibility of plate to plate variations. Plate to plate variations with the liquid 5 composition indicate that there may be other factors on the plate surface or during plate preparation which could interact with the liquid composition. Table 11 Factor SS df MS F Significance Plate 3.726 2 1.863 40.32 0.001 Strain 8.93 2 4.465 96.623 0.001 Plate-Strain 1.019 4 0.255 5.515 0.001 Table 12 Factor-within subjects effects F Significance composition-control 17.106 0.001 comp6sition-control-plate 6.496 0.001 composition-control-strain 50.165 0.001 composition-control-plate-strain 0.896 0.001 10 Discussion The ability, of the liquid composition of the present invention to change bacterial adherence through its altered surface adhesion was studied. The media with the liquid composition contained identical buffers and underwent identical autoclave 15 sterilization, as compared to control medium ruling out any organic or PH modification. Hydrophocity modification in the liquid composition can lead to an environmental preference for the slime to be less or more adherent. The change in surface characteristics may be explained by a new order, which is introduced by the nanostructures, leading to. a change in water hydrophobic ability. 20 EXAMPLE 9 Electrochemical Deposition Tests The liquid composition of the present invention has been subjected to a series of electrochemical deposition tests, in a quasi-two-dimensional cell.

WO 2005/079153 PCT/IL2005/000198 49 Experimental Setup The experimental setup is shown in Figures 16a-c. A quasi-two-dimensional cell 20, 125 mm in diameter, included a Plexiglas base 22 and a Plexiglas cover 24. When cover 24 was positioned on base 22 a quasi-two-dimensional cavity, about 5 1 mm in height, was formed. Two concentric electrodes 26 were positioned in cell 20 and connected.to a voltage source 28 of 12.4 ± 0.1 V. The external electrode was shaped as a ring, 90 mm in diameter, and made of a 0.5 mm copper wire. The internal electrode was shaped as a disc having a thickness of 0.1 mm and diameter of 28 mm. The external electrode was connected to the positive pole of the voltage source and the 10 internal electrode was connected to the negative pole thereof First, the experimental setup was used to perform an electrochemical deposition process directly on the liquid composition of the present invention and, for comparison, on a control solution composed of Reverse Osmosis (RO) water. Second, the experimental setup was used to examine the capability of the liquid 15 composition to leave an electrochemical deposition signature, as follows. The liquid composition was placed in cell 20. After being in contact with base 22 for a period of 30 minutes, the liquid composition was replaced with RO water and an electrochemical deposition process was performed on the RO water. 20 Results Figures 17a-b show electrochemical deposition of the liquid composition of the present invention (Figure 17a) and the control (Figure 17b). A transition between dense branching morphology and dendritic growth were observed in the liquid composition. The dense branching morphology spanned over a distance of several 25 millimeters front the face of the negative electrode. In the control, the dense branching morphology was observed only in close proximity to the negative electrode and no morphology transitionwas observed. Figure 18 shows electrochemical deposition of RO water in a cell, which was in contact with the liquid composition of the present invention for a period of 30 30 minutes. Comparing Figures 18 and 17b, one can see that the liquid composition leaves a clear signature on the surface .of the cell, hence allowing the formation of the branching and dendritic morphologies thereon. Such formation is absent in Figure 17b where the RO water was placed in a clean cell.

WO 2005/079153 PCT/IL2005/000198 50 The capability of the liquid composition to preserve an electrochemical deposition signature on the cell can be explained as a long range order which is induced on the RO water by the cell surface after incubation with the liquid composition. 5 EXAMPLE 10 Bacterial Colonies Growth Colony growth of Bacillus subtilis was investigated in the presence of the liquid composition of the present invention. The control group included the same 10 bacteria in thepresence of RO water. Figures 19a-b show results of Bacillus subtilis colony growth after 24 hours, for the liquid composition (Figure 19a) and the control (Figure 19b). As shown, the liquid composition of the present invention significantly accelerates the colony growth. 15 To further demonstrate the unique feature of the liquid composition of the present invention, an additional experiment was performed using a mixture of the raw powder, from which the nanostructure of the liquid composition is formed, and RO water, withoutthe manufacturing process as further detailed above. This mixture is referred to hereinafter as Source Powder (SP) water. 20 Figures 20a-c show the results of Bacillus subtilis colony growth, for the SP water (Figure 20a),. RO water (Figure 20b) and the liquid composition (Figure 20c). As shown, the, colony growth in the presence of the SP water is even slower than the colony growth in the RO water, indicating that the raw material per se has a negative effect on the bacteria. On the other hand, the liquid composition of the present 25 invention significantly accelerates the colony growth, although, in principle, the liquid composition is' cmposed of the same material. EXAMPLE 11 Macromolecule Binding to Solid Phase Matrix 30 A myriad of biological treatments and reactions are performed on solid phase matrices such as Microtitration plates, membranes, beads, chips and the like. Solid phase matrices may have different physical and chemical properties, including, for WO 2005/079153 PCT/IL2005/000198 51 example, hydrophobic properties, hydrophilic properties, electrical (e.g., charged, polar) properties and affinity properties. The objectives of the experiments described in this example were to investigate the effect of the liquid composition of the present invention on the binding of 5 biological material to microtitration plates and membranes having different physical and chemical properties. Methods The following microtitration plates, all produced by NUNCT were used: (i) MaxiSorp

TM

, which contains mixed hydrophilic/hydrophobic regions and is 10 characterized by high binding capacity of and affinity for IgG and other molecules (binding capacity of IgG equals 650 ng/cm 2 ); (ii) PolySorp

TM

, which has a hydrophobic surface and is characterized by high binding capacity of and affinity for lipids; (iii) MedimSorp T M , which has a surface chemistry between PolySorpTM and MaxiSorp T M , and is characterized by high binding capacity of and affinity for proteins; 15 (iv) Non-Sorp T h4 , which is a non-treated microtitration plate characterized by low binding capacity of and affinity for biomolecules; and (v) MultiSor T M , which has a hydrophilic surface and is characterized by high binding capacity of and affinity for Glycans. The following m-icrotitration plates of CORNINGTM (Costar) were used: (i) a 20 medium binding microtitration plate, which has a hydrophilic surface and a binding capacity to IgG of .250 ng/cm 2 ; (ii) a carbon binding microtitration plate, which covalently couples to carbohydrates; (iii) a high binding microtitration plate, which has a high adsorption capacity; and (iv) a high binding black microtitration plate, also having high adsorption capacity. 25 The binding, efficiency of bio-molecules to the above microtitration plates was tested in four categories: ionic strengths, buffer pH, temperature and time. The binding experiments were conducted by coating the microtitration plate with fluorescent-labeled bio-molecules or with a mixture of labeled and non-labeled bio-molecules of the same type, removal of the non-bound molecules by washing and 30 measuring the fluorescent signal remaining on the plate.

WO 2005/079153 PCT/IL2005/000198 52 The following protocol was employed: 1) Pre-diluting the fluorescent labeled bio-molecules to different concentrations (typically 0.4 - 0.02 ptg/ml) in a binding buffer. Each set of dilutions was performed in two binding buffers: (i) the liquid 5 composition of the present invention; and (ii) control RO water. 2) Dispensing (in triplicates) 100 pl samples from each concentration to the microtitration plates, and measuring the initial fluorescence level. 3) Incubating the plates overnight at 4 IC or 2 hours at 37 'C. 4) Discarding the coating solution. 10 5) Adding 150 pl of washing solution to each well and agitating at room temperature for 5 minutes. This washing step was repeated three times. Typical- washing solution includes 1 x PBS, pH 7.4; 0.05 % Tween20"; and 0.06 M NaCl. 6) Adding 200 pLl fluorescence reading solution including 0.01 M NaOH 15 and incubating for 180 minutes or overnight at room temperature. 7) Reading the fluorescence using a fluorescence bottom mode, with excitation wavelength of 485 nm, emission wavelength of 535 and optimal gain of 10 flashes. The effect of the liquid composition of the present invention on the biding 20 efficiency of glycoproteins (IgG of 150,000 D either labeled with Fluorescein isothiocyanate (FITX) or non-labeled) to the above described plates was investigated. IgG is a polyclonal antibody composed of a mixture of mainly hydrophilic molecules. The molecules have a carbohydrate hydrophilic region, at the universal region and are slightly hydrophobic at the variable region. Such types of molecules are known to bind 25 to MaxiSorp T M plates with very high efficiency (650 ng/cm 2 ). The following types of liquid composition of the present invention were used: LC1, LC2, LC3, LC4, LC5 and LC6, as further detailed hereinabove. Table 13 below summarizes six assays which were conducted for IgG. In Table 13, assays in which only labeled antibodies were used are designated Ab*, and assays 30 in which a mixture of labeled and non-labeled antibodies were used are designated Ab*/Ab.

WO 2005/079153 PCT/IL2005/000198 53 Table 13 Assay Plate tvpe Coating Washing buffer Reading buffer Read condition time Ab*/Ab Medium 0.05M carbonate 0.1M phosphate 0.O1M NaOH 120' Ab* (Costar) buffer buffer+0.2MNaCl+ LC1 LC1 0.05%tween C-5(1C) C- lot 5 (1 C) LC1 O.N. 4 C C-5 (1C) Ab*/Ab Medium 0.05M Carbonate 0.1M phosphate 0.O1M NaOH 120' Ab* (Costar) buffer buffer+0.2MNaCl+ LC1 LC1 0.05% tween C-5 (IC) C-3 (C) LC1 C-5 (IC) O.N. 4 "C lxPBS+0.06MNaCl +0.05% tween LC2 C- (2C) Ab*/Ab Medium 0.05M Carbonate 1xPBS+0.06MNaCl 0.O1M NaOH 120' Ab* (Costar) buffer +0.05% tween LCl Polysorp LC1 LC2 C-5(1C) Maxisorp C-5 (IQ C- (2C) Non-sorp O.N. 4 0 C/ RT O.N. Ab*/Ab Medium 0.05M Carbonate 1xPBS+0.06MNaCl 0.O1M NaOH 120' Ab* (Costar) buffer +0.05% tween LC5 Polysorp LC3 LC5 C-5(1C) Makisorp C- (2C) C- (2C) Nonsorp O.N.:4 C Ab*/Ab Black 0.05MCarbonate 1xPBS+0.06MNaCl 0.01M NaOH 120' Ab* (Costar). buffer +0.05% tween C- (2C) White C-(2C) C- (2C) (Costar) Transparen O.N. 4 0 C t (Costar), Ab* Medium: 0.05MCarbonate 1xPBS+0.06MNaCl 0.O1M NaOH 120' (Costai) buffer +0.05% tween LC3 Polysorp LC3 LC4 C-3C-5C Maxisbrp C-2C C- (2C) Non-sorp O.N. 4 C/ 2h 37 CO The effect of the liquid composition of the present invention on the binding efficiency of Peauiut (Arachis hypogaea) agglutinin (PNA) was investigated on the 5 MaxiSorpT and Non-Sorp.plates. PNA is a 110,000 Dalton lectin, composed of WO 2005/079153 PCT/IL2005/000198 54 four identical glycoprotein subunits of approximately 27,000 Daltons each. PNA lectin binds glycoproteins and glycolipids with a specific configuration of sugar residues through hydrophilic regions. PNA also possesses hydrophobic regions. The assay, designated PNA*, included the use of three coating buffers: (i) carbonate buffer, pH 5 9.6, (ii) acetate buffer, pH 4.6 and (iii) phosphate buffer, pH 7.4. Table 14, below summarizes the experiment. Table 14 Assay Plate Coating Washing buffer Reading buffer Read Ype condition time PNA* Maxisorp 0.05M 1xPBS+0.06MNaC1 0.O1MNaOH 120' Non-sorp Carbonate +0.05% tween LC3 buffer LC4 C-3C-5C LC6 C- (2C) C- (2C) 0.1M acetate buffer LC6 C- (2C) 0.1 Mpho sphat buffer LCl C-5 (iC) -_O.N. 4 C I The. effect. of the liquid composition of the present invention on binding 10 efficiency of nucleic acid was investigated on the MaxiSorp

T

N1, PolysorpT and Non SorpTM plates. Generally, DNA molecules do not bind well to polystyrene plates. Even more problematic is the binding of oligonucleotides, which are small single stranded DNA molecules, having a molecular weight of several thousand Daltons. Table 15 below summarizes the experiments which were conducted for labeled oligonucleotide 15 binding. The assays are designated by Oligo*.

WO 2005/079153 PCT/IL2005/000198 55 Table 15 Assay Plate Coating Washing buffer Reading buffer Read type condition time Oligo* Maxisorp 0.05M 1xPBS+0.06MNaCl 0.OlMNaOH 180' Non-sorp Carbonate +0.05% tween LC3 buffer LC4 C-3C-5C LC6 C- (2C) C-(2C) 0.1M acetate buffer LC6 C-(2C) * 0.1M phosphat buffer LC1 C-5 (IC) 2h 37 C Oligo* Polysorp 0.05M 1xPBS+0.06MNaC1 0.O1MNaOH 180' Maxisorp Carbonate +0.05% tween LC3 buffer LC2 C-3C-5C LC6 C- (2C) C- (2C) 0.1M acetate buffer LC6 C- (2C) 0.1 Mphosphat "buffer LC1 C-5 (IC) O.N. 4 0 C Oligo* Polysorp 0.1M acetate 1xPBS+0.06MNaCl 0.O1M NaOH 180' Maxisorp buffer + 0.2M +0.05% tween LC3 sodium LC4 C-3C-5C acetate C- (2C) LC6 C- (2C) 0.1M phosphate buffer +0.2M sodium acetate LC1 C-5 (I C) 2h 37 C WO 2005/079153 PCT/IL2005/000198 56 IgG Results and Discussion Figures 21a-22d show the results of the Ab*/Ab assays (Figures 21a-d) and the Ab* assays (Figure 22a-d) to the medium CostarThM (a), Non-SorpTM (b), Maxisorp T M (c) and PolysorpTM (d) plates. The results obtained using the liquid composition of the 5 present invention are marked with filled symbols (triangles, squares, etc.) and the control results are marked with empty symbols. The lines correspond to linear regression fits. The binding efficiency can be estimated by the slope of the lines, whereby a larger slope corresponds to a better binding efficiency. As shown in Figures 21a-22d, the slopes obtained using the liquid composition 10 of the present invention are steeper than the slopes obtained in the control experiments. Thus, the liquid composition of the present invention is capable of enhancing the binding efficiency. The enhancement binding capability of the liquid composition of the present invention, is designated Sr and defined as the ratio of the two slopes in each Figure, such that Sr > 1 corresponds to binding enhancement and Sr 15 < 1 corresponds to binding suppression. The values of the Sr parameter calculated for the slopes obtained in Fgures 21a-d were, 1.32, 2.35, 1.62 and 2.96, respectively, and the values of the Sr parameter calculated for the slopes obtained in Figures 22a-d were, 1.42, 1.29, 1.10 and 1.71, respectively. Figures 23a-24d show the results of the Ab* assays for the overnight 20 incubation at 4 0 C(Figures 23a-d) and the 2 hours incubation at 37 'C (Figure 24a-d) in NonSorpM (a ),medium CostarTM (b), PolySorpTM (c) and MaxiSorpTM (d) plates. Similar to Figures 21a-22d, the results obtained using the liquid composition of the present invention and the control are marked with filled and empty symbols, respectively. As:shown in Figures 23a-24d, except for two occurrences (overnight 25 incubation in the NonSorp

T

M' 'plate, and 2 hours in the PolySorpTM plate), the slopes obtained using the liquid composition of the present invention are steeper than the slopes obtained in the control experiments. Specifically, the calculated values of the Sr parameter obtained for Figures 23a-d were, 0.94, 1.10, 1.20 and 1.27, respectively, while the calculated values of the Sr parameter obtained for Figures 24a-d were, 1.16, 30 1.35, 0.94 and 1.11,, respectively. Figures 25a-26d show the results of the Ab*/Ab assays for the overnight incubation at,4 'C (Figures 25a-d) and the overnight incubation at room temperature (Figure 26a-d) in the medium CostarTM (a), PolySorpTMI (b), MaxiSorp

TM

(c) and Non- WO 2005/079153 PCT/IL2005/000198 57 SorpTM (d) plates. As shown in Figures 25a-26d, except for one occurrence (incubation at room temperature in the non-sorp plate) the slopes obtained using the liquid composition of the present invention are steeper than the slopes obtained in the control. Specifically, the calculated values of the Sr parameter obtained for Figures 5 25a-d were, 1.15, 1.25, 1.07 and 2.10, respectively, and the calculated values of the Sr parameter obtained for Figures 26a-d were, 1.30, 1.48, 1.38 and 0.84, respectively. Different washing protocols are compared in Figures 27a-d using the medium. CostarTm plate. Figures 27a-b show the results of the Ab*/Ab (Figure 27a) and Ab* (Figure 27b) assays when phosphate buffer was used as the washing buffer, and 10 Figures 27c-d show the results of Ab*/Ab (Figure 27c) and Ab* (Figure 27d) assays using PBS. The calculated values of the Sr parameter for the Ab*/Ab and Ab* assays (Figures 27a-d), were, respectively, 1.03, 0.97, 1.04 and 0.76. Figures 28a-b show the results of a single experiment in which the medium CostarTM plate was used for an overnight incubation at 4 'C (see the first experiment in 15 Table 13). As shown in this experiment, the calculated values of the Sr parameter were 0.37 for the Ab*/Ab assay (Figure 28a) and 0.67 for the Ab* assay (Figure 28b). Table 17 below, summarizes the results of Figures 21a-28b in terms of binding enhancement (Sr > 1) and binding suppression (Sr < 1) for each of the aforementioned plates. 20 Table 17 Medrm Polysorp Maxisorp Non-soip S~~~costar _______ >1 8/12 5/6 6/6 4/6 >1.05 5/12 5/6 6/6 4/6 >1.1 4/12 5/6 5/6 3/6 <1. 4/12. 1/6 0/6 2/6 <0.95 3/12 1/6 0/6 2/6 <0.9 3/12 0/6 0/6 1/6 As demonstrated in Table 17 and Figures 21a-28b, the liquid composition of the present invention enhances IgG binding, with a more pronounced effect on the MaxiSorpTMI and PolySorpTM plates.

WO 2005/079153 PCT/IL2005/000198 Lectin Results and Discussion Figures 29a-c show.the results of the PNA absorption assay to the Non-Sorp

T

IM plate for the acetate (Figure 29a), carbonate (Figure 29b) and phosphate (Figure 29c) buffers. In Figures 29a-c, the results obtained using the liquid composition of the 5 present invention are marked with open symbols and results of the control are marked with filled symbols. The calculated values of the Sr parameter for the acetate, carbonate and phosphate buffers were 0.65, 0.75 and 0.78, respectively,. Thus, in all three buffers the liquid composition of the present invention significantly inhibits the binding of PNA. 10 Figures 30a-d show the results of PNA absorption assay in which MaxiSorpTM plates in carbonate (Figure 30a-b), acetate (Figure 30c) and phosphate (Figure 30d) coating buffers were used. Similar- symbols as in Figures 29a-c were used for presentation. Referring to Figure 3.0a, with the carbonate buffer, a two-phase curve was obtained, with a linear part in low protein concentration in which no effect was 15 observed and a nonlinear part in high protein concentration (above about 0.72) in which the liquid composition of the present invention significantly inhibits the binding of PNA. Figure 30b presents the linear part of the graph, and a calculated value of Sr parameter of 1:01 for the carbonate buffer. The calculated values of the Sr parameter for the acetate and phosphate buffers were 0.91 and 0.83, respectively, indicating a 20 similar trend in which the liquid composition of the present invention inhibits the binding of PNA. The results of the PNA* assay are summarized in Table 18, below, in terms of binding enhancement (Sr> 1) and binding suppression (Sr < 1). Table 18 Sr MayiSor1pi Non-SoiT >1 1/3** 0/3 >1.05 0/3 0/3 >1.1 0/3 0/3 <1 2/3 0/0 <0.95 2/3 0/3 <0.9 1/3 3/3 **Sr was calculated for the liner part of the graph. 25 Hence, in the Non-SorpTM plate, the inhibition was not effected by the different buffers (pH). On the other hand, in the MaxiSorpTM plate, a pronounced effect was observed in the carbonate buffer were the curve saturated.. This can be explained by WO 2005/079153 PCT/IL2005/000198 59 the dissociation of the four subunits, which effectively increases the number of competing molecules. Note that the two proteins, IgG and PNA, behave in opposite ways on the MaxiSorpTM plates. This indicates that the liquid composition of the present invention 5 effects the molecular structure of the proteins. Oligonucleotides Results and Discussion The olig6nucleotide was bound only to the MaxiSorpT' plates in acetate coating buffer. Table 19 below summarizes the obtained values of the Sr parameter, for nine 10 different concentrations of the oligonucleotide and four different experimental conditions, averaged over the assays in which MaxiSorpTM plates in acetate coating buffer were used. Table 19 conditions 37 0 C 4 *C 37 C+Na 4 TC+Na average 0.4 1.32 1.20 1.75 2.17 1.61 0.36 1.33 1.44 1.30 1.17 1.31 0.32 0.98 1.31 1.17 1.30 1.19 .0.28 1.38 1.47 1.27 1.34 1.36 0.24 1.16 1.16 1.13 1.26 1.18 0.20 1.26 1.23 0.94 1.09 1.13 0.16 1.08 1.16 1.22 1.20 1.16 0.12 0.89 1.18 1.34 1.57 1.24 0.08 1.21 1.03 0.93 1.29 1.11 Figures 3 la-b show the average values of the Sr parameter quoted in Table 19, 15 where Figure 31 a shows the average values for each experimental conditions and Figure 31b shows the' overall average, with equal weights for all the experimental conditions. As shown in Figure 31a-b, the average values of the Sr parameter were significantly larger then 1, with a higher binding efficiency for higher concentrations of 20 oligonucleotides. Thus, it can be concluded the liquid composition of the present invention is capable of enhanding binding efficiency with and without the addition of salt to the coatinglbuffer. It is a conmon knowledge that acetate buffer is used to precipitate DNA in aqua's solutions. Under such conditions the DNA molecules interact to forn "clumps" WO 2005/079153 PCT/IL2005/000198 60 which precipitate at the bottom of the plate, creating regions of high concentration, thereby increasing the probability to bind and generating higher signal per binding event. Intra-molecular.interactions compete with the mechanism of clump formations. In contrast to the control water, the liquid composition of the present invention is 5 capable of suppressing the enhancement of clump formations for higher concentration. The higher binding efficiency of DNA on MaxiSorp T M plates using acetate buffer composed of the liquid composition of the present invention, demonstrates the capability of the liquid composition of the present invention to at least partially de-fold DNA molecules. This feature. of the present invention was also observed in DNA 10 electrophoresis experiments, as further detailed in Example 14, below. EXAMPLE 12 Isolation and Purification of DNA Nucleic acids (DN A and RNA) are the basic and most important material used 15 by researchers in the life sciences. Gene function, biomolecule production and drug development (pharmacogenomics) are all fields that routinely apply nucleic acids techniques. Typically, PCR techniques are required for the expansion of a particular sequence of DNA or RNA. Extracted DNA or RNA is initially purified. Following amplification of a particular region under investigation, the sequence is purified from 20 oligonucleotide primers, primer dimers, deoxinucleotide bases (A, T, C, G) and salt and subsequently verified. Materials and Methods: The effect of liquid composition of the present invention on the purification of the PCR product was studied by reconstitution of the Promega kit "Wizard - PCR preps 25 DNA purification system" (A7170). The use of Promega WizardTM kit involves the following steps: 1) Mix the purification buffer with the PCR sample to create conditions for binding the DNA to the Resin. 2) Mix the Resin suspension with the PCR mixture, for binding the DNA 30 to the Resin, applies the resin samples to syringes and generate vacuum. 3) Add Isopropanol and suck the solution by vacuum to remove non bound DNA. 4) Elute the bound DNA with water.

WO 2005/079153 PCT/IL2005/000198 61 5) Performing gel electrophoresis as further detailed hereinbelow. Reconstitution of the kit was performed with the original water supplied with the kit (hereinafter control) or by replacing aqua solutions of the kit with either RO water or the liquid composition of the present invention for steps 1, 2 and 4. In step 3 5 the identical 80 % isopropanol solution as found in the kit was used in all experiments. The following protocol was used for gel electrophoresis: (a) Gel solution: 8 % PAGE (+ Urea) was prepared with either RO water or the liquid composition of the present invention according to Table 20, below. Table 20 Total volume 250 (ml) 40 % acrylamid 50 10 x TBE 25 Urea 84.1 g RO/liquid composition about 105 10 (b) Add polymerization reagents containing 405pl 10 % APS and 55 ptl TEMED (Sigma T-7024) to 50 ml of gel solution. (c) Pour the gel solution into the gel cassette (Rhenium Ltd, Novex NC2015, 09-01505-C2), place the plastic combs and allow to 15 polymerize for 30 minutes at room temperature. (d) Remove the combs and strip off tape to allow assembling of two gels on two opposite sides of a single device. (e) Fill in the inner chamber to the top of the gel and the outer chamber to about fifth of the gel height with running buffer-TBE x1 in either RO 20 - water or the liquid composition of the present invention. (f) Prepare samples by diluting them in sample buffer containing TBE Ficoll, Bromophenol blue and urea (SBU), and mix 1:1 with the DNA ''Sample. (g) Load 8 -10 p.tl of the mix into each well. 25 (h) Set the power supply to 100 V and let the DNA migrate continue until the color dye (Bromophenol blue) reaches 1 cm from the bottom. The following protocol was used for gel staining visualization photographing and analyzing: WO 2005/079153 PCT/IL2005/000198 62 (a) Place the gels in staining solution containing 1 U/ptl GelStarTM in lxTBE for 15 minutes whilst shaking. (b) Destain the gels for 30 minutes in 1xTBE buffer. (c) Place the gels on U.V. table; use 365 nm light so as to see the DNA. 5 (d) 'Using DC20TM digital camera, photograph the gels and store the digital information for further analysis. PCR was prepared from Human DNA (Promega G 3041) using ApoE gene specific primers (fragment size 265 bp), according to the following protocol (for 100 reactions): 10 (a) Mark.0.2 [pl PCR-tubes according to the appropriate serial number. (b) Add 2.5 pl of 40 pg/ml Human DNA (Promega G 3041) or water to the relevant tubes. (c) Adjust to 17 ptl with 14.5 pl DDW. (d) Prepare 3630 pl of the PCR mix according to Table 21 (see below). 15 (e) Add 33 p1 of the mix to each tube. (f) 'Place the samples in the PCR machine. (g) Run a PCR program according to Table 22 (see below). (h) Analyze 5 pl of each product on S % PAGE gel. (i) Store reactions at -20 'C. 20 Table 21: PCR Mix DDW 836 DMSO100 % 550 fw primer 1* (10 pM) 550 rv primer 2* (10 pM) 550 10 x PCR buffer (15 mM 550 MgCl) dNTPs (2 mM) 550 MgCl (25 mM) 0 Taq polymerase (5 p/ul) 44 total in t1 3630 *primer 15'TCCAAGGAGCTGCAGGCGGCGCA (SEQ ID NO:1) *primer 1 6-fam 5'mTCCAAGGAGCTGCAGGCGGCGCA (SEQ ID NO:2) *primer 1 biotin5'bTCCAAGGAGCTGCAGGCGGCGCA (SEQ ID NO:3) 25 *primer 2 5GGCGCTCGCGGATGGCGCTGAG (SEQ ID NO:4).

WO 2005/079153 PCT/IL2005/000198 63 Table 22: PCR program Temperature time 1 95 0 C 5 min 2 94 C 1 min 3 68 C 1 min 4 72 0 C 30 see 5 go back to step 2 x34 times 6 72 C 5 min 7 4 OC hold Results: For clarity, in the present and following Examples, control is abbreviated to "CO," Reverse Osmosis water is abbreviated to "RO," and the liquid composition of 5 the present invention is abbreviated to "LC." Figure 32 is an image of 50 ptl PCR product samples in an experiment, referred to herein as Experiment 3. There are 11 lanes in Figure 32, in which lane 1 correspond to the PCR product before purification, lane 7 is a ladder marker, and lanes 2-6, 8-11 correspond to the following combinations of the aforementioned steps 1, 2 and 4: 10 CO/CO/CO elution 1 (lane 2), RO/RO/RO elution 1 (lane 3), LC/LC/LC elution 1 (lane 4), CO/CO/CO elution 2 (lane 5), RO/RO/RO elution 2 (lane 6), LC/LC/LC elution 2 (lane 8), CO/CO/CO elution 3 (lane 9), RO/RO/RO elution 3 (lane 10), and LC/LC/LC elution 3 (lane 11). All three assays systems exhibit similar purification features. Efficient removal 15 of the low M.W molecules (smaller than 100 bp) is demonstrated. The unwanted molecules include primers and their dimers as well as nucleotide bases. Figures 33a-b are images of 50 pl PCR product samples in an experiment, referred to herein as' Experiment 4, for elution 1 (Figure 33a) and elution 2 (Figure 33b). There are 13 lanes in Figures 33 a-b, in which lane 6 is a ladder marker, and lanes 20 1-5, 7-13 correspond to the following combinations: CO/CO/CO (lane 1), RO/RO/RO (lane 2), LC/LC/LC (lane 3), CO/LC/LC (lane 4), CO/RO/RO (lane 5), CO/CO/LC (lane 7), CO/CO/RO (lane 8), CO/LC/CO (lane 9), CO/RO/CO (lane 10), LC/LC/CO (lane 11), RO/RO/CO (lane 12), LC/LC/LC (lane 13), where in lane 13 a different concentration wasused for the liquid composition of the present invention. 25 Figures .34a-b are images of 50 ptl PCR product samples in an experiment, referred to herein as Experiment 5,- for elution 1 (Figure 34a) and elution 2 (Figure WO 2005/079153 PCT/IL2005/000198 64 34b). In Figures 34a-b, lane 4 is a ladder marker, and lanes 1-3, 5-13 correspond to the following combinations: CO/CO/CO (lane 1), RO/RO/RO (lane 2), LC/LC/LC (lane 3), CO/LC/LC (lane 5), CO/RO/RO (lane 6), CO/CO/LC (lane 7), CO/CO/RO (lane 8), CO/LC/CO (lane 9), CO/RO/CO (lane 10), LC/LC/CO (lane 11), RO/RO/CO (lane 5 12), and LC/CO/CO (lane 13). Lane 14 in Figure 34a corresponds to the combination RO/CO/CO. Figures. 35a-b are, images of 50 pl PCR product samples in an experiment, referred to herein as Experiment 6, for elution 1 (Figure 35a) and elution 2 (Figure 35b). In-Figures 35a-b, lanes 1-13 correspond to the same combinations as in Figure 10 34a, and lane 15 corresponds to the PCR product before purification. EXAMPLE 13 Column Capacity In this example, the effect of the liquid composition of the present invention on 15 column capacity was examined. 100 PCR reactions, each prepared according to the protocols of Example 12 were prepared and combined to make a 5 ml stock solution. The experiment, referred to herein as Experiment 7, included two steps, in which in a preliminary step (hereinafter step A) was directed at examining the effect of volume applied to the columns on binding and elution, and a primary step (hereinafter step B) 20 was directed at investigating the effect of the liquid composition of the present invention on the column capacity. In Step A,:four columns (columns 1-4) were applied with 50, 150, 300 or 600 ptl stock PCR product solution, and 13 columns (5-17) were applied with 300 l of stock PCR solution. All columns were eluted with 50 pl of water. The eluted solutions 25 were loaded in lanes 7-10 in the following order: lane 7 (original PCR, concentration factor x 1), lane 8 (original x 3), lane 9 (x 6) and lane 10 (x 12). A "mix" of all elutions from columns 5-17 (x 6) was loaded in lane 11. Lanes 1-5 were loaded with elutions from columns 1-4 and the "mix" of columns 5-17, pre-diluted to the original concentration (x 1). Lane 6 was the ladder marker. 30 The following protocol was employed in Step A: 1) Mark the Wizard TM minicolumn and the syringe for each sample, and insert into the Vacuum Manifold.

WO 2005/079153 PCT/IL2005/000198 65 2) Dispense 100 pl of each direct PCR purification buffer solution into a micro-tube., 3) Vortex briefly. 4) Add 1 ml of each resin solution and vortex briefly 3 times for 1 minute. 5 5) Add the Resin/DNA mix to the syringe and apply vacuum. 6) Wash by adding 2ml of 80 % isopropanol solution to each syringe and apply vacuum.. 5) Dry the resin by maintaining the vacuum for 30 seconds. 6) Transfer the minicolumn to a 1.5 ml microcentrifuge tube. 10 7.) Centrifuge at 10000 g for 2 minutes. 8) Transfer the minicolumn to a clean 1.5 ml tube. 9) Add 50 pl of the relevant water (nuclease free or the liquid composition of the present invention). 10) Centrifuge at 10000 g for 20 second. 15 11) Transfer to 50 p1 storage microtube and store at -20 *C. 12) Repeat steps 9711 for a second elution cycle. Visualization steps: 13) Mix 16 pl of each sample with 6 pl loading buffer. 14) Load 10 pl of each mix in acrylamide urea gel (AAU) and run the gel at 20 70 V 10mAmp. 15) Stain the gel with Gel Star T M solution (5 pl of 10000 u solution in 50ml TBE), shake for 15 minutes at room temperature. 16). Shake in TBE buffer at room temperature for 30 minutes to destain the gel. 25 17) photograph the gel. In Step B the "mixed" elution of Step A was used as "concentrated PCR solution" and applied to 12. columns. Columns 1-5 were applied with 8.3 p1, 25 pl, 50 pl, 75 p1 and 100 pl respectively using the kit reagents. The columns were eluted by 50 pl kit water and 5 pl of each elution was applied to the corresponding lane on the 30 gel. Columns 7-11 were treated as column 1-5 but with the liquid composition of the present invention as .binding and elution buffers. The samples were applied to the corresponding, g~l laes.-Columin 13 served as a control with the "mix" of columns 5 17 of Step A.

WO 2005/079153 PCT/IL2005/000198 66 The following protocol was employed in Step B: 1) Mark the WizardTM minicolumn and syringe to be used for each sample and insert into the vacuum manifold. 2) Dispense 100, pl of each direct PCR purification buffer solution into 5 micro-tube. 3) Vortex briefly. 4) Add 1 ml of each resin solution and vortex briefly 3 times for 1 minute. 5) Add the Resin/DNA mix to the syringe and apply vacuum. 6) Wash by adding 2 ml of 80 % isopropanol solution to each syringe and 10 apply vacuum. 5) Dry the resin by continuing to apply the vacuum for 30 seconds. 6) Transfer the minicolumn to 1.5 ml microcentrifuge tube. 7) Centrifuge at 10000 g for 2 minutes. 8) Transfer the minicolumn to a clean 1.5 ml tube. 15 9) Add 50 pl of nuclease free or the liquid composition of the present invention. 10) Centrifuge at 10000 g for 20 seconds. 11) Transfer to a 50 l storage micro-tube and store at -20 'C. 12) Repeat steps 9-11 for a second elution cycle. 20 Visualization steps were the same as in Step A. Results: Figures'. 36-37 show image (Figure 36) and quantitative analysis using Sionlmagem software (Figure 37) of lanes 1-11 of Step A. As shown in Figure 36a, lanes 8-11 are overloaded. Lanes 3 and 4 contain less DNA because columns 3 and 4 25 were overloaded and as a result less DNA was recovered after dilution of the eluted samples. As shown in Figure 37, DNA losing is higher when the DNA loading volume is bigger. Figures 38a-c show images of lanes 1-12 of Step B, for elution 1 (Figure 38a), elution 2 (figure 38b) and elation 3 (Figure 38c). The first elution figure shows that 30 the columns were similarly overloaded,. The differences in binding capacity are clearly seenin the second elution The band intensity increases correspondingly with the number of the lane..

WO 2005/079153 PCT/IL2005/000198 67 Comparing the intensity of corresponding lanes 1-5 and 7-11, indicates that the liquid composition of the present invention is capable of binding more DNA than the kit reagents. Figures 39a-b show quantitative analysis using SionlmageTM software, where 5 Figure 39a represents the area of the control (designated CO in Figures 39a-b) and the liquid composition, of the present invention (designated LC in Figures 39a-b) as a function of the loading volume for each of the three elutions, and Figure 39b shows the ratio LC/CO. As shown in Figures 39a-b in elution 3, the area is larger for the liquid composition of the present invention. 10 EXAMPLE 14 Isolation of DNA by Gel Electrophoresis Gel Electrophoresis is a routinely used method for determination and isolation of DNA molecules based on size and shape. DNA samples are applied to an upper part 15 of the gel, serving as a running buffer surrounding the DNA molecules. The gel is positively charged and forces the negatively charged DNA fragments to move downstream the gel when electric current is applied. The migration rate is faster for smaller and coiled or folded molecules and slower for large and unfolded molecules. Once the migration is completed, DNA can be tagged by fluorescent label and is 20 visualized under UV:illunination. The DNA can be also transferred to a membrane and visualized by enzymatic coloration at high sensitivity. DNA is evaluated according to its position on the gel and the band intensity. Following is a description of experiments in which the effect of the liquid composition of the present invention on DNA migration by gel electrophoresis was 25 examined. Materials and Methods: Two types of DNA were used: (i) PCR product, 280 base pair; and (ii) ladder DNA composed of eleven DNA fragments of the following sizes: 80, 100, -200, 300, 400, 500, 600, 700, 800, 900 and 1030 bp. The gel was prepared according to the 30 protocols of Example 12. Three experiments were performed. In Experiment 1, PCR batch number 181103 was loaded into lanes 2-10,, with the ladder DNA in lane 1; in Experiment 2, PCR batch number 31203 was loaded into lanes 2-11 with the ladder DNA in lane 1; WO 2005/079153 PCT/IL2005/000198 68 and in Experiment 3, PCR batch number 31203 was loaded into lanes 1-5 and 7-11, with the ladder DNA in lane 6. Results: Figures 40a-42b are DNA images comparing the migration speed in the 5 presence of RO water (Figures 40a, 41a and 42a) and in the presence of the liquid composition of thepresent invention (Figures 40b, 41b and 42b) for Experiments 1, 2 and 3, respectively. In the images of Figures 40a-42b both the running buffers and the gel buffers were composed of the same type of liquid, i.e., in Figures 40a, 41a and 42a both the running buffer and the gel buffer were composed of RO water, while in 10 Figures 40b, 41b and 42b both the running buffer and the gel buffer were composed of the liquid composition of the present invention. As shown in Figures 40a-42b, both types of DNA (PCR product and the ladder DNA) migrated significantly faster in RO water in comparison to the liquid composition of the present invention. 15 In an attempt to separate the effect of the liquid composition of the present invention on the gel content and its effect on the running buffer, the above experiments were repeated in all possible combinations of running and gel buffers. Hence, Figures 43a-45d are images of Experiments 1 (Figure 43a-d), 2 (Figure 44a-d) and 3 (Figure 45a-d), in which the effect of the liquid composition of the present 20 invention on the running buffer are investigated. In each pair of figures (i.e., pairs a-b and c-d) the gels are .composed: of the same liquid and the running buffer is different. Using the abbreviations introduced in Example 12, the following combinations of gel/running buffers are shown in Figures 43a-45d: Figures 43a-b are images of RO/RO and RO/LC, respectively; Figures 43.c-d are images of LC/LC and LC/RO respectively, 25 Figures 44a-b are images of RO/RO and RO/LC, respectively; Figures 44c-d are images of LC/RO and LC/LC respectively, Figures 45a-b are images of RO/LC and RO/RO, respectively; and Figures 45c-d are images of LC/LC and LC/RO respectively. Figures 46a-48d are images of Experiments 1 (Figure 46a-d), 2 (Figure 47a-d) and 3 (Figure 48a-d), in which the effect of the liquid composition of the present 30 invention on the gel buffer are investigated. In each pair of figures (a-b, c-d) the running buffers are composed of the same liquid but the gel buffers are different. Specifically, Figures 46a-b are images of RO/RO and LC/RO, respectively; Figures 46c-d are images of LC/LC and RO/LC respectively, Figures 47a-b are images of WO 2005/079153 PCT/IL2005/000198 69 RO/RO and LC/RO, respectively; Figures 47c-d are images of RO/LC and LC/LC respectively, Figures 48a-b are images of RO/RO and LC/RO, respectively; and Figures 48c-d are images of RO/LC and LC/LC respectively. As shown in Figures 43a-48d, the liquid composition of the present invention, 5 causes the retardation of DNA migration as compared to RO water. Note that no significant change in the electric field was observed. This effect is more pronounced when the gel buffer is composed of the liquid composition of the present invention and the running buffer:is composed of RO water. Thus, the above experiments demonstrate that under the influence of the liquid 10 composition of the present invention, the DNA configuration is changed, in a manner that the folding of the DNA is decreased (un-folding). The un-folding of DNA in the liquid composition of the present invention may indicate that stronger hydrogen boned interactions exists between the DNA molecule and the liquid composition of the present invention in comparison to RO water. 15 EXAMPLE 15 Enzyme Activity and Stability Increasing both enzyme activity and stability are important for enhancing efficiency and reducing costs of any process utilizing enzymes. During long term 20 storage, prolonged activity and also when over-diluted, enzymes are typically exposed to stress which may contribute to loss of stability and ultimately to loss of activity. In this example, the effect of the liquid composition of the present invention on the activity. and stability of. enzymes is demonstrated. This study relates to two commonly used enzymes in the biotechnological industry: Alkaline Phosphatase (AP), 25 and -Galactosidase. Two forms of AP were used: an unbound form and a bound form in which AP was bound to Strept-Avidin (ST-AP). Following is, a description of experiments in which the effect of the liquid composition of the. present invention on diluted enzymes was investigated. The dilutions were performed either in RO water or in the liquid composition of the present 30 invention.without additives and in neutral pH (7.4).

WO 2005/079153 PCT/IL2005/000198 70 Unbound Form of Alkaline Phosphatase Materials and Methods: Alkaline Phpsphatase (Jackson INC) was serially diluted in either RO water or the liquid composition of the present invention. Diluted samples 1:1,000 and 1:10,000 5 were incubated in tubes at room temperature. At different time intervals, enzyme activity was determined by mixing 10 pl of enzyme with 90 pLl.pNPP solution (AP specific colorimetric substrate). The assay was performed in microtitration plates (at least 4 repeats for each test point). Color intensity was determined by an ELISA reader at wavelength of 405 nm. 10 Enzyme activity was determined at time t=0 for each dilution, both in RO water and in three different concentrations of the liquid composition of the present invention: LC3, LC7 and LC8 as further detailed hereinbelow. Stability was determined as the activity after 22 hours (t=22) and 48 hours (t=48) divided by the activity at t=0. Results & Discussion: 15 Tables 23-25 below summarize the average activity values of six experiments, numbered 1-6, for t=0 (Table 23), t=22 (Table 24) and t=48 (Table 25). All experiments 1-5 were conducted at room temperature. Table 23 liquid dilution average activity 1 2 3 4 5 6 1:1000 '3.27 2.91 2.72 1.74 2.46 2.89 RO 1:10000 0.49 0.35 0.37 0.29 0.45 0.42 - 0 0.07 0.08 0.08 0.10 0.08 0.08 1:1000 3.55 3.51 3.39 3.39 0.08 3.43 LC7 1:10000 0.62 0.56 0.55 0.63 0.08 0.58 0- "0.08 0.08 0.08 0.08 0.08 0.08 1:1000 3.44 3.34 3.45 3.54 3.37 3.55 LC8 1:10000 0.58 0.45 0.56 0.58 0.48 0.59 0 0.08 0.08 0.08 0.09 0.08 0.08 1:1000 3.47 3.39 3.44 3.60 2.87 3.48 LC3 1:10000 0.63 0.68 0.80 0.67 0.41 0.55 0 0.08 0.08 0.08 0.08 0.09 0.08 WO 2005/079153 PCT/IL2005/000198 71 Table 24 liquid dilution average activity 1 2 3 4 5 6 1:1000 1.78 0.77 2.01 0.36 1.46 1.70 RO 1:10000 0.28 0.14 0.17 0.17 0.19 0.22 0 . ,. 0.04 0.07 0.07 0.06 0.04 0.08 1:1000 3.42 2.47 3.10 2.64 2.57 LC7 1:10000 0.45 0.32 0.40 0.40 0.47 0 0.04 0.08 0.09 0.06 0.08 1:1000 3.27 2.23 3.42. 3.39 3.02 3.30 LC8 1:10000 0.45 0.27 0.08 0.47 0.47 0.47 0 0.04 0.04 0.05 0.04 0.04 0.08 1:1000 3.50 3.31 3.36 3.15 3.08 3.31 LC3 1:10000 0.56 0.55 0.61 0.58 0.46 0.48 0 F0.08 0.04 0.08 0.08 0.05 0.08 Table 25 liquid diltution average activity 1 2 3 4 5 6 1:1000 1.34 0.49 0.86 0.22 0.60 1.34 RO 1:10000 0.22 0.12 0.11 0.13 0.13 0.22 1 0 0.08 0.08 0.08 0.08 0.08 0.08 1:1000 3.03 2.43 2.05 2.16 3.03 LC7 1:10000 0.37 0.31 0.23 0.29 0.37 0 0.08 0.08 0.08 0.08 0.08 1:1000 2.48 2.32 2.07 2.67 1.78 2.48 LC8 1:10000 0.37 0.25 0.27 0.41 0.26 0.37 0 0.05 0.07 0.07 0.08 0.08 0.05 1:1000 3.27 3.57 2.22 2.58 1.83 3.27 LC3 1:10000 0.46 0.45 0.42 0.45 0.31 0.46 0 0.08 0.08 0.07 0.08 0.08 0.08 As shown in Tables 23-25 the activity in the presence of LC7, LC8 and LC3 is 5 consistently above the activity in the presence of RO water. To quantify the effect of the liquid composition of the present invention on the stability, a stability enhancement parameter, Se, was defined as the stability in the presence of the liquid composition of the present invention divided by the stability in RO water. Figure 49 shows the values of Se, for 22 hours (full triangles) and 48 hours (full 10 squares), as a function of the dilution. The values of S, for LC7, LC8 and LC3 are shown in.Figure 49 iii blue, red, and green, respectively). As shown in Figure 49, the measured stabilizing effect is in the range of about 2 to 3.6 for enzyme dilution of WO 2005/079153 PCT/IL2005/000198 72 1:10,000, andin the range of about 1.5 to 3 for dilution of 1:1,000. The same phenomena were observed at low temperatures, although to a somewhat lesser extent. Bound Form of Alkaline Phosphatase 5 Binding an enzyme to another molecule typically increases its stability. Enzymes are typically stored at high concentrations, and only diluted prior to use to the desired dilution. The following experiments are directed at investigating the stabilization effect of the liquid composition of the present invention in which the enzymes are stored at high concentrations for prolonged periods of time. 10 Materials and Methods: Strept-Avidin Alkaline Phosphatase (Sigma) was diluted 1:10 and 1:10,000 in RO water and in the aforementioned liquid compositions LC7, LC8 and LC3 of the present invention The diluted samples were incubated in tubes for 5 days at room temperature. 15 All samples were diluted to a final enzyme concentration of 1:10,000 and the activity was determined as further detailed hereinabove. Enzyme activity was determined at time t=0 and after 5 days. Results and Discussion: Figure 50 is a chart showing the activity of the conjugated enzyme after 5 days 20 of storage in a dilution of 1:10 (blue) and in a dilution of 1:10,000 (red), for the RO water and the liquid composition of the present invention. In RO water, the enzyme activity is about 0.150 OD for both dilutions. In contrast, in the presence of the liquid composition of the present invention the activity is about 3.5 times higher in the 1:10 dilution than in the 1:10,000 dilution. However, for both dilutions, the enzyme is 25 substantially more active in the liquid composition of the present invention than in RO water. 5-Galactosidase materials and Methods: The experiments with p-Galactosidase were performed according to the same 30 protocol used 'for the Alkaline Phosphatase experiments described above with the exception of enzyne type concentration and in incubation time. P-Galactosidase (Sigma) was serially diluted in RO water and in the liquid composition of the present WO 2005/079153 PCT/IL2005/000198 73 invention. The samples were diluted to 1:330 and 1:1000 and were incubated at room temperature. The enzyme activity was determined at time intervals 0, 24 hours, 48 hours, 72 hours and 120 hours, by mixing 10 pl of enzyme with 100 pl of ONPG solution (p-Gal 5 specific colorimetric substrate) for 15 minutes at 37 0 C and adding 50 pl stop solution (1M Na 2 Hco 3 ). The assay was performed in microtitration plates (8 repetitions from each test point). An ELISA reader at wavelength of 405 nm was used to determine color intensity The enzyme activity was determined at time t=0 for each dilution, for the RO 10 water and for the aforementioned liquid compositions LC7, LC8 and LC3 of the present invention. Five experiments were performed under identical conditions. The enzyme stability and the stability enhancement parameter, Se, were calculated as further detailed hereinab6ve. Results and Discussion: 15 Figures 51 a-d show the stability (the activity at time tw0, divided by the activity at t=0), at t =24 hours (Figure 51a), t = 48 hours (Figure 51b), t = 72 hours (Figure 51c) and t= 120 hours (Figure 51d). The liquids RO, LC7, LCS, LC3 and LC4 are shown in Figures 51a-d in blue, red, green and purple, respectively, and average values of the stability are shown as circles. As shown in Figures 51 a-5 1 d, the activity in the 20 presence of LC7, LCS and LC3 is consistently above the activity in the presence of RO water. Figures 52a-d show the stability enhancement parameter, Se, at t = 24 hours (Figure 52a), t = 48 hours (Figure 52b), t = 72 hours (Figure 52c) and t = 120 hours (Figure 52d), with similar color notations as in Figures 51a-d. As shown in Figure 52a 25 d, the measured stabilizing effect is in the range of about. 1.3 to 2.21 for enzyme dilution of 1:1000, and.in the range of about 0.83 to 1.3 for dilution of 1:330. Thus, the stabilizing effect liquid composition of the present invention on p Galactosidase is 'similar to the stabilizing effect found for AP. The extent of stabilization is somewhat lower. This can be explained by the relatively low specific 30 activity (464 u/mg) .having high protein concentration in the assay, which has attenuated activity lost over time.

WO 2005/079153 PCT/IL2005/000198 74 Activity and stability of dry alkaline phosphatase Many enzymes are dried before storage. The drying process and the subsequent storage in a dry state for a prolonged period of time are known to effect enzyme activity. The following experiments are directed at investigating the effect of the liquid 5 composition of the present invention on the activity and stability of dry alkaline phosphatase. Materials and Methods: Alkaline Phosphatase (Jackson INC) was diluted 1:5000 in RO water and in the aforementioned liquid compositions LC7, LC8 and LC3 of the present invention, as 10 further detailed hereinabove. Nine microtitration plates were filled with aliquots of 5 l solution. One plate was tested for enzyme activity at time t=0, as further detailed hereinabove, and the remaining 8 plates were dried at 37 'C overnight. The drying process was performed in a dessicated environment for 16 hours. 15 Two, plates were tested for enzyme activity by initial cooling to room temperature and subsequent, addition. of 100 pl pNPP solution at room temperature. Color intensity was determined by an ELISA reader at a wavelength of 405 nm and the stability was calculated as further detailed hereinabove. Six plates were transferred to 60 'C for 30 minutes and the enzyme activity was determined thereafter. 20 Results: Figure 53a shows the activity of the enzymes after drying (two repeats) and after 30 minutes of heat treatment at 60 'C (6 repeats). Average values are shown in Figure 53a by a 1"+ symbol. Both treatments substantially damaged the enzyme and their effect was additiVe. 25 Figure 53b shows the stability enhancement parameter, Se. In spite of the relatively small database and the extreme conditions to which the enzyme was exposed, the liquid composition of the present invention has evidently stabilized the activity of the enzyme. For example, for LC7 the average value of the stability enhancement parameter was increased from 1.16 to 1.22. 30 WO 2005/079153 PCT/IL2005/000198 75 EXAMPLE 16 Anchoring of DNA In this.example, the effect of anchoring DNA with glass beads in the presence or absence of the liquid composition of the present invention was examined. 5 Anchoring polynucleotides to a solid support such as glass beads can be of utmost benefit in the field of molecular biology research and medicine. Typically, DNA manipulations, comprise a sequence of reactions, one following the other, including PCR, ligation,.restriction and transformation. Each reaction is preferably performed under its own suitable reaction conditions requiring its own specific buffer. Typically, 10 in between each reaction, the DNA or RNA sample must be precipitated and then reconstituted in its new appropriate buffer. Repeated precipitations and reconstitutions takes time .and more importantly leads to loss of starting material, which can be of utmost relevance when this material is rare. As an example, the inventors chose to investigate. what effect the liquid composition of the present invention has on DNA in 15 the presence of glass beads during a PCR reaction. Materials and Methods: PCR was prepared from a pBS plasmid cloned with a 750 base pair gene using a T7 forward primer (TAATACGACTCACTATAGGG) and an M13 reverse primer (GGAAACAGCTATGACCATGA) such that the fragment size obtained is 750 bp. 20 The primers were constituted in PCR-grade water at a concentration of 200pM (200pmol/pl). These were subsequently diluted 1:20 in NeowaterTm, to a working concentration of 10pM each to make a combined mix. For example 1 pl of each primer (from 200pM stock) is combined and diluted with 18 pl of Neowater

T

m, mixed and spun down The concentrated DNA was diluted 1:500 with NeowaterTm to a working 25 concentration of 2pg/pgl. The PCR was performed in a Biometra T- Gradient PCR machine. The enzyme used was SAWADY Taq DNA Polymerase (PeqLab 01-1020) in buffer Y. A PCR mix was prepared as follows: Table 26 Final conc X1 Concentration Reagents Xl 1l. X10 Buffer Y 0.2mM 0.2 gl 10mM each dNTPs WO 2005/079153 PCT/IL2005/000198 76 0.4 units 0.08pl 5u/pl Taq 3.22pl Neowater Pick a few beads with a tip end and gently tap on the Glass Beads - without tip on top of an open tube - a few glass beads will fall any treatment. into the tube. Important - the amount of the powder in the mix. should be almost invisible. Too much glass powder will inhibit the PCR reaction The samples were mixed but not vortexed. They were placed in a PCR machine at 94'C for exactly 1 min and then removed. 4

.

5 pl of the PCR mix was then aliquoted into clean tubes to which 0.5tl of primer mix and 5 pl of diluted DNA were 5 added in that order. After mixing, but not vortexing or centrifugation, the samples were placed in the PCR machine and the following PCR program used: Table 27 Time Temp Step 10 see 94'C Step 1 10 see 50'C Step2 10 see 74 0 C Step3 The products of the PCR reaction were run on 8 % PAGE gels for analysis as 10 described herein above. The PCR products loaded onto the gel were as follows: Lane 1: DNA diluted in Neowaterm, Primers (mix) diluted in H 2 0, vol (to 10pil) with NeowaterTm (with glass beads). Lane 2: DNA diluted in NeowaterTm, Primers (mix) diluted in NeowaterTm, vol 15 (to 1Opl) with Neowiter T (with glass beads). Lane 3: All in H 2 0 (positive control) (with glass beads). Lane 4: Negative control. No DNA, Primers in NeowaterTm (to. 10pl) with H 2 0 (with glass beads). Lane 5: DNA diluted in Neowaterrm, Primers (mix) diluted in H 2 0, vol (to 20 10 tl) with Neowater (without glass beads).

77 Lane 6: DNA diluted in NeowaterTM, Primers (mix) diluted in NeowaterTM, vol (to 10 pl) with NeowaterTM (without glass beads). Lane 7: All in H20 (positive control)(without glass beads). Lane 8: Negative control. No DNA, Primers in NeowaterTM (to 10 pl) with H20 5 (without glass beads). Results and conclusion Fig. 54 is a DNA image. As can be seen, when PCR is performed in the presence of glass beads, neowater is required for the reaction to take place. When neowater is not included in the reaction, no PCR product is observed (see lane 3). 10 In conclusion, the liquid composition of the present invention is required during a PCR reaction in the presence of glass beads. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for 15 brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in that art. Accordingly, it is intended to embrace all 20 such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification and herein incorporated in the entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by 25 reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. The term 'comprise' and variants of the term such as 'comprises' or 'comprising' are used herein to denote the inclusion of a stated integer or stated integers 30 but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Claims

1. A method of making nanoparticles, comprising the steps of: (a) heating a solid powder; (b) immersing said solid in a liquid that is colder than said hot powder; and (c) during said immersing, irradiating said liquid with radiofrequency ('RF') radiation, wherein said immersing and said irradiating serve to break up said powder into the nanoparticles.

2. A method of producing a liquid composition from a solid powder, the method comprising: (a) heating the solid powder, thereby providing a heated solid powder; (b) immersing said heated solid powder in a liquid that is colder than said heated solid powder; and (c) substantially contemporaneously with said step (b), irradiating said liquid and said heated solid powder by electromagnetic radiation, said electromagnetic radiation being characterized by a frequency selected such that nanostructures are formed from particles of the solid powder.

3. The method according to claim I or claim 2, wherein said solid powder includes micron-size particles.

4. The method according to any one of claims I to 3, wherein both said powder and the nanoparticles are crystalline.

5. The method according to any one of claims 1 to 4, wherein said liquid includes water.

6. The method according to any one of claims I to 5, wherein said solid is selected from the group consisting of ferroelectric solids and ferromagnetic solids.

7. The method according to any one of claims I to 6, wherein said solid is selected from the group consisting of BaTiO 3 and W0 3 .

8. The method according to any one of claims I to 7, wherein said solid is selected from the group consisting of minerals, ceramics, glasses, metals and synthetic polymers. 79

9. The method according to any one of claims 1 to 8, wherein said RF radiation is continuous wave ('CW') RF radiation.

10. The method according to claim 9, wherein said CW RF radiation has a frequency of at least about 500 MHz.

11. The method according to claim 9, wherein said CW RF radiation has a frequency of at least about 800 MHz.

12. The method according to any one of claims I to 11, wherein said powder is heated to at least about 700 *C.

13. The method according to any one of claims I to 11, wherein said powder is heated to at least about 880 *C.

14. Nanoparticles produced by the method according to any one of claims I to 13.

15. Ferroelectric crystalline nanoparticles produced by the method according to claim 4.

16. Ferromagnetic crystalline nanoparticles produced by the method according to claim 4.

17. Piezoelectric crystalline nanoparticles produced by the method according to claim 4.

18. An apparatus for converting a solid powder into nanoparticles, comprising: (a) a furnace for heating the powder; (b) a container of a liquid into which said hot powder is dropped to cool and break up the hot powder; and (c) a mechanism for irradiating said liquid with radiofrequency ('RF') radiation while said hot powder is dropped into said liquid.

19. The apparatus according to claim 18, wherein said furnace is a tube furnace.

20. The apparatus according to claim 18 or claim 19, further comprising: (d) a meshed sieve for selecting a particle size of the powder, only particles of said selected size then being heated in said furnace. 80

21. The apparatus according to any one of claims 18 to 20, wherein said furnace is operative to heat the powder to at least about 700 *C.

22. The apparatus according to any of claims 18 to 21, wherein said RF radiation is CW RF radiation having a frequency of at least about 500 MHz. Date:

23 March 2010