EP1776469A2 - Composition solide-fluide et utilisations de celle-ci - Google Patents
Composition solide-fluide et utilisations de celle-ciInfo
- Publication number
- EP1776469A2 EP1776469A2 EP05703238A EP05703238A EP1776469A2 EP 1776469 A2 EP1776469 A2 EP 1776469A2 EP 05703238 A EP05703238 A EP 05703238A EP 05703238 A EP05703238 A EP 05703238A EP 1776469 A2 EP1776469 A2 EP 1776469A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition
- core material
- liquid
- nanostructures
- nanostractures
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C01G41/00—Compounds of tungsten
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- B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
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- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B13/00—Oxygen; Ozone; Oxides or hydroxides in general
- C01B13/14—Methods for preparing oxides or hydroxides in general
- C01B13/145—After-treatment of oxides or hydroxides, e.g. pulverising, drying, decreasing the acidity
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B13/00—Oxygen; Ozone; Oxides or hydroxides in general
- C01B13/14—Methods for preparing oxides or hydroxides in general
- C01B13/32—Methods for preparing oxides or hydroxides in general by oxidation or hydrolysis of elements or compounds in the liquid or solid state or in non-aqueous solution, e.g. sol-gel process
- C01B13/322—Methods for preparing oxides or hydroxides in general by oxidation or hydrolysis of elements or compounds in the liquid or solid state or in non-aqueous solution, e.g. sol-gel process of elements or compounds in the solid state
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
- C01F11/00—Compounds of calcium, strontium, or barium
- C01F11/20—Halides
- C01F11/22—Fluorides
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G23/00—Compounds of titanium
- C01G23/003—Titanates
- C01G23/006—Alkaline earth titanates
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/0018—Mixed oxides or hydroxides
- C01G49/0036—Mixed oxides or hydroxides containing one alkaline earth metal, magnesium or lead
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
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- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2006/00—Physical properties of inorganic compounds
- C01P2006/12—Surface area
Definitions
- the present invention relates to a solid-fluid composition and, more 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 modern science. These small particles are of interest from a fundamental view pomt 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 nanoelectronics, nanofluidics, coatings and paints and biotechnology.
- MEMS Micro Electro Mechanical Systems
- MEMS are fabricated using integrated circuit 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.
- nanoparticles are frequently used in nanometer-scale equipment for probing the real-space structure and function of biological molecules.
- Auxiliary nanoparticles such as calcium alginate nanospheres, have also been used to help improve gene transfection protocols.
- resonant collective oscillations of conduction electrons also known as particle plasmons
- the resonance frequency of a particle plasmon is determined mainly by the dielectric function of the 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.
- the laser wavelength is tuned to the plasmon resonance frequency of the particle, the local electric field in proximity to the nanoparticles can be enhanced by several orders of magnitude.
- 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.
- 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.
- 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.
- 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.
- 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 ⁇ m.
- a material having the carbon hexagonal mesh sheet of 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.
- nanoshells which are nanoparticles having a dielectric core and a conducting shell layer. Similar to carbon nanotubes, nanoshells are also 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.
- 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.
- nanoparticles retaining physical properties of larger, micro-sized, particles are of utmost importance.
- the diversity of fields in which the present invention finds uses is the field of molecular biology based research and diagnostics.
- PCR polymerase chain reaction
- PCR amplification is being used to carry out a variety of tasks in molecular cloning and analysis of DNA. These tasks 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 RNA, the generation of large amounts of
- 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.
- Adenine, Thymine, Cytosine and Guanine respectively designated as A, T, C, G.
- Each strand of DNA matches up with a homologous strand in which A pairs with T, and C pairs with G.
- a specific sequence of bases which codes for a protein 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 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.
- 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 sequence which is complementary to the other oligonucleotide.
- 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 great number of times resulting in an exponential amplification of the target fragment in between the primers.
- a DNA polymerase cofactor is a non- protein compound on which the enzyme depends for activity. Without the presence of 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 of a buffer with a low concentration of cofactors results in mispriming and amplification of non-target sequences.
- thermostable DNA polymerases such as Thermus aquaticus (Taq) DNA polymerase
- Taq Thermus aquaticus
- a precise concentration of magnesium ions is necessary to both maximize the efficiency of the polymerase and the specificity of the reaction.
- 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 amplificate, concentration of buffers reaction supplements and the like.
- the efficiency of nucleic acid amplification techniques can be significantly improved with the aid of a liquid composition incorporating nanostructures therein.
- a nanostructure 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 steady physical state.
- 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 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 surface.
- a liquid composition comprising a liquid and nanostructures, the liquid composition facilitates increment of bacterial colony expansion rate, 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition facilitates increment of phage-bacteria or virus-cell interaction, 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition is 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.
- 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 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.
- 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.
- 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 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition 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.
- the composition wherein the solid phase matrix is hydrophilic.
- the solid phase matrix is hydrophobic.
- the solid phase matrix comprises hydrophobic regions and hydrophilic regions.
- the macromolecule is an antibody.
- the antibody is a polyclonal antibody.
- the macromolecule comprises at least one carbohydrate hydrophilic region.
- the macromolecule comprises at least one carbohydrate hydrophobic region.
- the macromolecule is a lectin.
- the macromolecule is a DNA molecule.
- the macromolecule is an RNA molecule.
- 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 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition is 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.
- the composition of the present invention decreases its adherence to biomaterial.
- the biomaterial is selected from the group consisting of plastic, polyester and cement.
- the biomaterial is suitable for being surgically implanted in a subject.
- the bacterial adherence is Staphylococcus epidermidis adherence.
- the Staphylococcus epidermidis adherence is selected from the group consisting of Staphylococcus epidermidis RP 62 A adherence , Staphylococcus epidermidis M7 adherence and Staphylococcus epidermidis (API-6706112) adherence.
- a liquid composition comprising a liquid and nanostructures, the liquid composition is capable of stabilizing enzyme activity, 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.
- the enzyme 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 Phosphatase, and -Galactosidase.
- 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 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition is 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.
- a 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 molecules being in a steady physical state.
- the nucleic acid amplification process is a polymerase chain reaction.
- the composition is capable of enhancing catalytic activity of a DNA polymerase of said polymerase chain reaction.
- the polymerase chain reaction is magnesium free.
- the polymerase chain reaction is manganese free.
- a kit for polymerase chain reaction comprising, in separate packaging (a) a thermostable 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.
- the kit further comprises at least one dNTP.
- the kit further comprises at least one control template DNA.
- the kit further comprises at least one control primer.
- a method of amplifying a DNA sequence 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 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.
- a liquid composition comprising a liquid and nanostructures, the liquid composition 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, the core material and the envelope of ordered fluid molecules being in a steady physical state.
- the macromolecule is a polynucleotide.
- the polynucleotide is selected from the group consisting of DNA and RNA.
- the solid support comprises glass beads.
- the glass beads are between about 80 and 150 microns in diameter.
- the manipulation is effected by a chemical reaction.
- the chemical reaction is selected from the group consisting of an amplification reaction, a ligation reaction, a transformation reaction, transcription reaction, reverse transcription reaction, restriction digestion and transfection reaction.
- a liquid composition comprising a liquid, beads and nanostructures, the liquid composition being capable of allowing the manipulation of at least one macromolecule 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.
- 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.
- at least a portion of the fluid molecules are in a gaseous state.
- the nanostructures are capable of clustering with at least one additional nanostructure.
- the nanostructures are capable of maintaining long range interaction with at least one additional nanostructure.
- the fluid molecules are identical to molecule of the liquid.
- a concentration of the nanostructures is lower than 10 20 nanostructures per liter, more preferably lower than 10 15 nanostructures per liter.
- the nanostructures are capable of maintaining long range interaction thereamongst.
- the core material is selected from the group consisting of a ferroelectric core material, a ferromagnetic core material and a piezoelectric core material.
- the core material is a crystalline core material.
- the liquid is water.
- the nanostructures are designed such that a contact angle between the composition and a solid surface is smaller than a contact angle between the liquid and the solid surface.
- a method of producing a liquid composition from a solid powder comprising: (a) heating the solid powder, thereby providing a heated solid powder; (b) ilmmersing the heated solid powder in a cold liquid; and (c) substantially 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.
- the solid powder comprises micro-sized particles.
- the micro-sized particles are crystalline particles.
- the nanostructures are crystalline nanostructures.
- the solid powder is selected from the group consisting of a ferroelectric material and a ferromagnetic material.
- the solid powder is selected from the group consisting of BaTiO 3 , WO 3 and
- the solid powder comprises a material selected from the group consisting of a mineral, a ceramic material, glass, metal and synthetic polymer.
- 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. 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 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 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.
- FIG. 1 is a schematic illustration of a nanostructure, according to a preferred 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
- 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 shows signals recorded of an upper portion of the tube;
- FIG. 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
- 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);
- 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);
- 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 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
- 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
- FIG. 17a-b show electrochemical deposition of the liquid composition of the present invention ( Figure 17a) and the control ( Figure 17b);
- 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);
- 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. 21a-d show bindings of labeled and non-labeled antibodies to medium costar microtifration plate ( Figure 21a), non-sorp microtifration plate ( Figure 21b), maxisorp microtifration plate (Figure 21c) and polysorp microtifration plate ( Figure 2 Id), using the liquid composition of the present invention or control buffer;
- FIGs. 22a-d show bindings of labeled antibodies to medium costar microtifration plate ( Figure 22a), non-sorp rnicrotiiration plate ( Figure 22b), maxisorp microtifration plate (Figure 22c) and polysorp microtifration plate (Figure 22d), using the liquid composition of the present invention or control buffer;
- FIGs. 23a-d show bindings of labeled antibodies after overnight incubation at
- FIGs. 24a-d show bindings of labeled antibodies 2 hours post incubation at 37 °C, to non-sorp microtitration plate (Figure 24a), medium costar microtifration plate ( Figure 24b), polysorp microtitration plate (Figure 24c) and maxisorp microtitration plate (Figure 24d), using the liquid composition of the present invention or control buffer; FIGs.
- FIGs. 25a-d show binding of labeled and non-labeled antibodies after overnight incubation at 4 °C, to medium costar microtitration plate (Figure 25a), polysorp microtitration plate (Figure 25b), maxisorp microtitration plate (Figure 25c) and non- sorp microtifration plate (Figure 25d), using the liquid composition of the present invention or control buffer; FIGs.
- FIGs. 26a-d show binding of labeled and non-labeled antibodies after overnight incubation at room temperature, to medium costar microtitration plate (Figure 25a), 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 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) and only labeled antibodies ( Figure 28a), after overnight incubation at 4 °C, to medium costar microtitration plate, using the liquid composition of the present invention or control buffer;
- FIGs. 29a-c show binding of labeled lectin to non-sorp microtifration plate for acetate ( Figure 29a), carbonate (Figure 29b) and phosphate (Figure 29c) buffers, using the liquid composition of the present invention or control buffer;
- Figures 30a-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;
- FIGs. 31a-b show an average binding enhancement capability of the liquid composition of the 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;
- FIGs. 36-37 are an image ( Figure 36) and quantitative analysis (Figure 37) of
- FIGs. 38a-c are images of PCR products columns having been passed through 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 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);
- 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;
- FIG. 46a-4Sd are lane images captured in gel electrophoresis experiments in which the effect of the liquid composition of the present invention on the gel buffer was investigated;
- Figure 49 shows values of a stability enhancement parameter, S e , 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;
- 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 an 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;
- FIG. 51a-d show stability of ⁇ -Galactosidase after 24 hours (Figure 51a), 48 hours (Figure 51b), 72 hours (Figure 51c) and 120 hours (Figure 5 Id), in an experiment in which the effect of the liquid composition of the present invention on the activity and stability of ⁇ -Galactosidase was investigated;
- FIG. 52a-d shows values of a stability enhancement parameter, S e , after 24 hours ( Figure 52a), 48 hours (Figure 52b), 72 hours (Figure 52c) and 120 hours ( Figure
- FIG. 53a shows remaining activity of alkaline phosphatase after drying and heat treatment
- FIG. 53b show values of the stability enhancement parameter, S e , of alkaline phosphatase after drying and heat treatment
- FIG. 54 shows lane images captured in gel electrophoresis experiments in 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.
- the present invention is of a nanostructure and liquid composition having the 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 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 description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways.
- Figure 1 illustrates a nanostructure 10 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.
- 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,
- the fluid molecules of envelope 14 may be either in a liquid state or in a gaseous state.
- 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 be selected in accordance with the application for which the nanostructure is designed.
- core material 12 may also have a crystalline structure.
- 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 an electric 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 ferroelectric or ferromagnetic, nanostructure 10 retains its ferroelectric or ferromagnetic properties.
- nanostructure 10 has a particular feature in which macro scale physical properties are brought into a nanoscale environment.
- nanostructure 10 is capable of clustering with at least one additional nanostructure. More specifically, 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 capable of maintaining long range interaction (about 0.5-10 ⁇ m), with the other nanostructures.
- nanostructure 10 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.
- 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 ⁇ m or above 10 ⁇ m in diameter, which are broken in a controlled manner, to provide nanometer-sized particles. Typically, such a process is performed in a cold liquid (preferably, but not obligatorily, water) into which high-temperature raw powder is inserted, under condition of electromagnetic radiofrequency (RF) radiation.
- RF electromagnetic radiofrequency
- 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 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.
- 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 maximum (and molar volume minimum) is brought about by the opposing effects of increasing temperature, causing both structural collapse that 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 entropy and enthalpy due to the less ordered structure and greater hydrogen bond bending, respectively.
- 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 dependent competition between the ordering effects of hydrogen bonding and the disordering kinetic effects.
- 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 enviromnent around these biomolecules are sfrongly involved in biological function with regards to infracellular function including, for example, signal fransduction from receptors to cell nuclei. Additionally these water structures are stable and can protect the surface of the molecule.
- the method 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.
- a solid powder e.g., a mineral, a ceramic powder, a glass powder, a metal powder, a synthetic polymer, etc.
- a sufficiently high temperature preferably more than about 700 °C.
- Representative examples of solid powders which are contemplated include, without limitation, BaTiO 3 , WO 3 and Ba 2 F Oi 2 .
- the heated powder is immersed in a cold liquid, preferably water, below its density anomaly temperature, e.g., 3 °C or 2 °C.
- the cold liquid and the powder are irradiated by electromagnetic RF radiation, preferably above 500 MHz, which may be either continuous wave RF radiation or modulated RF radiation.
- electromagnetic RF radiation preferably above 500 MHz, which may be either continuous wave RF radiation or modulated RF radiation.
- the combination of cold liquid, and RF radiation influences the interface between the particles and the liquid, thereby breaking the liquid molecules and the particles.
- the broken liquid molecules are in the form of free radicals, which envelope the (nano-sized) debris of the particles.
- a small size perturbation may contribute to a pure Casimir effect, which is manifested by long-range interactions.
- the above method according to present invention successfully produces the nanostructure of the present invention.
- the above method allows the formation of envelope 14 as further detailed hereinabove.
- envelope 14 of nanostructure 10 is preferably made of molecules which are identical to the molecule of the liquid.
- the nanostructure may be furtlier 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.
- the concentration of the nanostructures is not limited. A preferred concentration is below 10 20 nanostructures per liter, more preferably below 10 15 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.
- 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. 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 learn" from the environment for improving the growth rate.
- 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, 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 elecfrochemical 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 (for example usmg two electrodes), so that an electrochemical process is initiated.
- a particular property of the ECD process is the material distribution obtained thereby.
- 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 the distance between the elecfrodes.
- 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.
- 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 dendritic morphology.
- a predetermined morphology e.g., dense branching and/or dendritic
- the liquid composition of the present invention is capable of preserving an elecfrochemical signature on the surface of the cell even when replaced by a different liquid (e.g., water).
- 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.
- the contact angle between the liquid composition and the surface is smaller than a contact angle between liquid (without the nanostructures) and the surface.
- this feature of the present invention is not limited to large concentrations of the nanostructures in the liquid, but rather also 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 Examples 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 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.
- ⁇ 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 liquid having different behaviors.
- the elecfrophoretic mobility of particles (the ratio of the velocity of particles to the field strength) is proportional to the ⁇ potential.
- 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 behavior.
- 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 the nanostructures in the liquid, hence, it may contribute, for example, to the formation of special morphologies in the electrochemical deposition process.
- ⁇ potential of the liquid composition including, without limitation, microelecfrophoresis, light scattering, light diffraction, acoustics, electroacoustics etc.
- one method of measuring ⁇ potential is disclosed in U.S. Patent No, 6,449,563, the contents of which are hereby incorporated by reference.
- the present invention also relates to the field of molecular biology research and diagnosis, particularly to nucleic 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 (SSSR).
- nucleic 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 (SSSR).
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand displacement amplification
- SSSR self- sustained sequence replication
- the ability to employ a magnesium-free or manganese-free PCR is highly 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.
- 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.
- 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 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, but not limited to, dATP, dCTP, dGTP, dTTP. Analogues such as dlTP and 7-deaza- dGTP are also contemplated.
- 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 performance.
- a method of amplifying a DNA sequence comprises the following method steps illustrated in the flowchart of Figure 2b.
- the liquid composition of the present invention is provided, and in a second step, a plurality of 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.
- the DNA sequence is first treated to form single-stranded complementary strands.
- pair of oligonucleotide primers which are specific to the DNA sequence are added to the liquid composition.
- the 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.
- Anchoring polynucleotide to a solid support such as glass beads can be of utmost benefit in the field of molecular biology research and medicine.
- 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 research and medical applications, including, but not limited to amplification, transcription, reverse transcription, ligation, restriction digestion, transfection and transformation.
- 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 defined as the synthesis of messenger RNA from DNA.
- DNA manipulations comprise a sequence of reactions, one following the other.
- 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.
- the DNA or RNA sample must be precipitated and then reconstituted in its new appropriate buffer.
- 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 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 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.
- 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.
- the liquid composition of the present invention can be used as a buffer or an add-on to an existing buffer, for improving many chemical and biological assays and reactions.
- the liquid composition of the present invention can be used to at least partially de-fold DNA molecules.
- the liquid composition of the present invention can be used to facilitate isolation and purification of DNA.
- 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.
- the liquid composition of the present invention can also be used for enhancing binding of macromolecule to a solid phase matrix. As 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.
- 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 enhanced, including, witiiout limitation macromolecule having one or more carbohydrate hydrophilic or carbohydrate hydrophobic regions, antibodies, polyclonal antibodies, lectin, DNA molecules, RNA moleculs and the like. Additionally, as demonsfrated in the Examples section that follows (see
- liquid composition of the present invention can be used for increasing a capacity of a column, binding of nucleic acids to a resin and improving gel electrophoresis separation.
- 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 sudden cooling causes the micro-sized particles of the powder to break mto nanostructures. Subsequently, the liquid composition (nanostructure and water) was allowed to heat to room temperature.
- FIGS. 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.
- 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 Figure 3 a, suggest a crystalline structure thereof.
- Figure 3b is an image of a single nanostructure, about 20 nm in diameter.
- a briglit corona marked 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 the center of the nanostructure, as compared to the briglit 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 3 c.
- Figure 3c furtlier demonstrates, in a larger magnification than in 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.
- 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 with a Ru based dye (N3) dissolved in ethanol. One cuvette containing the liquid composition of the present invention (LCI) was exposed to the dye solution for 24 hours.
- a Ru based dye N3
- 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 to the above tests as control groups.
- Figure 4 shows the results of the four tests.
- 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.
- the interaction with the nanostructures affects the dye 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 “dye S-R” was dyed (dye in ethanol) and the sample designated “dye S-dry R” was dried and remeasured.
- FIG. 5a-b show results of five integrated light scattering (ILS) measurements of the liquid composition of the present invention (LCI) after centiifugation.
- Figure 5a shows signals recorded at the lower portion of the tubes. As shown, no signal from structures less that 1 ⁇ m 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 ⁇ m is shown. In all the measurements, the location of the peaks are consistent with nanostructures of about 200-300 nm. This experiment demonstrated that the nanostructures have a specific gravity which is lower than the specific gravity of the host liquid (water).
- EXAMPLE 4 pH Tests The liquid composition of the present invention was subjected to two pH tests.
- caraminic indicator was added to the liquid composition of the present invention (LCI) so as to provide an indication of affective pH.
- Figure 6a shows the spectral change of the caraminic indicator during titration. 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.
- EXAMPLE 5 Zeta Potential Measurement Zeta ( ⁇ ) potential measurements were performed on the liquid composition of the present invention.
- Figure 8 shows ⁇ potential of 6 samples: extra pure water, extra pure water shifted to pH S, exfra pure water shifted to pH 10, two samples of the liquid composition with positive quality and one sample of the liquid composition with negative quality.
- the measurement of the ⁇ potential was performed using a Zeta
- the ⁇ potential of the liquid composition of the present invention is significantly higher, indicating a high mobility of the nanostructures in the liquid.
- EXAMPLE 6 Bacteriophage Reaction The effect of the liquid composition of the present invention (LC9) on bacteriophage typing was investigated. 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. 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.
- SA staphylococcus aureus
- Figures 9a-b illustrate the bacteriophage reaction in the tested media, as follows:
- Figure 9a shows Bacteriophages No. 6 in a confrol medium (right hand side) and in the liquid composition of the present invention (left hand side);
- Figure 9b shows
- the bacteriophage reaction in the liquid composition of the present invention demonsfrated an accelerated lysis of bacteria
- FIG. 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.
- RTD determination Figure 11 shows increased dilution by 10 times in each increment. Increased concenfration 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 reaction are given in Tables 3 and 4, respectively. The ANOVA for repeated measures is presented in Table 5.
- the liquid composition of the present invention accelerates the phage reaction time (x3); and increases the bacteriolysis surface area; increases the RTD (xlOO or more)
- the bacteriophage reactions in the liquid composition of the present invention demonstrate opposite trends compare to confrol in OD measurements, and increased potency with time. Discussion
- the kinetics of phage-host interaction has been enhanced in media containing 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 concenfration 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 present invention are still effective.
- the propagating strains pre-treated with the liquid composition are much more effective.
- EXAMPLE 7 Effect of the Liquid Composition on Phage-Bacteria Interaction
- ⁇ phage is used in molecular biology for representing the genome DNA of organisms.
- the following experiment relies on standard ⁇ phage interaction applications.
- the materials in the test groups were prepared with the liquid composition as a solvent.
- the materials in confrol groups were prepared as described hereinbelow.
- the pH of the confrol groups was adjusted to the pH of the liquid composition solutions, which was between 7.2 and 7.4. Materials and Methods 1) LB medium 10 g.
- MgSO 4 - 1 M 120.37 g of MgSO were dissolved in 1000 ml distilled water and sterilized by autoclaving.
- SM buffer (phage storage buffer) 5.8 g of NaCl, 2 g of MgSO 4 , 50 ml of IM Tris HCl (pH 7.5), 5 ml of 2 % (w/v) gelatin were dissolved in distilled water, to a final volume of 1000 ml, and then, sterilized by autoclaving.
- Phage ⁇ GEM 11 (Promega).
- 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; (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.
- PFU Plaque Forming Unit
- 1/10 dilutions one in SM buffer based on liquid composition of tlie present invention and one in SM buffer based on ddH 2 O.
- 1 ⁇ l of each dilution was incubated with 200 ⁇ l of competent bacterial host (see methods, item 13). The suspension was incubated at 37 °C for 15 minutes to allow tlie bacteriophage to inject its DNA into the host bacteria. After incubation a hot (45-
- Increased compatibility can be established by the observation of either larger plaques than those of confrol (a greater distance from the initial infection site), or a greater number of phage particles 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 liquid composition of the present invention was used (about 2-fold greater than the confrol) indicates that the liquid composition of the present invention affects the infectivity.
- Pre-infection treatments are required for increasing probability of infection by preparing competent bacteria, which are easier infected by phage than non-treated bacteria.
- 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 assumed that the liquid composition increases the affinity between bacterial receptors and phage particles.
- EXAMPLE 8 Effect of the Liquid Composition on the Adherence of Coagulase-Negative Staphylococci to Microtiter Plate Production of slime polys accharide, 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 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.
- 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 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 developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. Further efforts are done to find better protection from slime adherence.
- surface interaction can modify slime adherence. For example,
- 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. 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.
- 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 Figures 13a-c show the OD in all the slime-producing Staphylococcus epidermidis (see Table 8, above).
- 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 interaction between time and type of water used (p 0.787).
- FIG 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 confrol. Significant adherence differences in the liquid composition and control, between the micro titer plates, and, among the strains were found (p ⁇ 0.001). Significant interactions were found between plates, strain and the type of water used. The extent of adherence is dependent on the sfrain, 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).
- Figure 15 shows slime adherence differences in the liquid composition of the present invention and the confrol on the same micro titer plate.
- Tables 11-12 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 ⁇ .001), strain (p ⁇ .001), and water ( ⁇ .001) 4S were also found, confirming that the adherence differences in the liquid composition depend also on the plate, sfrain 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 composition indicate that there may be other factors on the plate surface or during plate preparation which could interact with the liquid composition.
- 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 sterilization, as compared to confrol 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.
- EXAMPLE 9 Electrochemical Deposition Tests The liquid .composition of the present invention has been subjected to a series of elecfrochemical deposition tests, in a quasi-two-dimensional cell. Experimental Setup The experimental setup is shown in Figures 16a-c.
- 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 internal elecfrode was connected to the negative pole thereof.
- the experimental setup was used to perform an elecfrochemical deposition process directly on the liquid composition of the present invention and, for comparison, on a control solution composed of Reverse Osmosis (RO) water.
- the experimental setup was used to examine the capability of the liquid composition to leave an elecfrochemical deposition signature, as follows. The liquid composition was placed in cell 20. After being in contact with base 22 for a period of
- FIGS. 17a-b show electrochemical deposition of the liquid composition of the present invention (Figure 17a) and the confrol ( 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 millimeters from the face of the negative electrode. In the confrol, the dense branching morphology was observed only in close proximity to the negative electrode and no morphology transition was 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 minutes.
- the confrol group included the same bacteria in the presence of RO water.
- Figures 19a-b show results of Bacillus subtilis colony growth after 24 hours, for the liquid composition ( Figure 19a) and the confrol ( Figure 19b).
- the liquid composition of the present invention significantly accelerates the colony growth.
- 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, without the manufacturing process as further detailed above. This mixture is referred to hereinafter as Source Powder (SP) water.
- SP Source Powder
- 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).
- 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.
- the liquid composition of the present invention significantly accelerates the colony growth, although, in principle, the liquid composition is composed of the same material.
- Solid phase Matrix 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 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 biological material to microtitration plates and membranes having different physical and chemical properties.
- microtitration plates of CORNINGTM (Costar) were used: (i) a medium binding microtifration plate, which has a hydrophilic surface and a binding capacity to IgG of 250 ng/cm 2 ; (ii) a carbon binding microtifration 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.
- the binding efficiency of bio-molecules to the above microtifration plates was tested in four categories: ionic strengths, buffer pH, temperature and time.
- the binding experiments were conducted by coating the microtifration 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 measuring the fluorescent signal remaining on the plate.
- the following protocol was employed: 1) Pre-diluting the fluorescent labeled bio-molecules to different concentrations (typically 0.4 - 0.02 ⁇ g/ml) in a binding buffer. Each set of dilutions was performed in two binding buffers: (i) the liquid composition of the present invention; and (ii) control RO water. 2) Dispensing (in triplicates) 100 ⁇ l samples from each concenfration to the microtitration plates, and measuring the initial fluorescence level.
- IgG is a polyclonal antibody composed of a mixture of mainly hydrophilic molecules.
- the molecules have a carbohydrate hydrophilic region, at tlie universal region and are slightly hydrophobic at the variable region.
- Such types of molecules are known to bind to MaxiSorpTM plates with very high efficiency (650 ng/cm 2 ).
- the following types of liquid composition of the present invention were used: LCI, LC2, LC3, LC4, LC5 and LC6, as further detailed hereinabove.
- Table 13 summarizes six assays which were conducted for IgG.
- assays in which only labeled antibodies were used are designated Ab*
- assays in which a mixture of labeled and non-labeled antibodies were used are designated Ab*/Ab.
- PNA agglutinin
- Figures 21a-22d show the results of the Ab*/Ab assays ( Figures 21a-d) and the
- the results obtained using the liquid composition of the 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 of the present invention are steeper than the slopes obtained in the confrol 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 ⁇ 1 corresponds to binding suppression.
- Sr > 1 corresponds to binding enhancement
- Sr ⁇ 1 corresponds to binding suppression.
- the values of the Sr parameter calculated for the slopes obtained in Figures 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 incubation at 4 °C ( Figures 23a-d) and the 2 hours incubation at 37 °C ( Figure 24a-d) in NonSorpTM (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 incubation in the NonSorpTM 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.
- 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), PolySorpTM (b), MaxiSorpTM (c) and Non- SorpTM (d) plates. As shown in Figures 25a-26d, except for one occurrence
- Figures 27a-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
- Figures 27c-d show the results of Ab*/Ab (Figure 27c) and Ab* (Figure 27d) assays using PBS.
- 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.
- Figure 30a with the carbonate buffer, a two-phase curve was obtained, with a linear part in low protein concenfration in which no effect was observed and a nonlinear part in high protein concenfration (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 similar frend 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
- Table 19 summarizes the obtained values of the Sr parameter, for nine different concenfrations of the oligonucleotide and four different experimental conditions, averaged over the assays in which MaxiSorpTM plates in acetate coating buffer were used. Table 19
- Figures 31a-b show the average values of the Sr parameter quoted in Table 19, where Figure 31a shows the average values for each experimental conditions and Figure 31b shows the overall average, with equal weights for all the experimental conditions.
- the average values of the Sr parameter were significantly larger then 1, with a higher binding efficiency for higher concenfrations of oligonucleotides.
- the liquid composition of the present invention is capable of enhancing binding efficiency with and without the addition of salt to the coating buffer. It is a common knowledge that acetate buffer is used to precipitate DNA in aqua's solutions.
- the liquid composition of the present invention is capable of suppressing the enhancement of clump formations for higher concenfration.
- the higher binding efficiency of DNA on MaxiSorpTM 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 and RNA are the basic and most important material used 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 oligonucleotide primers, primer dimers, deoxinucleotide bases (A, T, C, G) and salt and subsequently verified.
- step 3 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
- PCR was prepared from Human D ⁇ A (Promega G 3041) using ApoE gene specific primers (fragment size 265 bp), according to the following protocol (for 100 reactions): (a) Mark 0.2 ⁇ l PCR-tubes according to the appropriate serial number. (b) Add 2.5 ⁇ l of 40 ⁇ g/ml Human D ⁇ A (Promega G 3041) or water to the relevant tubes. (c) Adjust to 17 ⁇ l with 14.5 ⁇ l DDW. (d) Prepare 3630 ⁇ l of tlie PCR mix according to Table 21 (see below).
- lane 1 corresponds to the PCR product before purification
- lane 7 is a ladder marker
- lanes 2-6, 8-11 correspond to the following combinations of the aforementioned steps 1, 2 and 4: 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).
- Figures 33a-b are images of 50 ⁇ l PCR product samples in an experiment, referred to herein as Experiment 4, for elution 1 ( Figure 33 a) and elution 2 ( Figure 33b).
- lanes in Figures 33a-b there are 13 lanes in Figures 33a-b, in which lane 6 is a ladder marker, and lanes 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 concenfration was used for the liquid composition of the present invention.
- Figures 34a-b are images of 50 ⁇ l PCR product samples in an experiment, referred to herein as Experiment 5, for elution 1 (Figure 34a) and elution 2 (Figure 34b).
- lane 4 is a ladder marker
- lanes 1-3, 5-13 correspond to the following combinations: CO/CO/CO (lane 1), RO/RO/RO (lane 2), LC/LC/LC (lane 1)
- Lane 14 in Figure 34a corresponds to the combination RO/CO/CO.
- Figures 35a-b are images of 50 ⁇ l PCR product samples in an experiment, referred to herein as Experiment 6, for elution 1 (Figure 35a) and elution 2 (Figure 35b).
- Lanes 35a-b lanes 1-13 correspond to the same combinations as in Figure 34a, and lane 15 corresponds to the PCR product before purification.
- EXAMPLE 13 Column Capacity
- the effect of the liquid composition of the present invention on 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.
- step A was directed at examining the effect of volume applied to the columns on binding and elution
- step B was directed at investigating the effect of the liquid composition of the present invention on the column capacity.
- Step A four columns (columns 1-4) were applied with 50, 150, 300 or 600 ⁇ l stock PCR product solution, and 13 columns (5-17) were applied with 300 ⁇ l of stock PCR solution. All columns were eluted with 50 ⁇ l of water. The eluted solutions 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.
- Step A 1) Mark the WizardTM minicolumn and the syringe for each sample, and insert into the Vacuum Manifold. 2) Dispense 100 ⁇ l 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) 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 micro centrifuge tube. 7) Centrifuge at 10000 g for 2 minutes.
- Step B Shake in TBE buffer at room temperature for 30 minutes to destain the gel. 17) photograph the gel.
- 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 ⁇ l, 25 ⁇ l, 50 ⁇ l, 75 ⁇ l and 100 ⁇ l respectively using the kit reagents.
- the columns were eluted by 50 ⁇ l kit water and 5 ⁇ l of each elution was applied to the corresponding lane on the 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 gel lanes.
- Step B 1) Mark the WizardTM minicolumn and syringe to be used for each sample and insert into the vacuum manifold. 2) Dispense 100 ⁇ l of each direct PCR purification buffer solution into 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 apply vacuum. 5) Dry the resin by continuing to apply the vacuum for 30 seconds.
- Lanes 3 and 4 contain less DNA because columns 3 and 4 were overloaded and as a result less DNA was recovered after dilution of the eluted samples.
- 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 elution 3 (Figure 38c). The first elution figure shows that the columns were similarly overloaded,. The differences in binding capacity are clearly seen in the second elution. The band intensity increases correspondingly with the number of the lane. 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
- Figure 39a represents the area of the confrol (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.
- 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 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.
- DNA can be tagged by fluorescent label and is visualized under UV illumination.
- 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.
- 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; and in Experiment 3, PCR batch number 31203 was loaded into lanes 1-5 and 7-11, with the ladder DNA in lane 6.
- Figures 40a-42b are DNA images comparing the migration speed 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) for Experiments 1, 2 and 3, respectively.
- both the ranting 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 Figures 40b, 41b and 42b both the running buffer and the gel buffer were composed of the liquid composition of the present invention.
- both types of DNA PCR product and the ladder DNA
- 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 invention on the running buffer are investigated.
- the gels are composed of the same liquid and the running buffer is different.
- Figures 43a-45d are images of RO/RO and RO/LC, respectively;
- Figures 43 c-d are images of LC/LC and LC/RO respectively,
- 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
- 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 invention on the gel buffer are investigated.
- the running buffers are composed of the same liquid but the gel buffers are different.
- 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 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;
- Figures 48c-d are images of RO/LC and LC/LC respectively.
- the liquid composition of the present invention 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.
- the above experiments demonstrate that under the influence of the liquid 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.
- 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 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 demonsfrated.
- This study relates to two commonly used enzymes in the biotechnological industry: Alkaline Phosphatase (AP), and / 3-Galactosidase. Two forms of AP were used: an unbound form and a bound form in which AP was bound to Sfrept-Avidin (ST-AP).
- a stability enhancement parameter, S e 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 S e , for 22 hours (full triangles) and 48 hours (full squares), as a function of the dilution. Tlie values of S e for LC7, LC8 and LC3 are shown in Figure 49 in blue, red, and green, respectively).
- the measured stabilizing effect is in the range of about 2 to 3.6 for enzyme dilution of 1:10,000, and in 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 Binding an enzyme to another molecule typically increases its stability.
- Enzymes are typically stored at high concenfrations, 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.
- Materials and Methods Sfrept-Avidin Alkaline Phosphatase (Sigma) was diluted 1 :10 and 1 :10,000 in RO water and in the aforementioned liquid compositions LC7, LCS and LC3 of the present invention. The diluted samples were incubated in tubes for 5 days at room temperature. All samples were diluted to a final enzyme concenfration of 1:10,000 and the activity was determined as further detailed hereinabove.
- Figure 50 is a chart showing the activity of the conjugated enzyme after 5 days 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.
- the enzyme activity is about 0.150 OD for both dilutions.
- the activity is about 3.5 times higher in the 1:10 dilution than in the 1:10,000 dilution.
- the enzyme is substantially more active in the liquid composition of the present invention than in RO water.
- ⁇ -Galactosidase Materials and Methods The experiments with ⁇ -Galactosidase were performed according to the same protocol used for the Alkaline Phosphatase experiments described above with the exception of enzyme type, concentration and in incubation time. ⁇ -Galactosidase
- the measured stabilizing effect is in tlie 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.
- the stabilizing effect liquid composition of the present invention on ⁇ - 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 activity (464 u mg) having high protein concenfration in the assay, which has attenuated activity lost over time.
- Activity and stability of dry alkaline phosphatase Many enzymes are dried before storage.
- Color intensity was determined by an ELIS A reader at a wavelength of 405 nm and the stability was calculated as further detailed hereinabove. Six plates were transferred to
- 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
- FIG 53a by a "+" symbol. Both treatments substantially damaged the enzyme and their effect was additive.
- Figure 53b shows the stability enhancement parameter, S e .
- 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.
- EXAMPLE 16 Anchoring of DNA
- the effect of anchoring DNA with glass beads in the presence or absence of the liquid composition of the present invention was examined. Anchoring polynucleotides to a solid support such as glass beads can be of utmost benefit in the field of molecular biology research and medicine.
- 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.
- 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.
- the inventors chose to investigate what effect the liquid composition of the present invention has on DNA in the presence of glass beads during a PCR reaction.
- PCR was prepared from a pBS plasmid cloned with a 750 base pair gene using a T7 forward primer (TAATACGACTCACTATAGGG) and an Ml 3 reverse primer (GGAAACAGCTATGACCATGA) such that the fragment size obtained is 750 bp.
- the primers were constituted in PCR-grade water at a concenfration of 200 ⁇ M (200pmol/ ⁇ l). These were subsequently diluted 1 :20 in Neowater Tm , to a working concentration of lO ⁇ M each to make a combined mix.
- each primer from 200 ⁇ M stock
- 18 ⁇ l of Neowater Tm mixed and spun down
- the concentrated DNA was diluted 1 :500 with Neowater Tm to a working concentration of 2pg/ ⁇ l.
- the PCR was performed in a Biomefra 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:
- 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 ⁇ l of the PCR mix was then aliquoted into clean tubes to which 0.5 ⁇ l of primer mix and 5 ⁇ l of diluted DNA were added in that order. After mixing, but not vortexing or centrifugation, the samples were placed in tlie PCR machine and the following PCR program used:
- the products of the PCR reaction were run on 8 % PAGE gels for analysis as described herein above.
- the PCR products loaded onto the gel were as follows: Lane 1: DNA diluted in Neowater Tm , Primers (mix) diluted in H 2 O, vol (to lO ⁇ l) with Neowater Tm (with glass beads). Lane 2: DNA diluted in Neowater Tm , Primers (mix) diluted in Neowater Tm , vol (to lO ⁇ l) with Neowater (with glass beads). Lane 3: All in H 2 O (positive control) (with glass beads). Lane 4: Negative confrol. No DNA, Primers in Neowater Tm (to lO ⁇ l) with H 2 O (with glass beads).
- Lane 5 DNA diluted in Neowater Tm , Primers (mix) diluted in H 2 O, vol (to 1 O ⁇ l) with Neowater Tm (without glass beads). Lane 6: DNA diluted in Neowater Tm , Primers (mix) diluted in Neowater Tm , vol
- 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). In conclusion, the liquid composition of the present invention is required during a PCR reaction in the presence of glass beads.
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