Patent Grant
10441608
This invention relates generally to novel methods and novel devices for the continuous manufacture of nanoparticles, microparticles and nanoparticle/liquid solution(s). The nanoparticles (and/or micron-sized particles) comprise a variety of possible compositions, sizes and shapes. The particles (e.g., nanoparticles) are caused to be present (e.g., created and/or the liquid is predisposed to their presence (e.g., conditioned)) in a liquid (e.g., water) by, for example, preferably utilizing at least one adjustable plasma (e.g., created by at least one AC and/or DC power source), which plasma communicates with at least a portion of a surface of the liquid. At least one subsequent and/or substantially simultaneous adjustable electrochemical processing technique is also preferred. Multiple adjustable plasmas and/or adjustable electrochemical processing techniques are preferred. The continuous process causes at least one liquid to flow into, through and out of at least one trough member, such liquid being processed, conditioned and/or effected in said trough member(s). Results include constituents formed in the liquid including micron-sized particles and/or nanoparticles (e.g., metallic-based nanoparticles) of novel size, shape, composition, zeta potential and properties present in a liquid.
Many techniques exist for the production of nanoparticles including techniques set forth in “Recent Advances in the Liquid-Phase Syntheses of Inorganic Nanoparticles” written by Brian L. Cushing, Vladimire L. Kolesnichenko and Charles J. O'Connor; and published in Chemical Reviews, volume 104, pages 3893-3946 in 2004 by the American Chemical Society; the subject matter of which is herein expressly incorporated by reference.
Further, the article “Chemistry and Properties of Nanocrystals of Different Shapes” written by Clemens Burda, Xiaobo Chen, Radha Narayanan and Mostafa A. El-Sayed; and published in Chemical Reviews, volume 105, pages 1025-1102 in 2005 by the American Chemical Society; discloses additional processing techniques, the subject matter of which is herein expressly incorporated by reference.
The article “Shape Control of Silver Nanoparticles” written by Benjamin Wiley, Yugang Sun, Brian Mayers and Younan Xia; and published in Chemistry—A European Journal, volume 11, pages 454-463 in 2005 by Wiley-VCH; discloses additional important subject matter, the subject matter of which is herein expressly incorporated by reference.
Still further, U.S. Pat. No. 7,033,415, issued on Apr. 25, 2006 to Mirkin et al., entitled Methods of Controlling Nanoparticle Growth; and U.S. Pat. No. 7,135,055, issued on Nov. 14, 2006, to Mirkin et al., entitled Non-Alloying Core Shell Nanoparticles; both disclose additional techniques for the growth of nanoparticles; the subject matter of both are herein expressly incorporated by reference.
Moreover, U.S. Pat. No. 7,135,054, which issued on Nov. 14, 2006 to Jin et al., and entitled Nanoprisms and Method of Making Them; is also herein expressly incorporated by reference.
The present invention has been developed to overcome a variety of deficiencies/inefficiencies present in known processing techniques and to achieve a new and controllable process for making nanoparticles of a variety of shapes and sizes and/or new nanoparticle/liquid materials not before achievable.
This invention relates generally to novel methods and novel devices for the continuous manufacture of a variety of constituents in a liquid including micron-sized particles, nanoparticles, ionic species and nanoparticle/liquid(s) solution(s). The constituents and nanoparticles produced can comprise a variety of possible compositions, sizes and shapes, which exhibit a variety of novel and interesting physical, catalytic, biocatalytic and/or biophysical properties. The liquid(s) used and created/modified during the process play an important role in the manufacturing of, and/or the functioning of the micron-sized particles and the nanoparticles. The particles (e.g., nanoparticles) are caused to be present (e.g., created and/or the liquid is predisposed to their presence (e.g., conditioned)) in at least one liquid (e.g., water) by, for example, preferably utilizing at least one adjustable plasma (e.g., created by at least one AC and/or DC power source), which adjustable plasma communicates with at least a portion of a surface of the liquid. Metal-based electrodes of various composition(s) and/or unique configurations are preferred for use in the formation of the adjustable plasma(s), but non-metallic-based electrodes can also be utilized. Utilization of at least one subsequent and/or substantially simultaneous adjustable electrochemical processing technique is also preferred. Metal-based electrodes of various composition(s) and/or unique configurations are preferred for use in the electrochemical processing technique(s). Electric fields, magnetic fields, electromagnetic fields, electrochemistry, pH, zeta potential, etc., are just some of the variables that can be positively effected by the adjustable plasma(s) and/or adjustable electrochemical processing technique(s). Multiple adjustable plasmas and/or adjustable electrochemical techniques are preferred to achieve many of the processing advantages of the present invention, as well as many of the novel compositions which result from practicing the teachings of the preferred embodiments. The overall process is a continuous process, having many attendant benefits, wherein at least one liquid, for example water, flows into, through and out of at least one trough member and such liquid is processed, conditioned, modified and/or effected by said at least one adjustable plasma and/or said at least one adjustable electrochemical technique. The results of the continuous processing include new constituents in the liquid, micron-sized particles, nanoparticles (e.g., metallic-based nanoparticles) of novel size, shape, composition, zeta potential and/or properties suspended in a liquid, such nanoparticle/liquid mixture being produced in an efficient and economical manner.
Certain processing enhancers may also be added to or mixed with the liquid(s). The processing enhancers include both solids and liquids. The processing enhancer may provide certain processing advantages and/or desirable final product characteristics.
The phrase “trough member” is used throughout the text. This phrase should be understood as meaning a large variety of fluid handling devices including, pipes, half pipes, channels or grooves existing in materials or objects, conduits, ducts, tubes, chutes, hoses and/or spouts, so long as such are compatible with the process disclosed herein.
Additional processing techniques such as applying certain crystal growth techniques disclosed in copending patent application entitled Methods for Controlling Crystal Growth, Crystallization, Structures and Phases in Materials and Systems; which was filed on Mar. 21, 2003, and was published by the World Intellectual Property Organization under publication number WO 03/089692 on Oct. 30, 2003 and the U.S. National Phase application, which was filed on Jun. 6, 2005, and was published by the United States Patent and Trademark Office under publication number 20060037177 on Feb. 23, 2006 (the inventors of each being Bentley J. Blum, Juliana H. J. Brooks and Mark G. Mortenson). The subject matter of both applications is herein expressly incorporated by reference. These applications teach, for example, how to grow preferentially one or more specific crystals or crystal shapes from solution. Further, drying, concentrating and/or freeze drying can also be utilized to remove at least a portion of, or substantially all of, the suspending liquid, resulting in, for example, dehydrated nanoparticles.
An important aspect of one embodiment of the invention involves the creation of an adjustable plasma, which adjustable plasma is located between at least one electrode positioned adjacent to (e.g., above) at least a portion of the surface of a liquid and at least a portion of the surface of the liquid itself. The liquid is placed into electrical communication with at least one second electrode (or a plurality of second electrodes) causing the surface of the liquid to function as an electrode helping to form the adjustable plasma. This configuration has certain characteristics similar to a dielectric barrier discharge configuration, except that the surface of the liquid is an active electrode participant in this configuration.
Each adjustable plasma utilized can be located between the at least one electrode located above a surface of the liquid and a surface of the liquid due to at least one electrically conductive electrode being located somewhere within (e.g., at least partially within) the liquid. At least one power source (in a preferred embodiment, at least one source of volts and amps such as a transformer) is connected electrically between the at least one electrode located above the surface of the liquid and the at least one electrode contacting the surface of the liquid (e.g., located at least partially, or substantially completely, within the liquid). The electrode(s) may be of any suitable composition and suitable physical configuration (e.g., size and shape) which results in the creation of a desirable plasma between the electrode(s) located above the surface of the liquid and at least a portion of the surface of the liquid itself.
The applied power (e.g., voltage and amperage) between the electrode(s) (e.g., including the surface of the liquid functioning as at least one electrode for forming the plasma) can be generated by any suitable source (e.g., voltage from a transformer) including both AC and DC sources and variants and combinations thereof. Generally, the electrode or electrode combination located within (e.g., at least partially below the surface of the liquid) takes part in the creation of a plasma by providing voltage and current to the liquid or solution, however, the adjustable plasma is actually located between at least a portion of the electrode(s) located above the surface of the liquid (e.g., at a tip or point thereof) and one or more portions or areas of the liquid surface itself. In this regard, the adjustable plasma can be created between the aforementioned electrodes (i.e., those located above at least a portion of the surface of the liquid and a portion of the liquid surface itself) when a breakdown voltage of the gas or vapor around and/or between the electrode(s) and the surface of the liquid is achieved or maintained.
In one preferred embodiment of the invention, the liquid comprises water, and the gas between the surface of the water and the electrode(s) above the surface of the water (i.e., that gas or atmosphere that takes part in the formation of the adjustable plasma) comprises air. The air can be controlled to contain various different water content(s) or a desired humidity which can result in different compositions, sizes and/or shapes of nanoparticles being produced according to the present invention (e.g., different amounts of certain constituents in the adjustable plasma and/or in the solution can be a function of the water content in the air located above the surface of the liquid) as well as different processing times, etc.
The breakdown electric field at standard pressures and temperatures for dry air is about 3 MV/m or about 30 kV/cm. Thus, when the local electric field around, for example, a metallic point exceeds about 30 kV/cm, a plasma can be generated in dry air. Equation (1) gives the empirical relationship between the breakdown electric field “Ec” and the distance “d” (in meters) between two electrodes:
Of course, the breakdown electric field “Ec” will vary as a function of the properties and composition of the gas located between electrodes. In this regard, in one preferred embodiment where water is the liquid, significant amounts of water vapor can be inherently present in the air between the “electrodes” (i.e., between the at least one electrode located above the surface of the water and the water surface itself which is functioning as one electrode for plasma formation) and such water vapor should have an effect on at least the breakdown electric field required to create a plasma therebetween. Further, a higher concentration of water vapor can be caused to be present locally in and around the created plasma due to the interaction of the adjustable plasma with the surface of the water. The amount of “humidity” present in and around the created plasma can be controlled or adjusted by a variety of techniques discussed in greater detail later herein. Likewise, certain components present in any liquid can form at least a portion of the constituents forming the adjustable plasma located between the surface of the liquid and the electrode(s) located adjacent (e.g., along) the surface of the liquid. The constituents in the adjustable plasma, as well as the physical properties of the plasma per se, can have a dramatic influence on the liquid, as well as on certain of the processing techniques (discussed in greater detail later herein).
The electric field strengths created at and near the electrodes are typically at a maximum at a surface of an electrode and typically decrease with increasing distance therefrom. In cases involving the creation of an adjustable plasma between a surface of the liquid and the at least one electrode(s) located adjacent to (e.g., above) the liquid, a portion of the volume of gas between the electrode(s) located above a surface of a liquid and at least a portion of the liquid surface itself can contain a sufficient breakdown electric field to create the adjustable plasma. These created electric fields can influence, for example, behavior of the adjustable plasma, behavior of the liquid, behavior of constituents in the liquid, etc.
In this regard,
The adjustable plasma region 4, created in the embodiment shown in
The composition of the electrode(s) 1 involved in the creation of the adjustable plasma(s) 4 of
Still further, the electrode(s) 1 and 5 may be of similar chemical composition and/or mechanical configuration or completely different compositions in order to achieve various compositions and/or structures of liquids and/or specific effects discussed later herein.
The distance between the electrode(s) 1 and 5; or 1 and 1 (shown later herein) or 5 and 5 (shown later herein) is one important aspect of the invention. In general, the location of the smallest distance “y” between the closest portions of the electrode(s) used in the present invention should be greater than the distance “x” in order to prevent an undesirable arc or formation of an unwanted corona or plasma occurring between the electrode (e.g., the electrode(s) 1 and the electrode(s) 5) (unless some type of electrical insulation is provided therebetween). Features of the invention relating to electrode design, electrode location and electrode interactions between a variety of electrodes are discussed in greater detail later herein.
The power applied through the power source 10 may be any suitable power which creates a desirable adjustable plasma 4 under all of the process conditions of the present invention. In one preferred mode of the invention, an alternating current from a step-up transformer (discussed in greater detail later herein) is utilized. In another preferred embodiment, a rectified AC source creates a positively charged electrode 1 and a negatively charged surface 2 of the liquid 3. In another preferred embodiment, a rectified AC source creates a negatively charged electrode 1 and a positively charged surface 2 of the liquid 3. Further, other power sources such as RF power sources are also useable with the present invention. In general, the combination of electrode(s) components 1 and 5, physical size and shape of the electrode(s) 1 and 5, electrode manufacturing process, mass of electrodes 1 and/or 5, the distance “x” between the tip 9 of electrode 1 above the surface 2 of the liquid 3, the composition of the gas between the electrode tip 9 and the surface 2, the flow rate and/or flow direction “F” of the liquid 3, the amount of liquid 3 provided, type of power source 10, frequency of power source 10, all contribute to the design, and thus power requirements (e.g., breakdown electric field) required to obtain a controlled or adjustable plasma 4 between the surface 2 of the liquid 3 and the electrode tip 9.
In further reference to the configurations shown in
Preferred techniques for automatically raising and/or lowering the electrodes 1, 5 are discussed later herein. The power source 10 can be connected in any convenient electrical manner to the electrodes 1 and 5. For example, wires 11a and 11b can be located within at least a portion of the electrode holders 6a, 6b (and/or electrical insulating portions 7a, 7b) with a primary goal being achieving electrical connections between the portions 11a, 11b and thus the electrodes 1, 5.
Likewise, a set of manually controllable electrode configurations, corresponding generally to
Moreover, it should be understood that in alternative preferred embodiments of the invention, well defined sharp points are not always required for the tip 9. In this regard, the electrode 1 shown in
Likewise,
Likewise,
The electrode configurations shown generally in
Likewise, several additional alternative cross-sectional embodiments for the liquid-containing trough member 30 are shown in
Also, the initial temperature of the liquid 3 input into the trough member 30 can also affect a variety of properties of products produced according to the disclosure herein. For example, different temperatures of the liquid 3 can affect particle size, concentration or amounts of various formed constituents (e.g., transient, semi-permanent or permanent constituents), pH, zeta potential, etc. Likewise, temperature controls along at least a portion of, or substantially all of, the trough member 30 can have similar effects.
Further, certain processing enhancers may also be added to or mixed with the liquid(s). The processing enhancers include both solids and liquids. The processing enhancer may provide certain processing advantages and/or desirable final product characteristics. Examples of processing enhancers may include certain acids, certain bases, salts, nitrates, etc. Processing enhancers may assist in one or more of the electrochemical reactions disclosed herein; and/or may assist in achieving one or more desirable properties in products formed according to the teachings herein.
It should be understood that a variety of different shapes can exist for the trough member 30, any one of which can produce desirable results as a function of a variety of design and production considerations. For example, one or more constituents produced in the portion(s) 30a, 30b and/or 30c could be transient and/or semi permanent. If such constituent(s) produced, for example, in portion 30a is to be desirably and controllably reacted with one or more constituents produced in, for example, portion 30b, then a final product (e.g., properties of a final product) which results from such mixing could be a function of when constituents formed in the portions 30a and 30b are mixed together. For example, final properties of products made under similar sets of conditions experienced in, for example, the portions 30a and 30b, if combined in, for example, the section 30d (or 30d′), could be different from final properties of products made in the portions 30a and 30b and such products are not combined together until minutes or hours or days later. Also, the temperature of liquids entering the section 30d (or 30d′) can be monitored/controlled to maximize certain desirable properties of final products and/or minimize certain undesirable products. Still further, processing enhancers may be selectively utilized in one or more of the portions 30a, 30b, 30c, 30d and/or 30o (or at any point in the trough member 30).
In particular,
In contrast,
Each of the electrode configurations shown in
The embodiments disclosed herein relate generally to novel methods and novel devices for the continuous manufacture of a variety of constituents in a liquid including nanoparticles, and nanoparticle/liquid(s) solution(s). The nanoparticles produced in the various liquids can comprise a variety of possible compositions, sizes and shapes, zeta potential (i.e., surface change), conglomerates, composites and/or surface morphologies which exhibit a variety of novel and interesting physical, catalytic, biocatalytic and/or biophysical properties. The liquid(s) used and/or created/modified during the process play an important role in the manufacturing of and/or the functioning of the nanoparticles and/or nanoparticle/liquid(s) solutions(s). The atmosphere(s) used play an important role in the manufacturing and/or functioning of the nanoparticle and/or nanoparticle/liquid(s) solution(s). The nanoparticles are caused to be present (e.g., created) in at least one liquid (e.g., water) by, for example, preferably utilizing at least one adjustable plasma (e.g., formed in one or more atmosphere(s)), which adjustable plasma communicates with at least a portion of a surface of the liquid. The power source(s) used to create the plasma(s) play(s) an important role in the manufacturing of and/or functioning of the nanoparticles and/or nanoparticle/liquid(s) solution(s). For example, the voltage, amperage, polarity, etc., all can influence processing and/or final properties of produced products. Metal-based electrodes of various composition(s) and/or unique configurations are preferred for use in the formation of the adjustable plasma(s), but non-metallic-based electrodes can also be utilized. Utilization of at least one subsequent and/or substantially simultaneous adjustable electrochemical processing technique is also preferred. Metal-based electrodes of various composition(s) and/or unique configurations are preferred for use in the adjustable electrochemical processing technique(s).
Adjustable Plasma Electrodes and Adjustable Electrochemical Electrodes
An important aspect of one embodiment of the invention involves the creation of an adjustable plasma, which adjustable plasma is located between at least one electrode (or plurality of electrodes) positioned above at least a portion of the surface of a liquid and at least a portion of the surface of the liquid itself. The surface of the liquid is in electrical communication with at least one second electrode (or a plurality of second electrodes). This configuration has certain characteristics similar to a dielectric barrier discharge configuration, except that the surface of the liquid is an active participant in this configuration.
The adjustable plasma region 4, created in the embodiment shown in
The composition of the electrode 1 can also play an important role in the formation of the adjustable plasma 4. For example, a variety of known materials are suitable for use as the electrode(s) 1 of the embodiments disclosed herein. These materials include metals such as platinum, gold, silver, zinc, copper, titanium, and/or alloys or mixtures thereof, etc. However, the electrode(s) 1 (and 5) can be made of any suitable material which may comprise metal(s) (e.g., including appropriate oxides, carbides, nitrides, carbon, silicon and mixtures or composites thereof, etc.). Still further, alloys of various metals are also desirable for use with the present invention. Specifically, alloys can provide chemical constituents of different amounts, intensities and/or reactivities in the adjustable plasma 4 resulting in, for example, different properties in and/or around the plasma 4 and/or different constituents being present transiently, semi-permanently or permanently within the liquid 3. For example, different spectra can be emitted from the plasma 4 due to different constituents being excited within the plasma 4, different fields can be emitted from the plasma 4, etc. Thus, the plasma 4 can be involved in the formation of a variety of different nanoparticles and/or nanoparticle/solutions and/or desirable constituents, or intermediate(s) present in the liquid 3 required to achieve desirable end products. Still further, it is not only the chemical composition and shape factor(s) of the electrode(s) 1, 5 that play a role in the formation of the adjustable plasma 4, but also the manor in which any electrode(s) 1, 5 have been manufactured can also influence the performance of the electrode(s) 1, 5. In this regard, the precise shaping technique(s) including forging, drawing and/or casting technique(s) utilized to from the electrode(s) 1, 5 can have an influence on the chemical and/or physical activity of the electrode(s) 1, 5, including thermodynamic and/or kinetic and/or mechanical issues.
The creation of an adjustable plasma 4 in, for example, air above the surface 2 of a liquid 3 (e.g., water) will, typically, produce at least some gaseous species such as ozone, as well as certain amounts of a variety of nitrogen-based compounds and other components. Various exemplary materials can be produced in the adjustable plasma 4 and include a variety of materials that are dependent on a number of factors including the atmosphere between the electrode 1 and the surface 2 of the liquid 3. To assist in understanding the variety of species that are possibly present in the plasma 4 and/or in the liquid 3 (when the liquid comprises water), reference is made to a 15 Jun. 2000 thesis by Wilhelmus Frederik Laurens Maria Hoeben, entitled “Pulsed corona-induced degradation of organic materials in water”, the subject matter of which is expressly herein incorporated by reference. The work in the aforementioned thesis is directed primarily to the creation of corona-induced degradation of undesirable materials present in water, wherein such corona is referred to as a pulsed DC corona. However, many of the chemical species referenced therein, can also be present in the adjustable plasma 4 of the embodiments disclosed herein, especially when the atmosphere assisting in the creation of the adjustable plasma 4 comprises humid air and the liquid 3 comprises water. In this regard, many radicals, ions and meta-stable elements can be present in the adjustable plasma 4 due to the dissociation and/or ionization of any gas phase molecules or atoms present between the electrode 1 and the surface 2. When humidity in air is present and such humid air is at least a major component of the atmosphere “feeding” the adjustable plasma 4, then oxidizing species such as hydroxyl radicals, ozone, atomic oxygen, singlet oxygen and hydropereoxyl radicals can be formed. Still further, amounts of nitrogen oxides like NOx and N2O can also be formed. Accordingly, Table 1 lists some of the reactants that could be expected to be present in the adjustable plasma 4 when the liquid 3 comprises water and the atmosphere feeding or assisting in providing raw materials to the adjustable plasma 4 comprises humid air.
An April, 1995 article, entitled “Electrolysis Processes in D.C. Corona Discharges in Humid Air”, written by J. Lelievre, N. Dubreuil and J.-L. Brisset, and published in the J. Phys. III France 5 on pages 447-457 therein (the subject matter of which is herein expressly incorporated by reference) was primarily focused on DC corona discharges and noted that according to the polarity of the active electrode, anions such as nitrites and nitrates, carbonates and oxygen anions were the prominent ions at a negative discharge; while protons, oxygen and NOx cations were the major cationic species created in a positive discharge. Concentrations of nitrites and/or nitrates could vary with current intensity. The article also disclosed in Table I therein (i.e., Table 2 reproduced herein) a variety of species and standard electrode potentials which are capable of being present in the DC plasmas created therein. Accordingly, one would expect such species as being capable of being present in the adjustable plasma(s) 4 of the present invention depending on the specific operating conditions utilized to create the adjustable plasma(s) 4.
An article published 15 Oct. 2003, entitled, “Optical and electrical diagnostics of a non-equilibrium air plasma”, authored by XinPei Lu, Frank Leipold and Mounir Laroussi, and published in the Journal of Physics D: Applied Physics, on pages 2662-2666 therein (the subject matter of which is herein expressly incorporated by reference) focused on the application of AC (60 Hz) high voltage (<20 kV) to a pair of parallel electrodes separated by an air gap. One of the electrodes was a metal disc, while the other electrode was a surface of water. Spectroscopic measurements performed showed that light emission from the plasma was dominated by OH (A-X, N2 (C-B) and N2+ (B-X) transitions. The spectra from
An article by Z. Machala, et al., entitled, “Emission spectroscopy of atmospheric pressure plasmas for bio-medical and environmental applications”, published in 2007 in the Journal of Molecular Spectroscopy, discloses additional emission spectra of atmospheric pressure plasmas. The spectra from
An article by M. Laroussi and X. Lu, entitled, “Room-temperature atmospheric pressure plasma plume for biomedical applications”, published in 2005 in Applied Physics Letters, discloses emission spectra from OH, N2, N2+, He and O. The spectra from
Also known in the art is the generation of ozone by pulsed-corona discharge over a water surface as disclosed by Petr Lukes, et al, in the article, “Generation of ozone by pulsed corona discharge over water surface in hybrid gas-liquid electrical discharge reactor”, published in J. Phys. D: Appl. Phys. 38 (2005) 409-416 (the subject matter of which is herein expressly incorporated by reference). Lukes, et al, disclose the formation of ozone by pulse-positive corona discharge generated in a gas phase between a planar high voltage electrode (made from reticulated vitreous carbon) and a water surface, said water having an immersed ground stainless steel “point” mechanically-shaped electrode located within the water and being powered by a separate electrical source. Various desirable species are disclosed as being formed in the liquid, some of which species, depending on the specific operating conditions of the embodiments disclosed herein, could also be expected to be present.
Further, U.S. Pat. No. 6,749,759 issued on Jun. 15, 2004 to Denes, et al, and entitled Method for Disinfecting a Dense Fluid Medium in a Dense Medium Plasma Reactor (the subject matter of which is herein expressly incorporated by reference), discloses a method for disinfecting a dense fluid medium in a dense medium plasma reactor. Denes, et al, disclose decontamination and disinfection of potable water for a variety of purposes. Denes, et al, disclose various atmospheric pressure plasma environments, as well as gas phase discharges, pulsed high voltage discharges, etc. Denes, et al, use a first electrode comprising a first conductive material immersed within the dense fluid medium and a second electrode comprising a second conductive material, also immersed within the dense fluid medium. Denes, et al then apply an electric potential between the first and second electrodes to create a discharge zone between the electrodes to produce reactive species in the dense fluid medium.
All of the constituents discussed above, if present, can be at least partially (or substantially completely) managed, controlled, adjusted, maximized, minimized, eliminated, etc., as a function of such species being helpful or harmful to the resultant nanoparticles and/or nanoparticle/solutions produced, and then may need to be controlled by a variety of different techniques (discussed in more detail later herein). As shown in
Additionally, by controlling the temperature of the liquid 3 in contact with the adjustable plasma 4, the amount(s) of certain constituents present in the liquid 3 (e.g., for at least a portion of the process and/or in final products produced) can be maximized or minimized. For example, if a gaseous species such as ozone created in the adjustable plasma 4 was desired to be present in relatively larger quantities, the temperature of the liquid 3 could be reduced (e.g., by a chilling or refrigerating procedure) to permit the liquid 3 to contain more of the gaseous species. In contrast, if a relatively lesser amount of a particular gaseous species was desired to be present in the liquid 3, the temperature of the liquid 3 could be increased (e.g., by thermal heating, microwave heating, etc.) to contain less of the gaseous species. Similarly, often species in the adjustable plasma 4 being present in the liquid 3 could be adjusting/controlling the temperature of the liquid 3 to increase or decrease the amount of such species present in the liquid 3.
Further, certain processing enhancers may also be added to or mixed with the liquid(s). The processing enhancers include both solids and liquids. The processing enhancer may provide certain processing advantages and/or desirable final product characteristics. Examples of processing enhancers may include certain acids, certain bases, salts, nitrates, etc. Processing enhancers may assist in one or more of the electrochemical reactions disclosed herein; and/or may assist in achieving one or more desirable properties in products formed according to the teachings herein.
Further, depending on, for example, electric, magnetic and/or electromagnetic field strength, polarity, etc., in and around the liquid 3, as well as the volume of liquid 3 present (e.g., a function of, for example, the cross-sectional size and shape of the trough member 30 and/or flow rate of the liquid 3) discussed in greater detail elsewhere herein), the physical and chemical construction of the electrode(s) 1 and 5, atmosphere (naturally occurring or supplied), liquid 3 composition, greater or lesser amounts of electrode(s) materials(s) (e.g., metal(s) or derivatives of metals) may be found in the liquid 3. Additional important information is disclosed in copending patent application entitled Methods for Controlling Crystal Growth, Crystallization, Structures and Phases in Materials and Systems; which was filed on Mar. 21, 2003, and was published by the World Intellectual Property Organization under publication number WO 03/089692 on Oct. 30, 2003 and the U.S. National Phase application, which was filed on Jun. 6, 2005, and was published by the United States Patent and Trademark Office under publication number 20060037177 on Feb. 23, 2006 (the inventors of each being Bentley J. Blum, Juliana H. J. Brooks and Mark G. Mortenson). The subject matter of both applications is herein expressly incorporated by reference. These published applications disclose (among other things) that the influence of, for example, electric fields, magnetic fields, electromagnetic energy, etc., have proven to be very important in the formation and/or control of various structures in a variety of solids, liquids, gases and/or plasmas. Such disclosed effects are also relevant in the embodiments disclosed herein. Further, the observation of extreme variations of, for example, pH in and around electrodes having a potential applied thereto (and current flow therethrough) also controls reaction products and/or reaction rates. Thus, a complex set of reactions are likely to be occurring at each electrode 1, 5 and electrode assemblies or electrode sets (e.g., 1, 5; 1, 1; 5, 5; etc.).
In certain situations, the material(s) (e.g., metal(s), metal ion(s), metal composite(s) or constituents (e.g., Lewis acids, Bronsted-Lowry acids, etc.) and/or inorganics found in the liquid 3 (e.g., after processing thereof) may have very desirable effects, in which case relatively large amounts of such material(s) will be desirable; whereas in other cases, certain materials found in the liquid (e.g., undesirable by-products) may have undesirable effects, and thus minimal amounts of such material(s) may be desired in the final product. Further, the structure/composition of the liquid 3 per se may also be beneficially or negatively affected by the processing conditions of the present invention. Accordingly, electrode composition can play an important role in the ultimate material(s) (e.g., nanoparticles and/or nanoparticle/solutions) that are formed according to the embodiments disclosed herein. As discussed above herein, the atmosphere involved with the reactions occurring at the electrode(s) 1 (and 5) plays an important role. However, electrode composition also plays an important role in that the electrodes 1 and 5 themselves can become part of, at least partially, intermediate and/or final products formed. Alternatively, electrodes may have a substantial role in the final products. In other words, the composition of the electrodes may be found in large part in the final products of the invention or may comprise only a small chemical part of products produced according to the embodiments disclosed herein. In this regard, when electrode(s) 1, 5 are found to be somewhat reactive according to the process conditions of the various embodiments disclosed herein, it can be expected that ions and/or physical particles (e.g., metal-based particles of single or multiple crystals) from the electrodes can become part of a final product. Such ions and/or physical components may be present as a predominant part of a particle in a final product, may exist for only a portion of the process, or may be part of a core in a core-shell arrangement present in a final product. Further, the core-shell arrangement need not include complete shells. For example, partial shells and/or surface irregularities or specific desirable surface shapes on a formed nanoparticle can have large influence on the ultimate performance of such nanoparticles in their intended use.
Also, the nature and/or amount of the surface change (i.e., positive or negative) on formed nanoparticles can also have a large influence on the behavior and/or effects of the nanoparticle/solution of final products and their relative performance.
Such surface changes are commonly referred to as “zeta potential”. In general, the larger the zeta potential (either positive or negative), the greater the stability of the nanoparticles in the solution. However, by controlling the nature and/or amount of the surface changes of formed nanoparticles the performance of such nanoparticle solutions in a variety of systems can be controlled (discussed in greater detail later herein). It should be clear to an artisan of ordinary skill that slight adjustments of chemical composition, reactive atmospheres, power intensities, temperatures, etc., can cause a variety of different chemical compounds (both semi-permanent and transient) nanoparticles (and nanoparticle components) to be formed, as well as different nanoparticle/solutions (e.g., including modifying the structures of the liquid 3 (such as water) per se).
Still further, the electrode(s) 1 and 5 may be of similar chemical composition or completely different chemical compositions and/or made by similar or completely different forming processes in order to achieve various compositions of ions, compounds, and/or physical particles in liquid and/or structures of liquids per se and/or specific effects from final resultant products. For example, it may be desirable that electrode pairs, shown in the various embodiments herein, be of the same or substantially similar composition, or it may be desirable for the electrode pairs, shown in the various embodiments herein, to be of different chemical composition(s). Different chemical compositions may result in, of course, different constituents being present for possible reaction in the various plasma and/or electrochemical embodiments disclosed herein. Further, a single electrode 1 or 5 (or electrode pair) can be made of at least two different metals, such that components of each of the metals, under the process conditions of the disclosed embodiments, can interact with each other, as well as with other constituents in the plasma(s) 4 and or liquid(s) 3, fields, etc., present in, for example, the plasma 4 and/or the liquid 3.
Further, the distance between the electrode(s) 1 and 5; or 1 and 1 (e.g., see
The power applied through the power source 10 may be any suitable power which creates a desirable adjustable plasma 4 and desirable adjustable electrochemical reaction under all of the process conditions of the present invention. In one preferred mode of the invention, an alternating current from a step-up transformer (discussed in the “Power Sources” section and the “Examples” section) is utilized. In other preferred embodiments of the invention, polarity of an alternating current power source is modified by diode bridges to result in a positive electrode 1 and a negative electrode 5; as well as a positive electrode 5 and a negative electrode 1. In general, the combination of electrode(s) components 1 and 5, physical size and shape of the electrode(s) 1 and 5, electrode manufacturing process, mass of electrodes 1 and/or 5, the distance “x” between the tip 9 of electrode 1 above the surface 2 of the liquid 3, the composition of the gas between the electrode tip 9 and the surface 2, the flow rate and/or flow direction “F” of the liquid 3, compositions of the liquid 3, conductivity of the liquid 3, temperature of the liquid 3, voltage, amperage, polarity of the electrodes, etc., all contribute to the design, and thus power requirements (e.g., breakdown electric field or “Ec” of Equation 1) all influence the formation of a controlled or adjustable plasma 4 between the surface 2 of the liquid 3 and the electrode tip 9.
In further reference to the configurations shown in
For example,
The portions 6a and 6b can be covered by, for example, additional electrical insulating portions 7a and 7b. The electrical insulating portions 7a and 7b can be any suitable electrically insulating material (e.g., plastic, rubber, fibrous materials, etc.) which prevent undesirable currents, voltage, arcing, etc., that could occur when an individual interfaces with the electrode holders 6a and 6b (e.g., attempts to adjust the height of the electrodes). Moreover, rather than the electrical insulating portion 7a and 7b simply being a cover over the electrode holder 6a and 6b, such insulating portions 7a and 7b can be substantially completely made of an electrical insulating material. In this regard, a longitudinal interface may exist between the electrical insulating portions 7a/7b and the electrode holder 6a/6b respectively (e.g., the electrode holder 6a/6b may be made of a completely different material than the insulating portion 7a/7b and mechanically or chemically (e.g., adhesively) attached thereto.
Likewise, the insulating member 8 can be made of any suitable material which prevents undesirable electrical events (e.g., arcing, melting, etc.) from occurring, as well as any material which is structurally and environmentally suitable for practicing the present invention. Typical materials include structural plastics such as polycarbonate plexiglass (poly (methyl methacrylate), polystyrene, acrylics, and the like. Certain criteria for selecting structural plastics and the like include, but are not limited to, the ability to maintain shape and/or rigidity, while experiencing the electrical, temperature and environmental conditions of the process. Preferred materials include acrylics, plexiglass, and other polymer materials of known chemical, electrical and electrical resistance as well as relatively high mechanical stiffness. In this regard, desirable thicknesses for the member 8 are on the order of about 1/16″-¾″ (1.6 mm-19.1 mm).
The power source 10 can be connected in any convenient electrical manner to the electrodes 1 and 5. For example, wires 11a and 11b can be located within at least a portion of the electrode holders 6a, 6b with a primary goal being achieving electrical connections between the portions 11a, 11b and thus the electrodes 1, 5. Specific details of preferred electrical connections are discussed elsewhere herein.
With regard to the adjustable plasmas 4 shown in
Still further, with regard to
Likewise, a set of manually controllable electrode configurations are shown in
Moreover, it should be understood that in alternative preferred embodiments of the invention, well defined sharp points for the tip 9 are not always required. In this regard, the electrode 1 shown in
Accordingly, it should be understood that a variety of sizes and shapes corresponding to electrode 1 can be utilized in accordance with the teachings of the present invention. Still further, it should be noted that the tips 9 of the electrodes 1 shown in various figures herein may be shown as a relatively sharp point or a relatively blunt end. Unless specific aspects of these electrode tips are discussed in greater contextual detail, the actual shape of the electrode tip(s) shown in the FIGs. should not be given great significance.
Still further, it should be understood that a trough member need not be only linear or “I-shaped”, but rather, may be shaped like a “Y” or like a “Ψ”, each portion of which may have similar or dissimilar cross-sections. One reason for a “Y” or “Ψ”-shaped trough member 30 is that two different sets of processing conditions can exist in the two upper portions of the “Y”-shaped trough member 30. For example, one or more constituents produced in the portion(s) 30a, 30b and/or 30c could be transient and/or semi permanent. If such constituent(s) produced, for example, in portion 30a is to be desirably and controllably reacted with one or more constituents produced in, for example, portion 30b, then a final product (e.g., properties of a final product) which results from such mixing could be a function of when constituents formed in the portions 30a and 30b are mixed together. For example, final properties of products made under similar sets of conditions experienced in, for example, the portions 30a and 30b, if combined in, for example, the section 30d (or 30d′), could be different from final properties of products made in the portions 30a and 30b and such products are not combined together until minutes or hours or days later. Also, the temperature of liquids entering the section 30d (or 30d′) can be monitored/controlled to maximize certain desirable properties of final products and/or minimize certain undesirable products. Further, a third set of processing conditions can exist in the bottom portion of the “Y”-shaped trough member 30. Thus, two different fluids 3, of different compositions and/or different reactants, could be brought together into the bottom portion of the “Y”-shaped trough member 30 and processed together to from a large variety of final products some of which are not achievable by separately manufacturing certain solutions and later mixing such solutions together. Still further, processing enhancers may be selectively utilized in one or more of the portions 30a, 30b, 30c, 30d and/or 30o (or at any point in the trough member 30).
It should be understood that a variety of different shapes can exist for the trough member 30, any one of which can produce desirable results.
Again with regard to
Likewise,
Accordingly, it should be clear from the disclosed embodiments that the various electrode configurations or sets shown in
Likewise,
As discussed herein, the electrode configurations or sets shown generally in
Likewise, several additional alternative cross-sectional embodiments for the liquid-containing trough member 30 are shown in
Similarly, the influence of many aspects of the electrode 5 on the liquid 3 (e.g., electrochemical interactions) is also, at least partially, a function of the amount of fluid juxtaposed to the electrode(s) 5, the temperature of the fluid 3, etc., as discussed immediately above herein.
Further, electric and magnetic field concentrations can also significantly affect the interaction of the plasma 4 with the liquid 3, as well as affect the interactions of the electrode(s) 5 with the liquid 3. For example, without wishing to be bound by any particular theory or explanation, when the liquid 3 comprises water, a variety of electric field, magnetic field and/or electromagnetic field influences can occur. Specifically, water is a known dipolar molecule which can be at least partially aligned by an electric field. Having partial alignment of water molecules with an electric field can, for example, cause previously existing hydrogen bonding and bonding angles to be oriented at an angle different than prior to electric field exposure, cause different vibrational activity, or such bonds may actually be broken. Such changing in water structure can result in the water having a different (e.g., higher) reactivity. Further, the presence of electric and magnetic fields can have opposite effects on ordering or structuring of water and/or nanoparticles present in the water. It is possible that unstructured or small structured water having relatively fewer hydrogen bonds relative to, for example, very structured water, can result in a more reactive (e.g., chemically more reactive) environment. This is in contrast to open or higher hydrogen-bonded networks which can slow reactions due to, for example, increased viscosity, reduced diffusivities and a smaller activity of water molecules. Accordingly, factors which apparently reduce hydrogen bonding and hydrogen bond strength (e.g, electric fields) and/or increase vibrational activity, can encourage reactivity and kinetics of various reactions.
Further, electromagnetic radiation can also have direct and indirect effects on water and it is possible that the electromagnetic radiation per se (e.g., that radiation emitted from the plasma 4), rather than the individual electric or magnetic fields alone can have such effects, as disclosed in the aforementioned published patent application entitled Methods for Controlling Crystal Growth, Crystallization, Structures and Phases in Materials and Systems which has been incorporated by reference herein. Different spectra associated with different plasmas 4 are discussed in the “Examples” section herein.
Further, by passing an electric current through the electrode(s) 1 and/or 5 disclosed herein, the voltages present on, for example, the electrode(s) 5 can have an orientation effect (i.e., temporary, semi-permanent or longer) on the water molecules. The presence of other constituents (i.e., charged species) in the water may enhance such orientation effects. Such orientation effects may cause, for example, hydrogen bond breakage and localized density changes (i.e., decreases). Further, electric fields are also known to lower the dielectric constant of water due to the changing (e.g., reduction of) the hydrogen bonding network. Such changing of networks should change the solubility properties of water and may assist in the concentration or dissolution of a variety of gases and/or constituents or reactive species in the liquid 3 (e.g., water) within the trough member 30. Still further, it is possible that the changing or breaking of hydrogen bonds from application of electromagnetic radiation (and/or electric and magnetic fields) can perturb gas/liquid interfaces and result in more reactive species. Still further, changes in hydrogen bonding can affect carbon dioxide hydration resulting in, among other things, pH changes. Thus, when localized pH changes occur around, for example, at least one or more of the electrode(s) 5 (or electrode(s) 1), many of the possible reactants (discussed elsewhere herein) will react differently with themselves and/or the atmosphere and/or the adjustable plasma(s) 4 as well as the electrode(s) 1 and/or 5, per se. The presence of Lewis acids and/or Bronsted-Lowry acids, can also greatly influence reactions.
Further, a trough member 30 may comprise more than one cross-sectional shapes along its entire longitudinal length. The incorporation of multiple cross-sectional shapes along the longitudinal length of a trough member 30 can result in, for example, a varying field or concentration or reaction effects being produced by the inventive embodiments disclosed herein. Additionally, various modifications can be added at points along the longitudinal length of the trough member 30 which can enhance and/or diminish various of the field effects discussed above herein. In this regard, compositions of materials in and/or around the trough (e.g., metals located outside or within at least a portion of the trough member 30) can act as concentrators or enhancers of various of the fields present in and around the electrode(s) 1 and/or 5. Additionally, applications of externally-applied fields (e.g., electric, magnetic, electromagnetic, etc.) and/or the placement of certain reactive materials within the trough member 30 (e.g., at least partially contacting a portion of the liquid 3 flowing thereby) can also result in: (1) a gathering, collecting or filtering of undesirable species; or (2) placement of desirable species onto, for example, at least a portion of an outer surface of nanoparticles already formed upstream therefrom. Further, it should be understood that a trough member 30 may not be linear or “I-shaped”, but rather may be “Y-shaped” or “Ψ-shaped”, with each portion of the “Y” or “Ψ” having a different (or similar) cross-section. One reason for a “Y” or “Ψ-shaped” trough member 30 is that two (or more) different sets of processing conditions can exist in the two (or more) upper portions of the “Y-shaped” or “Ψ-shaped” trough member 30. Additionally, the “Y-shaped” or “Ψ-shaped” trough members 30 permit certain transient or semi-permanent constituents present in the liquids 3 to interact; in contrast to separately manufactured liquids 3 in “I-shaped” trough members and mixing such liquids 3 together at a point in time which is minutes, hours or days after the formation of the liquids 3. Further, another additional set of processing conditions can exist in the bottom portion of the “Y-shaped” or “Ψ-shaped” trough members 30. Thus, different fluids 3, of different compositions and/or different reactants (e.g., containing certain transient or semi-permanent species), could be brought together into the bottom portion of the “Y-shaped” or “Ψ-shaped” trough members 30 and processed together to from a large variety of final products.
It should be understood that a variety of different shapes can exist for the trough member 30, any one of which can produce desirable results.
In general, the liquid transport means 40 may include any means for moving liquids 3 including, but not limited to a gravity-fed or hydrostatic means, a pumping means, a peristaltic pumping means, a regulating or valve means, etc. However, the liquid transport means 40 should be capable of reliably and/or controllably introducing known amounts of the liquid 3 into the trough member 30. Once the liquid 3 is provided into the trough member 30, means for continually moving the liquid 3 within the trough member 30 may or may not be required. However, a simple means includes the trough member 30 being situated on a slight angle θ (e.g., less than one degree to a few degrees) relative to the support surface upon which the trough member 30 is located. For example, the difference in vertical height between an inlet portion 31 and an outlet portion 32 relative to the support surface may be all that is required, so long as the viscosity of the liquid 3 is not too high (e.g., any viscosity around the viscosity of water can be controlled by gravity flow once such fluids are contained or located within the trough member 30). In this regard,
Further, when viscosities of the liquid 3 increase such that gravity alone is insufficient, other phenomena such as specific uses of hydrostatic head pressure or hydrostatic pressure can also be utilized to achieve desirable fluid flow. Further, additional means for moving the liquid 3 along the trough member 30 could also be provided inside the trough member 30, Such means for moving the liquid 3 include mechanical means such as paddles, fans, propellers, augers, etc., acoustic means such as transducers, thermal means such as heaters and or chillers (which may have additional processing benefits), etc. The additional means for moving the liquid 3 can cause liquid 3 to flow in differing amounts in different portions along the longitudinal length of the trough member 30. In this regard, for example, if liquid 3 initially flowed slowly through a first longitudinal portion of the trough member 30, the liquid 3 could be made to flow more quickly further downstream thereof by, for example, as discussed earlier herein, changing the cross-sectional shape of the trough member 30. Additionally, cross-sectional shapes of the trough member 30 could also contain therein additional fluid handling means which could speed up or slow down the rate the liquid 3 flows through the trough member 30. Accordingly, great flexibility can be achieved by the addition of such means for moving the fluid 3.
In particular,
In contrast,
As disclosed herein, each of the electrode configurations shown in
Possible ion exchange membranes 50 which function as a means for separating for use with the present invention include Anionic membranes and Cationic membranes. These membranes can be homogenous, heterogeneous or microporous, symmetric or asymmetric in structure, solid or liquid, can carry a positive or negative charge or be neutral or bipolar. Membrane thickness may vary from as small as 100 micron to several mm.
Some specific ionic membranes for use with certain embodiments of the present invention include, but are not limited to:
Electrode Control Devices
The electrode control devices shown generally in, for example,
First, specific reference is made to
The drive motors 21a/21b can be any suitable drive motor which is capable of small rotations (e.g., slightly below 1°/360° or slightly above 1°/360°) such that small rotational changes in the drive shaft 231a are translated into small vertical changes in the electrode assemblies. A preferred drive motor includes a drive motor manufactured by RMS Technologies model 1MC17-S04 step motor, which is a DC-powered step motor. This step motors 21a/21b include an RS-232 connection 22a/22b, respectively, which permits the step motors to be driven by a remote control apparatus such as a computer or a controller.
With reference to
The electrode assembly specifically shown in
With regard to the size of the control device 20 shown in
Further, in each of the embodiments of the invention shown in
A fan assembly, not shown in the drawings, can be attached to a surrounding housing which permits cooling air to blow across the cooling fins 282. The fan assembly could comprise a fan similar to a computer cooling fan, or the like. A preferred fan assembly comprises, for example, a Dynatron DF124020BA, DC brushless, 9000 RPM, ball bearing fan measuring about 40 mm×40 mm×20 mm works well. Specifically, this fan has an air flow of approximately 10 cubic feet per minute.
Power Sources
A variety of power sources are suitable for use with the present invention. Power sources such as AC sources of a variety of frequencies, DC sources of a variety of frequencies, rectified AC sources of various polarities, etc., can be used. However, in the preferred embodiments disclosed herein, an AC power source is utilized directly, or an AC power source has been rectified to create a specific DC source of variable polarity.
When a secondary coil 603 is positioned near the primary coil 601 and core 602, this flux will link the secondary coil 603 with the primary coil 601. This linking of the secondary coil 603 induces a voltage across the secondary terminals. The magnitude of the voltage at the secondary terminals is related directly to the ratio of the secondary coil turns to the primary coil turns. More turns on the secondary coil 603 than the primary coil 601 results in a step up in voltage, while fewer turns results in a step down in voltage.
Preferred transformer(s) 60 for use in various embodiments disclosed herein have deliberately poor output voltage regulation made possible by the use of magnetic shunts in the transformer 60. These transformers 60 are known as neon sign transformers. This configuration limits current flow into the electrode(s) 1/5. With a large change in output load voltage, the transformer 60 maintains output load current within a relatively narrow range.
The transformer 60 is rated for its secondary open circuit voltage and secondary short circuit current. Open circuit voltage (OCV) appears at the output terminals of the transformer 60 only when no electrical connection is present. Likewise, short circuit current is only drawn from the output terminals if a short is placed across those terminals (in which case the output voltage equals zero). However, when a load is connected across these same terminals, the output voltage of the transformer 60 should fall somewhere between zero and the rated OCV. In fact, if the transformer 60 is loaded properly, that voltage will be about half the rated OCV.
The transformer 60 is known as a Balanced Mid-Point Referenced Design (e.g., also formerly known as balanced midpoint grounded). This is most commonly found in mid to higher voltage rated transformers and most 60 mA transformers. This is the only type transformer acceptable in a “mid-point return wired” system. The “balanced” transformer 60 has one primary coil 601 with two secondary coils 603, one on each side of the primary coil 601 (as shown generally in the schematic view in
In alternating current (AC) circuits possessing a line power factor or 1 (or 100%), the voltage and current each start at zero, rise to a crest, fall to zero, go to a negative crest and back up to zero. This completes one cycle of a typical sinewave. This happens 60 times per second in a typical US application. Thus, such a voltage or current has a characteristic “frequency” of 60 cycles per second (or 60 Hertz) power. Power factor relates to the position of the voltage waveform relative to the current waveform. When both waveforms pass through zero together and their crests are together, they are in phase and the power factor is 1, or 100%.
The normal power factor of most such transformers 60 is largely due to the effect of the magnetic shunts 604 and the secondary coil 603, which effectively add an inductor into the output of the transformer's 60 circuit to limit current to the electrodes 1/5. The power factor can be increased to a higher power factor by the use of capacitor(s) 61 placed across the primary coil 601 of the transformer, 60 which brings the input voltage and current waves more into phase.
The unloaded voltage of any transformer 60 to be used in the present invention is important, as well as the internal structure thereof. Desirable unloaded transformers for use in the present invention include those that are around 9,000 volts, 10,000 volts, 12,000 volts and 15,000 volts. However, these particular unloaded volt transformer measurements should not be viewed as limiting the scope acceptable power sources as additional embodiments. A specific desirable transformer for use with various embodiments of the invention disclosed herein is made by Franceformer, Catalog No. 9060-P-E which operates at: primarily 120 volts, 60 Hz; and secondary 9,000 volts, 60 mA.
Accordingly, each transformer assembly 60a-60h (and/or 60a′-60h′; and/or 60a″-60h″) can be the same transformer, or can be a combination of different transformers (as well as different polarities). The choice of transformer, power factor, capacitor(s) 61, polarity, electrode designs, electrode location, electrode composition, cross-sectional shape(s) of the trough member 30, local or global electrode composition, atmosphere(s), local or global liquid 3 flow rate(s), liquid 3 local components, volume of liquid 3 locally subjected to various fields in the trough member 30, neighboring (e.g., both upstream and downstream) electrode sets, local field concentrations, the use and/or position and/or composition of any membrane 50, etc., are all factors which influence processing conditions as well as composition and/or volume of constituents produced in the liquid 3, nanoparticles and nanoparticle/solutions made according to the various embodiments disclosed herein. Accordingly, a plethora of embodiments can be practiced according to the detailed disclosure presented herein.
Electrode Height Control/Automatic Control Device
A preferred embodiment of the invention utilizes the automatic control devices 20 shown in various figures herein. The step motors 21a and 21b shown in, for example,
Each set of electrodes in each embodiment of the invention has an established target voltage range. The size or magnitude of acceptable range varies by an amount between about 1% and about 10%-15% of the target voltage. Some embodiments of the invention are more sensitive to voltage changes and these embodiments should have, typically, smaller acceptable voltage ranges; whereas other embodiments of the invention are less sensitive to voltage and should have, typically, larger acceptable ranges. Accordingly, by utilizing the circuit diagram shown in
The computer or logic control for the disclosed interrogation voltage adjustment techniques are achieved by any conventional program or controller, including, for example, in a preferred embodiment, standard visual basic programming steps utilized in a PC. Such programming steps include interrogating, reading, comparing, and sending an appropriate actuation symbol to increase or decrease voltage (e.g., raise or lower an electrode relative to the surface 2 of the liquid 3). Such techniques should be understood by an artisan of ordinary skill.
The following examples serve to illustrate certain embodiments of the invention but should not to be construed as limiting the scope of the disclosure.
In general, each of the 12 Examples utilize certain embodiments of the invention associated with the apparatuses generally shown in
Purified water (discussed later herein) was used as the liquid 3 in all of Examples 1-12. The depth “d” (refer to
The rate of flow of the water 3 in the trough member 30 was about 150-200 ml/minute, depending on which Example was being practiced. Specifically, for example, silver-based and copper-based nanoparticle/solution raw materials made in Examples 1-3 and 5 all utilized a flow rate of about 200 ml/minute; and a zinc-based nanoparticle/solution raw material made in Example 4 utilized a flow rate of about 150 ml/minute. Such flow of water 3 was obtained by utilizing a Masterflex® L/S pump drive 40 rated at 0.1 horsepower, 10-600 rpm. The model number of the Masterflex® pump 40 was 77300-40. The pump drive had a pump head also made by Masterflex® known as Easy-Load Model No. 7518-10. In general terms, the head for the pump 40 is known as a peristaltic head. The pump 40 and head were controlled by a Masterflex® LS Digital Modular Drive. The model number for the Digital Modular Drive is 77300-80. The precise settings on the Digital Modular Drive were, for example, 150 milliliters per minute for Example 4 and 200 ml/minute for the other Examples 1-3 and 5. Tygon® tubing having a diameter of ¼″ (i.e., size 06419-25) was placed into the peristaltic head. The tubing was made by Saint Gobain for Masterflex®. One end of the tubing was delivered to a first end 31 of the trough member 30 by a flow diffusion means located therein. The flow diffusion means tended to minimize disturbance and bubbles in water 3 introduced into the trough member 30 as well as any pulsing condition generated by the peristaltic pump 40. In this regard, a small reservoir served as the diffusion means and was provided at a point vertically above the end 31 of the trough member 30 such that when the reservoir overflowed, a relatively steady flow of water 3 into the end 31 of the V-shaped trough member 30 occurred.
Additionally, the plastic portions of the control devices 20 were also made from plexiglass having a thickness of about ⅛″ (about 3 mm). With reference to
With regard to
The size and shape of each electrode 1 utilized was about the same. The shape of each electrode 1 was that of a right triangle with measurements of about 14 mm×23 mm×27 mm. The thickness of each electrode 1 was about 1 mm. Each triangular-shaped electrode 1 also had a hole therethrough at a base portion thereof, which permitted the point formed by the 23 mm and 27 mm sides to point toward the surface 2 of the water 3. The material comprising each electrode 1 was 99.95% pure (i.e., 3N5) unless otherwise stated herein. When silver was used for each electrode 1, the weight of each electrode was about 2 grams. When zinc was used for each electrode 1, the weight of each electrode was about 1.1 grams. When copper was used for each electrode 1, the weight of each electrode was about 1.5 grams.
The wires used to attach the triangular-shaped electrode 1 to the transformer 60 were, for Examples 1-4, 99.95% (3N5) silver wire, having a diameter of about 1.016 mm. The wire used to attach the triangular shaped electrode 1 in Example 5 was 99.95% pure (3N5) copper wire, also having a diameter of about 1.016 mm. Accordingly, a small loop of wire was placed through the hole in each electrode 1 to electrically connect thereto.
The wires used for each electrode 5 comprised 99.95% pure (3N5) each having a diameter of about 1.016 mm. The composition of the electrodes 5 in Examples 1-3 was silver; in Example 4 was zinc and in Example 5 was copper. All materials for the electrodes 1/5 were obtained from ESPI having an address of 1050 Benson Way, Ashland, Oreg. 97520.
The water 3 used in Examples 1-12 as an input into the trough member 30 was produced by a Reverse Osmosis process and deionization process. In essence, Reverse Osmosis (RO) is a pressure driven membrane separation process that separates species that are dissolved and/or suspended substances from the ground water. It is called “reverse” osmosis because pressure is applied to reverse the natural flow of osmosis (which seeks to balance the concentration of materials on both sides of the membrane). The applied pressure forces the water through the membrane leaving the contaminants on one side of the membrane and the purified water on the other. The reverse osmosis membrane utilized several thin layers or sheets of film that are bonded together and rolled in a spiral configuration around a plastic tube. (This is also known as a thin film composite or TFC membrane.) In addition to the removal of dissolved species, the RO membrane also separates out suspended materials including microorganisms that may be present in the water. After RO processing a mixed bed deionization filter was used. The total dissolved solvents (“TDS”) after both treatments was about 0.2 ppm, as measured by an Accumet® AR20 pH/conductivity meter.
This Example utilizes 99.95% pure silver electrodes 1 and 5. Table 3 summarizes portions of electrode design, location and operating voltages. As can be seen from Table 3, the target voltages were set to a low of about 550 volts and to a high of about 2,100 volts.
Further, bar charts of the actual and target voltages for each electrode in each of the 8 electrode sets, Set #1-Set #8, are shown in
Table 4 contains information similar to that data shown in Table 3 relating to electrode set design, voltages, distances, etc. It is clear from Table 4 that the electrode configurations Set #1 and Set #2 were the same as of Set #'s 1-8 in Table 3 and Example 1. Further electrode Sets 3-8 are all configured in the same manner and corresponded to a different electrode configuration from Set #1 and Set #2 herein, which electrode configuration corresponds to that configuration shown in
5c′
5d′
5e′
5f′
5g′
5h′
The product produced according to Example 2 is referred to herein as “AT060”.
Table 5 herein sets forth electrode design and target voltages for each of the 16 electrodes in each of the eight electrode sets (i.e., Set #1-Set #8) utilized to form the product formed in this example referred to herein as “AT031”.
5b′
5c′
5e′
5f′
5g′
5h′
It should be noted that electrode Set #1 was the same in this Example 3 as in each of Examples 1 and 2 (i.e., an electrode configuration of 1/5). Another 1/5 configuration was utilized for each of the other electrode sets, namely Set #2 and Set #'s 5-8 were all configured in a manner according to a 5/5 configuration.
Material designated herein as “BT006” was manufactured in accordance with the disclosure of Example 4. Similar to Examples 1-3, Table 6 herein discloses the precise electrode combinations in each of the 8 electrode sets (i.e, Set #1-Set #8). Likewise, target and actual voltage, distances, etc., are also reported. It should be noted that the electrode set assembly of Example 4 is similar to the electrode set assembly used in Example 1, except that 99.95% pure zinc was used only for the electrodes 5. The triangular-shaped portion of the electrodes 1 also comprised the same purity zinc, however the electrical connections to the triangular-shaped electrodes were all 99.95% pure silver-wire, discussed above herein. Also, the flow rate of the reaction 3 was lower in this Example then in all the other Examples.
A copper-based nanoparticle solution designated as “CT006” was made according to the procedures disclosed in Example 5. In this regard, Table 7 sets forth pertinent operating parameters associated with each of the 16 electrodes in the 8 electrode sets.
Further,
Each of the silver-based nanoparticles and nanoparticle/solutions made in Examples 1-3 (AT-059/AT-038), (AT060/AT036) and (AT031), respectively; as well as the zinc nanoparticles and nanoparticle/solutions made in Example 4 (BT-004); and the copper nanoparticles and nanoparticle-based/solutions made in Example 5 (CT-006) were physically characterized by a variety of techniques. Specifically, Tables 8 and 9 herein show each of the 5 “raw materials” made according to Examples 1-5 as well as 10 solutions or mixtures made therefrom, each of the solutions being designated “GR1-GR10” or GR1B-GR10B”. The amount by volume of each of the “raw materials” is reported for each of the 10 solutions manufactured. Further, atomic absorption spectroscopy (“AAS”) was performed on each of the raw materials of Examples 1-5 as well as on each of the 10 solutions GR1-GR10 derived therefrom. The amount of silver constituents, zinc constituents and/or copper constituents therein were thus determined. The atomic absorption spectroscopy results (AAS) are reported by metallic-based constituent.
The AAS values were obtained from a Perkin Elmer AAnalyst 300 Spectrometer system. The samples from Examples 1-5 and Solutions GR1-GR10 were prepared by adding a small amount of nitric acid or hydrochloric acid (usually 2% of final volume) and then dilution to a desirable characteristic concentration range or linear range of the specific element to improve accuracy of the result. The “desireable” range is an order of magnitude estimate based on production parameters established during product development. For pure metals analysis, a known amount of feedstock material is digested in a known amount of acid and diluted to ensure that the signal strength of the absorbance will be within the tolerance limits and more specifically the most accurate range of the detector settings, better known as the linear range.
The specific operating procedure for the Perkin Elmer AAnalyst 300 system is as follows:
I) Principle
Further, the last 4 columns of Table 8 disclose “Metal PPM (Ionic)”; and O2 (ppm); NO3 (ppm); and “pH”. Each of these sets of numbers were determined by utilizing an ion selective electrode measurement technique. In particular, a NICO ion analyzer was utilized. Precise stabilization times and actual experimental procedures for collecting the data in each of these three columns of Table 8 (and Table 9) occurs immediately below.
Stabilization Times—After immersing the electrodes in a new solution, the mV reading normally falls rapidly at first by several mV, and then gradually, and increasingly slowly, falls to a stable reading as the ISE membrane equilibrates and the reference electrode liquid junction potential stabilizes. This equilibration may take up to 3 or 4 minutes to reach a completely stable value. Sometimes the reading begins to rise again after a short period of stability and it is important to ensure that the recording is made at the lowest point, before this rise has proceeded to any great extent. In this study it was found that it was not necessary to wait for a completely stable reading but that satisfactory results could be obtained by taking a reading after a pre-set time, so that each measurement was made at the same point of the decay curve. For optimum performance it was found that this delay time should be at least two minutes to ensure that the reading was in the shallower part of the curve.
Procedure:
Table 9 is also included herein which contains similar data to that data shown in Table 8 (and discussed in Examples 1-5) with the only exception being AT-031. The data in Table 9 comes from procedures copied from Examples 1-5 except that such procedures were conducted at a much later point in time (months apart). The raw materials and associated solutions, summarized in Table 9 show that the raw materials, as well as solutions therefrom, are substantially constant. Accordingly, the process is very reliable and reproducible.
Scanning Electron Microscopy/EDS
Scanning electron microscopy was performed on each of the new materials and solutions GR1-GR10 made according to Examples 1-5.
XEDS spectra were obtained using a EDAX Lithium drifted silicon detector system coupled to a IXRF Systems digital processor, which was interfaced with an AMRAY 1820 SEM with a LaB6 electron gun. Interpretation of all spectra generated was performed using IXRF EDS2008, version 1.0 Rev E data collection and processing software.
Instrumentation hardware and software setup entails positioning liquid samples from each Run ID on a sample stage in such a manner within the SEM to permit the area of interest to be under the electron beam for imaging purposes while allowing emitted energies to have optimum path to the XEDS detector. A sample is typically positioned about 18 mm beneath the aperture for the final lens and tilted nominally at 18° towards the XEDS detector. All work is accomplished within a vacuum chamber, maintained at about 10−6 torr.
The final lens aperture is adjusted to 200 to 300 μm in diameter and the beam spot size is adjusted to achieve an adequate x-ray photon count rate for the digital “pulse” processor. Data collection periods range between 200 and 300 seconds, with “dead-times” of less than 15%.
An aliquot of liquid sample solution is placed onto a AuPd sputtered glass slide followed by a dehydration step which includes freeze drying the solution or drying the solution under a dry nitrogen gas flow to yield particulates from the suspension. Due to the nature of the particulates, no secondary coating is required for either imaging or XEDS analysis.
Transmission Electron Microscopy
Transmission Electron Microscopy was performed on raw materials corresponding to the components used to manufacture GR5 and GR8, as well as the solutions GR5 and GR8. Specifically, an additional run was performed corresponding to those production parameters associated with manufacturing AT031 (i.e, the silver constituent in GR5); an additional run was performed corresponding to those production parameters associated with manufacturing AT060 (i.e., the silver constituent in GR8); and an additional run was performed corresponding to those production parameters associated with manufacturing BT006 (i.e., the zinc constituent used in both GR5 and GR8). The components were then mixed together in a similar manner as discussed above herein to result in solutions equivalent to previously manufactured GR5 and GR8.
The samples for each of the TEM photomicrographs were prepared at room temperature. Specifically, 4 microliters of each liquid sample were placed onto a holey carbon film which was located on top of filter paper (used to wick off excess liquid). The filter paper was moved to a dry spot and this procedure was repeated resulting in 8 total microliters of each liquid sample being contacted with one portion of the holey carbon film. The carbon film grids were then mounted in a single tilt holder and placed in the loadlock of the JEOL 2100 CryoTEM to pump for about 15 minutes. The sample was then introduced into the column and the TEM microscopy work performed.
The JEOL 2100 CryoTEM operated at 200 kv accelerating potential. Images were recorded on a Gatan digital camera of ultra high sensitivity. Typical conditions were 50 micron condenser aperture, spot size 2, and alpha 3.
These TEM photomicrographs show clearly that the average particle size of those particles in
TEM photomicrographs 43r do not show any significant crystallization of zinc.
TEM photomicrographs 43s (corresponding to solution GR5) also show similar silver features as shown in
Thus, these TEM photomicrographs suggest that the processing parameters utilized to manufacture GR5 resulted in somewhat smaller silver-based nanoparticles, when compared to those silver-based nanoparticles associated with GR8. The primary difference in production parameters between GR5 and GR8 was the location of the two adjustable plasmas 4 used to make the silver constituents in each solution. The zinc constituents in both of GR5 and GR8 are the same. However, the silver constituents in GR5 is made by adjustable plasmas 4 located at the First Electrode Set and the Fourth Electrode Set; whereas the silver constituent in GR8 is made by adjustable plasmas 4 located at the First and Second Electrode Sets.
UV-VIS Spectroscopy
Energy absorption spectra were obtained using US-VIS micro-spec-photometry. This information was acquired using dual beam scanning monochrometer systems capable of scanning the wavelength range of 190 nm to 1100 nm. Two UV-Vis spectrometers were used to collect absorption spectra; these were a Jasco V530 and a Jasco MSV350. Instrumentation was setup to support measurement of low-concentration liquid samples using one of a number of fuzed-quartz sample holders or “cuvettes”. The various cuvettes allow data to be collected at 10 mm, 1 mm or 0.1 mm optic path of sample. Data was acquired over the above wavelength range using both PMT and LED detectors with the following parameters; bandwidth of 2 nm, with data pitch of 0.5 nm, with and without a water baseline background. Both tungsten “halogen” and Hydrogen “D2” energy sources were used as the primary energy sources. Optical paths of these spectrometers were setup to allow the energy beam to pass through the samples with focus towards the center of the sample cuvettes. Sample preparation was limited to filling and capping the cuvettes and then physically placing the samples into the cuvette holder, within the fully enclosed sample compartment. Optical absorption of energy by the materials of interest was determined. Data output was measured and displayed as Absorbance Units (per Beer-Lambert's Law) versus wavelength and frequency.
Spectral signatures in a UV-Visible range were obtained for each of the raw materials produced in Examples 1-5 as well as in each of the solutions GR1-GR10 shown in Tables 8 and 9.
Specifically,
The UV-Vis spectral data for each of
In general, UV-Vis spectroscopy is the measurement of the wavelength and intensity of absorption of near-ultraviolet and visible light by a sample. Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels. UV-Vis spectroscopy can be applied to molecules and inorganic ions or complexes in solution.
The UV-Vis spectra have broad features that can be used for sample identification but are also useful for quantitative measurements. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law.
The dual beam UV-Vis spectrophotometer was used to subtract any signals from the solvent (in this case water) in order to specifically characterize the samples of interest. In this case the reference is the feedstock water that has been drawn from the outlet of the Reverse Osmosis process discussed in the Examples section herein.
Raman Spectroscopy
Raman spectral signatures were obtained using a Renishaw Invia Spectrometer with relevant operating information shown in
The reflection micro-spectrograph with Leica DL DM microscope was fitted with either a 20× (NA=0.5) water immersion or a 5× (NA=0.12) dry lens. The rear aperture of each lens was sized to equal or exceed the expanded laser beam diameter. Two laser frequencies were used, these being a multiline 50 mW Argon laser at ½ power setup for 514.5 nm and a 20 mW HeNe laser at 633 nm. High resolution gratings were fitted in the monochrometer optic path which allowed continuous scans from 50 to 4000 wavenumbers (1/cm). Ten to 20 second integration times were used. Sample fluid was placed below the lens in a 50 ml beaker. Both lasers were used to investigate resonance bands, while the former laser was primarily used to obtain Raman spectra. Sample size was about 25 ml. Measurements made with the 5× dry lens were made with the objective positioned about 5 mm above the fluid to interrogate a volume about 7 mm beneath the water meniscus. Immersion measurements were made with the 20× immersion lens positioned about 4 mm into the sample allowing investigation of the same spatial volume. CCD detector acquisition areas were individually adjusted for each lens to maximize signal intensity and signal-to-noise ratios.
Biological Characterization
A Bioscreen C microbiology reader was utilized to compare the effectiveness of the raw materials made in accordance with Examples 1-5, as well as the 10 solutions GR1-GR10 made therefrom. Specific procedure for obtaining Bioscreen results follows below.
Escherichia coli was obtained from the American Type Culture Collection (ATCC) under the accession number 25922. The initial pellets were reconstituted in trypticase soy broth (TSB, Becton Dickinson and Company, Sparks, Md.) and aseptically transferred to a culture flask containing 10 ml of TSB followed by overnight incubation at 37° C. in a Forma 3157 water-jacketed incubator (Thermo Scientific, Waltham, Mass., USA).
Bacterial strains were kept on trypticase soy agar (TSA, Becton Dickinson and Company, Sparks, Md.) plates and aliquots were cryogenically stored at −80° C. in MicroBank tubes (Pro-Lab Incorporated, Ontario, Canada).
Microbank tubes were thawed at room temperature and opened in a NuAire Labgard 440 biological class II safety cabinet (NuAire Inc., Plymouth, Minn., USA). Using a sterile inoculating needle, one microbank bead was aseptically transferred from the stock tube into 10 ml of either Trypticase Soy Broth (TSB, Becton Dickenson and Company, Sparks, Md.) for Bioscreen analysis or Mueller-Hinton Broth (MHB, Becton Dickinson and Company, Sparks, Md.) for MIC/MLC analysis. Overnight cultures of bacterial strains were grown at 37° C. for 18 hours in a Forma 3157 water-jacketed incubator (Thermo Scientific, Waltham, Mass., USA) and diluted to a 0.5 McFarland turbidity standard. Subsequently, a 10−1 dilution of the McFarland standard was performed, to give an approximate bacterial count of 1.0×107 CFU/ml. This final dilution must be used within 30 minutes of creation to prevent an increase in bacterial density due to cellular growth.
Nanoparticle solutions were diluted in MHB and sterile dH2O to a 2× testing concentration yielding a total volume of 1.5 ml. Of this volume, 750 μl consisted of MHB, while the other 750 μl consisted of varying amounts of sterile dH2O and the nanoparticle solution to make a 2× concentration of the particular nanoparticle solution being tested. Testing dilutions (final concentration in reaction) ranged from 0.5 ppm Ag to 6.0 ppm Ag nanoparticle concentration with testing performed at every 0.5 ppm interval.
To determine the minimum inhibitory concentration (MIC) of nanoparticle solutions, 100 μl of the diluted bacterial culture was added to 100 μl of a particular nanoparticle solution at the desired testing concentration in the separate, sterile wells of a 100 well microtiter plate (Growth Curves USA, Piscataway, N.J., USA). Wells inoculated with both 100 μl of the diluted bacterial culture and 100 μl of a 1:1 MHB/sterile ddH2O mix served as positive controls, while wells with 100 μl of MHB and 100 μl of a 1:1 MHB/sterile ddH2O mix served as negative controls for the reaction. Plates were placed inside the tray of a Bioscreen C Microbiology Reader (Growth Curves USA, Piscataway, N.J., USA) and incubated at a constant 37° C. for 15 hours with optical density (O.D.) measurements being taken every 10 minutes. Before each O.D. measurement, plates were automatically shaken for 10 seconds at medium intensity to prevent settling of bacteria and to ensure a homogenous reaction well.
All data was collected using EZExperiment Software (Growth Curves USA, Piscataway, N.J., USA) and analyzed using Microsoft Excel (Microsoft Corporation, Redmond, Wash., USA). The growth curves of bacteria strains treated with different nanoparticle solutions were constructed and the MIC determined. The MIC was defined as the lowest concentration of nanoparticle solution that prevented the growth of the bacterial culture for 15 hours, as measured by optical density using the Bioscreen C Microbiology Reader.
Once the MIC was determined, the test medium from the MIC and subsequent higher concentrations was removed from each well and combined according to concentration in appropriately labeled, sterile Eppendorf tubes. TSA plates were inoculated with 100 μl of test medium and incubated overnight at 37° C. in a Forma 3157 water-jacketed incubator (Thermo Scientific, Waltham, Mass., USA). The minimum lethal concentration (MLC) was defined as the lowest concentration of nanoparticle solution that prevented the growth of the bacterial culture as measured by colony growth on TSA.
The results of the Bioscreen runs are shown in
In contrast, each of the solutions GR1-GR10 showed superior performance, relative to each of the raw materials AT031, AT060 and AT059. Interestingly, the combination of the raw materials associated with silver nanoparticles with those raw materials associated with both zinc and copper nanoparticles produced unexpected synergistic results.
Additional Bioscreen results are shown in
Due to the unexpected favorable results shown in
GZA raw material was made in a manner similar to the BT-006 raw material except that a platinum electrode 1/5 configuration was utilized rather than zinc.
Freeze-Drying
Freeze-drying was accomplished by placing the GR5 and GR8 solution in a plastic (nalgene) container and placing the plastic container in a BenchTop 2K freeze dryer (manufactured by Virtis) which was maintained at a temperature of about −52° C. and a vacuum of less than 100 milliliter. About 10-20 ml of solution will freeze-dry overnight.
As is shown in
Viability/Cytoxicity Testing of Mammalian Cells
The following procedures were utilized to obtain cell viability and/or cytotoxicity measurements.
Mus musculus (mouse) liver epithelial cells (accession number CRL-1638) and Sus scrofa domesticus (minipig) kidney fibrobast cells (accession number CCL-166) were obtained from the American Type Culture Collection (ATCC).
Cell lines were thawed by gentle agitation in a Napco 203 water bath (Thermo Scientific, Waltham, Mass., USA) at 37° C. for 2 minutes. To reduce microbial contamination, the cap and O-ring of the frozen culture vial were kept above the water level during thawing. As soon as the contents of the culture vial were thawed, the vial was removed from the water, sprayed with 95% ethanol, and transferred into a NuAire Labgard 440 biological class II safety cabinet (NuAire Inc., Plymouth, Minn., USA). The vial contents were then transferred to a sterile 75 cm2 tissue culture flask (Corning Life Sciences, Lowell, Mass., USA) and diluted with the recommended amount of complete culture medium. Murine liver epithelial cell line CRL-1638 required propagation in complete culture media composed of 90% Dulbecco's Modified Eagle's Medium (ATCC, Manassas, Va., USA) and 10% fetal bovine serum (ATCC, Manassas, Va., USA), while minipig kidney fibroblast cell line CCL-166 was grown in complete culture media comprised of 80% Dulbecco's Modified Eagle's Medium and 20% fetal bovine serum. Cell line CRL-1638 was diluted with growth media in a 1:15 ratio, while cell line CCL-166 was diluted with growth media in a 1:10 ratio. The culture flasks were then incubated at about 37° C., utilizing a 5% CO2 and 95% humidified atmosphere in a NuAire, IR Autoflow water-jacketed, CO2 incubator (NuAire Inc., Plymouth, Minn., USA).
Every two days, old growth medium was removed from culturing flasks and replaced with fresh growth medium. Each day, observations for microbial growth, such as fungal colonies and turbidity in medium, were made with the naked eye. Additionally, cultured cells were observed under an inverted phase contrast microscope (VWR Vistavision, VWR International, and West Chester, Pa., USA) to check for both general health of the cells and cell confluency.
Once cells reached approximately 80% confluent growth, cells were deemed ready for subculturing. Old growth medium was removed and discarded and the cell sheet rinsed with 5 ml of prewarmed trypsin-EDTA dissociating solution (ATCC, Manassas, Va., USA). After 30 seconds of contact with the cell sheet, the trypsin-EDTA was removed and discarded. Ensuring that both the entire cell monolayer was covered and the flask was not agitated, a 3 ml volume of the prewarmed trypsin-EDTA solution was added to the cell sheet followed by incubation of the culture flask at 37° C. for about 15 minutes. After cell dissociation, trypsin-EDTA was inactivated by adding about 6 ml of complete growth medium to the cell culture flask followed by gentle pipetting to aspirate cells.
In order to count cells, 200 μl of the cell suspension was collected in a 15 ml centrifuge tube (Corning Life Sciences, Lowell, Mass., USA). Both 300 μL of phosphate buffered saline (ATCC, Manassas, Va., USA) and 500 μL of a 0.4% trypan blue solution (ATCC, Manassas, Va., USA) was added to the collected cell suspension and mixed thoroughly. After allowing to stand for about 15 minutes, 10 μl of the mixture was placed in each chamber of an iN Cyto, C-Chip disposable hemacytometer (INCYTO, Seoul, Korea) where the cells were counted with a VWR Vistavision inverted phase contrast microscope (VWR International, West Chester, Pa., USA) according to the manufacturer's instructions. The concentration of the cells in the suspension was calculated using a conversion formula based upon the cell count obtained from the hemacytometer.
The wells of black, clear bottom, cell culture-treated microtiter plates (Corning Life Sciences, Lowell, Mass., USA) were seeded with 200 μl of culture medium containing approximately 1.7×104 cells as shown in
Microtiter plates were incubated with the treatment compounds 37° C., utilizing a 5% CO2 and 95% humidified atmosphere for 24 hours. After incubation with nanoparticle solutions, the culture medium was removed and discarded from each well and replaced with 100 μl of fresh media containing Alamar Blue™ (Biosource International, Camarillo, Calif., USA) at a concentration of 50 μl dye/ml media. Plates were gently shaken by hand for about 10 seconds and incubated at about 37° C., utilizing a 5% CO2 and 95% humidified atmosphere for 2.5 hours. Fluorescence was then measured in each well utilizing an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Fluorescence measurements were carried out on the Fluoroskan II fluorometer produced by Labsystems (Thermo Scientific, Waltham, Mass., USA).
Cytotoxicity of the nanoparticle solutions was determined by measuring the proportion of viable cells after treatment when compared to the non-treated control cells. A percent viability of cells after treatment was then calculated and used to generate the concentration of nanoparticle at which fifty percent of cellular death occurred (LC50). All data was analyzed using GraphPad Prism software (GraphPad Software Inc., San Diego, Calif., USA).
Results of the viability/cytotoxicity tests are shown in
With regard to
Similarly,
In each of
With regard to
This Example utilizes the same basic apparatus used to make the solutions of Examples 1-5. However, this Example does not utilize any electrode(s) 1. This Example utilizes 99.95% pure silver electrodes for each electrode 5. Tables 11a, 11b and 11c summarize portions of electrode design, configuration, location and operating voltages. As shown in Tables 11a, 11b and 11c, the target voltages were set to a low of about 2,750 volts in Electrode Set #8 and to a high of about 4,500 volts in Electrode Sets #1-3. The high of 4,500 volts essentially corresponds to an open circuit which is due to the minimal conductivity of the liquid 3 between each electrode 5, 5′ in Electrode Sets #1-3
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
Atomic Absorption Spectroscopy (AAS) samples were prepared and measurement values were obtained. Slight process modifications were incorporated into those AAS procedures discussed earlier herein. These process changes are incorporated immediately below.
The AAS values were obtained from a Perkin Elmer AAnalyst 300 Spectrometer system, as in Examples 1-5. The samples manufactured in accordance with Examples 6-12 were prepared by adding a small amount of nitric acid or hydrochloric acid (usually 2-4% of final volume) and then dilution to a desirable characteristic concentration range or linear range of the specific element to improve accuracy of the result. The “desireable” range is an order of magnitude estimate based on production parameters established during product development. For pure metals analysis, a known amount of feedstock material is digested in a known amount of acid and diluted to ensure that the signal strength of the absorbance will be within the tolerance limits and more specifically the most accurate range of the detector settings, better known as the linear range.
The specific operating procedure for the Perkin Elmer AAnalyst 300 system is as follows:
I) Principle
Table 11d shows the results obtained from Example 6. Table 11d contains a column entitled “Electrode Configuration”. This column contains characters “0” and “X”. The character “0” corresponds to one electrode set 5, 5′. The character “X” represents that no electrodes were present. Thus, for Run ID “AT098”, only a single electrode set 5a, 5a′ was utilized. No detectable amount of silver was measurable by the AAS techniques disclosed herein. Run ID “AT099” utilized two electrode sets 5a, 5a′ and 5b, 5b′. The AAS techniques detected some amount of silver as being present, but that amount was less than 0.2 ppm. Run ID “AT100” utilized eight electrode sets, 5, 5′. This configuration resulted in a measured ppm of 7.1 ppm. Accordingly, it is possible to obtain metallic-based constituents (e.g., metallic-based nanoparticles/nanoparticle solution) without the use of an electrode 1 (and an associated adjustable plasma 4). However, the rate of formation of metallic-based constituents is much less than that rate obtained by using one or more plasmas 4. For example, Examples 1-3 disclosed silver-based products associated with Run ID's AT031, AT036 and AT038. Each of those Run ID's utilized two electrode sets that included adjustable plasmas 4. The measured silver ppm for each of these samples was greater than 40 ppm, which is 5-6 times more than what was measured in the product made according to Run ID AT100 in this Example 6. Thus, while it is possible to manufacture metallic-based constituents without the use of at least one adjustable plasma 4 (according to the teachings herein) the rates of formation of metallic based constituents are greatly reduced when no plasmas 4 are utilized as part of the production techniques.
Accordingly, even though eight electrode sets 5, 5′ were utilized to make the product associated with Run AT100, the lack of any electrode sets including at least one electrode 1 (i.e., the lack of plasma 4), severely limited the ppm content of silver in the solution produced.
This Example utilizes the same basic apparatus used to make the solutions of Examples 1-5, however, this Example uses only a single plasma 4. Specifically, for Electrode Set #1, this Example uses a “1a, 5a” electrode configuration. Subsequent Electrode Sets #2-#8 are sequentially added. Each of Electrode Sets #2-#8 have a “5, 5′” electrode configuration. This Example also utilizes 99.95% pure silver electrodes for each of electrodes 1 and 5 in each Electrode Set.
Tables 12a-12h summarize portions of electrode design, configuration, location and operating voltages. As shown in Tables 12a-12h, the target voltages were set to a low of about 900 volts (at Electrode Set #8) and a high of about 2,300 volts (at Electrode Set #1).
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
5b′
5c′
5d′
5e′
5f′
5g′
5b′
5c′
5d′
5e′
5f′
5g′
5h′
Atomic Absorption Spectroscopy (AAS) samples were prepared and measurement values were obtained, as discussed in Example 6. Table 12i shows the results. Note that Table 12i includes a column entitled “Electrode Configuration”. This column contains characters of “1” and “0” and “X”. The “1's” represent an electrode configuration corresponding to Electrode Set #1 (i.e., a 1, 5 combination). The “0's” represent an electrode combination of 5, 5′. The character “X” represents that no electrodes were present. Thus, for example, “AT084” is represented by “1000XXXX” which means a four electrode set combination was used to make “AT084” and the combination corresponded to Set #1=1, 5; Set #2=5, 5; Set #3=5, 5 and Set #4=5, 5 (there were no Sets after Set #4, as represented by “XXXX”).
Table 12i includes a column entitled “Measured Ag PPM (initial)”. This column corresponds to the silver content of each of the eight solutions measured within one hour of its production. As shown, the measured ppm increases with each added Electrode Set, wherein the Run AT080 produces a ppm level for silver comparable in amount to Run ID AT031 of Example 3. However, another column entitled, “Measured Ag PPM (10 days)” shows data which tells another story. Specifically, the “initial” and “10 day” PPM measurements are essentially the same (e.g., within operation error of the AAS) for samples corresponding to Run Id's AT097, AT086, AT085, AT084 and AT083. This means that essentially no significant settling of the constituent particles found in five of the eight runs occurred. However, once samples associated with Run ID AT082, AT081 and AT080 were examined after 10 days, a significant portion of the constituent particles had settled, with samples taken from Run AT080 losing about 10 ppm out of 40 ppm due to particulate settling.
In order to obtain an idea of what particle sizes were being produced in each of the eight samples associated with this Example 7, a dynamic light scattering (DLS) approach was utilized. Specifically, dynamic light scattering methods utilizing variations of scattered light intensities from an LED laser were measured over time to determine any changes in intensity from particle motion due to Brownian Motion. The instrument used to perform these measurements was a VISCOTEK 802 DLS with Dual Alternating Technology (D.A.T.).
All measurements were made using a 12 μL quartz cell, which was placed into a temperature controlled cell block. One 827.4 nm laser beam was passed through the solution to be measured. Scattering intensities were measured using a CCD detector with an optical view path mounted transversely to that of the laser. Experimental data was then mathematically transformed using variation of Einstein-Stokes and Rayleigh equations to derive values representative of particle size and distribution information. Data collection and math transforms were performed using Viscotek Omnisize version 3,0,0,291 software. This instrument hardware and software reliably provides measurements for particles with a radius from 0.8 nm to 2 μm.
This technique works best when the solution is free of micro-bubbles and particles subject to Stokes settling motion (some of which was clearly occurring in at least three of the samples in this Example 7). All vessels used to contain and prepare materials to be tested were rinsed and blow-dried to remove any debris. All water used to prepare vessels and samples was doubly de-ionized and 0.2 μm filtered. If solvent is needed, use only spectrographic grade isopropyl alcohol. All were rinsed with clean water after solvent exposure, and wiped only with clean lint-free cotton cloth.
An aliquot of solution sample, about 3 ml in total volume, was drawn into a small syringe and then dispensed into a clean about 4 dram glass sample vial. Two (2) syringe filters (0.45 μm) were fixed onto the syringe during this operation to doubly filter the sample, thus removing any large particles not intended as part of the solution. This sample was placed into a small vacuum chamber, where it was subjected to a 1 minute exposure to a low-level vacuum (<29.5 inches Hg) to boil the suspension, removing suspended micro-bubbles. The vacuum was drawn through a small dual-stage rotary vacuum pump such as a Varian SD-40. Using a glass Tuberculin syringe with a 20 gauge or smaller blunted needle, sample was withdrawn to fill the syringe and then rinsed, then placed into the 12 μL sample cell/cuvette. Additional like-type syringes were used to withdraw used sample and rinse fluids from this cell. The filled cuvette was inspected for obvious entrapped bubbles within the optical path.
This cell was inserted into the holder located in the VISCOTEK 802 DLS. Prior to this step, the instrument was allowed to fully warm to operating temperature for about 30 minutes and operating “OmniSIZE” software loaded in the controlling computer. This software will communicate and set-up the instrument to manufacturer prescribed conditions. Select a “new” measurement. Validate that the correct sample measurement parameters are selected, i.e.; temperature of 40° C., “Target” laser attenuation value of 300 k counts per second, 3 second measurement duration, water as the solvent, spike and drift respectively at 20% and 15%. Correct if needed. Then select “Tools-Options” from the controlling menu bar. Insure proper options are annotated; i.e. resolution at 200, ignore first 2 data points, peak reporting threshold of 0 and 256 correlator channels.
Once the sample was placed into the holder, the cover lid was securely closed causing the laser shutter to open. The sample was allowed to temperature stabilize for 5 to 10 minutes. On the menu tools bar, “Auto-Attenuate” was selected to cause the adjustment of laser power to fit the measurement requirements. Once the instrument and sample was set-up, the scatter intensity graphic display was observed. Patterns should appear uniform with minimal random spikes due to entrained nano/micro-bubbles or settling large particles.
A measurement was then performed. The developing correlation curve was also observed. This curve should display a shape as an “inverted S” and not “spike” out-of-limits. If the set-up was correct, parameters were adjusted to collect 100 measurements and “run” was then selected. The instrument auto-collected data and discarded correlation curves, not exhibiting Brownian motion behavior. At measurement series completion, retained correlation curves were inspected. All should exhibit expected shape and displayed between 30% and 90% expected motion behaviors. At this point, collected data was saved and software calculated particle size information. The measurement was repeated to demonstrate reproducibility. Resultant graphic displays were then inspected. Residuals should appear randomly dispersed and data measurement point must follow calculated theoretical correlation curve. The graphic distribution display was limited to 0.8 nm to 2 μm. The Intensity Distribution and Mass Distribution histograms were reviewed to find particle sizes and relative proportions of each, present in the suspension. All information was then recorded and documented.
In an effort to understand further the particles produced as a function of the different electrode combinations set forth in the Example 7, SEM photomicrographs of similar magnification were taken of each dried solution corresponding to each of the eight solutions made in this Example. These SEM photomicrographs are shown in
It should be noted that samples were prepared for the SEM by allowing a small amount of each solution produced to air dry on a glass slide. Accordingly, it is possible that some crystal growth may have occurred during drying. However, the amount of “growth” shown in each of samples AT082-AT080 is more than could possibly have occurred during drying alone. It is clear from the SEM photomicrographs that cubic-shaped crystals are evident in AT082, AT081 and AT080. In fact, nearly perfect cubic-shaped crystals are shown in
Accordingly, without wishing to be bound by any particular theory or explanation, when comparing the results of Example 7 with Example 6, it becomes clear that the creation of the plasma 4 has a profound impact on this inventive process. Moreover, once the plasma 4 is established, conditions favor the production of metallic-based constituents, including silver-based nanoparticles, including the apparent growth of particles as a function of each new electrode set 5, 5′ provided sequentially along the trough member 30. However, if the goal of the process is to maintain the suspension of metallic-based nanoparticles in solution, then, under the process conditions of this Example 7, some of the particles produced begin to settle out near the last three Electrode Sets (i.e., Run Id's AT082, AT081 and AT080). However, if the goal of the process is to achieve particulate matter settling, then that goal can be achieved by following the configurations in Runs AT082, AT081 and AT080.
UV-Vis spectra were obtained for each of the settled mixtures AT097-AT080. Specifically, UV-Vis spectra were obtained as discussed above herein (see the discussion in the section entitled, “Characterization of Materials of Examples 1-5 and Mixtures Thereof”).
UV-Vis spectra for these same eight samples are also shown in
In an effort to determine efficacy against an E. coli bacteria (discussed in greater detail earlier herein), each of the eight solutions made according to this Example 7 were all diluted to the exact same ppm for silver in order to compare their relative efficacies in a normalized approach. In this regard, the normalization procedure was, for each of the samples, based on the ppm measurements taken after ten days of settling. Accordingly, for example, samples made according to Run AT080 were diluted from 31.6 ppm down to 4 ppm; whereas the samples associated with Run AT083 were diluted from 28.1 ppm, down to 4 ppm. These samples were then further diluted to permit Bioscreen measurements to be performed, as discussed above herein.
This Example utilizes the same basic apparatus used to make the solutions of Examples 1-5, however, this Example uses only a single plasma 4 to make AT090 (i.e., similar to AT080); two plasmas 4 to make AT091 (i.e., similar to AT031); and two plasmas 4 to make AT089 (first time run), wherein Electrode Set #1 and Electrode Set #8 both utilize plasmas 4. This Example also utilizes 99.95% pure silver electrodes for each of electrodes 1 and 5 in each Electrode Set.
Tables 13a, 13b and 13c summarize portions of electrode design, configuration, location and operating voltages. As shown in Tables 13a-13c, the target voltages were on average highest associated with AT089 and lowest associated with AT091.
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
5b′
5c′
5d′
5e′
5f′
5g′
5h′
5b′
5c′
5e′
5f′
5g′
5h′
5b′
5c′
5d′
5e′
5f′
5g′
Atomic Absorption Spectroscopy (AAS) samples were prepared and measurement values were obtained, as discussed in Example 6. Table 13d shows the results. Note that Table 13d includes a column entitled “Electrode Configuration”. This column contains characters of “1” and “0”. The “1's” represent an electrode configuration corresponding to Electrode Set #1 (i.e., a 1, 5 combination). The “0's” represent an electrode combination of 5, 5′. Thus, for example, “AT089” is represented by “10000001” which means an eight electrode set combination was used to make “AT089” and the combination corresponded to Set #1=1, 5; Sets #2-#7=5, 5; and Set #8=1, 5.
Table 13d includes a column entitled “Measured Ag PPM (initial)”. This column corresponds to the silver content of each of the eight solutions measured within one hour of its production. As shown, the measured ppm for each of the three Runs were generally similar. However, another column entitled, “Measured Ag PPM (20 hours)” shows that the “initial” and “20 hours” PPM measurements are essentially the same (e.g., within operation error of the AAS) for samples corresponding to Run Id's AT089 and AT091. This means that essentially no significant settling of the constituent particles found in these runs occurred. However, the sample associated with Run ID AT090 was examined after 20 hours, a significant portion of the constituent particles had settled, with the samples taken from Run AT089 losing about 3.6 ppm out of 40 ppm due to particulate settling.
As discussed in Example 7, a dynamic light scattering (DLS) approach was utilized to obtain average particle size made in each of these three samples. The largest particles were made in AT090; and the smallest particles were made in AT091. Specifically,
In an effort to determine efficacy against an E. coli bacteria (discussed in greater detail earlier herein), each of the three solutions made according to this Example 8 were all diluted to the exact same ppm for silver in order to compare their relative efficacies in a normalized manner. In this regard, the normalization procedure was, for each of the samples, based on the ppm measurement taken after twenty hours of settling. Accordingly, for example, samples made according to Run AT090 were diluted from 37.2 ppm down to 4 ppm; whereas the samples associated with Run AT091 were diluted from 44.0 ppm, down to 4 ppm. These samples were then further diluted to permit Bioscreen measurements to be performed, as discussed above herein.
This Example utilizes essentially the same basic apparatus used to make the solutions of Examples 1-5, however, this Example uses two plasmas 4 occurring in a controlled atmosphere environment. Controlled atmospheres were obtained by using the embodiment shown in
Tables 14a-14e summarize portions of electrode design, configuration, location and operating voltages. As shown in Tables 14a-14e, the target voltages were set to a low of about 400-500 volts (reducing atmosphere and ozone) and a high of about 3,000 volts (helium atmosphere).
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
Likewise, the atmosphere of ozone (AT094) was achieved by creating a positive pressure of ozone created by an ozone generator and inputted into the atmosphere control device 35, as discussed above herein. It should be noted that significant nitrogen content was probably present in the supplied ozone.
Further, the atmosphere of helium (AT095) was achieved by creating a positive pressure of helium inputted into the atmosphere control device 35, as discussed above herein.
The atmosphere of air was achieved without using the atmosphere control device 35.
The reducing atmosphere (or air-deprived atmosphere) was achieved by providing the atmosphere control device 35 around each electrode 1, 5 in Electrode Sets #1 and #4 and not providing any gas into the inlet portion 37 of the atmosphere control devices 35. In this instance, the external atmosphere (i.e., an air atmosphere) was found to enter into the atmosphere control device 35 through the hole 37 and the plasma 4 created was notably much more orange in color relative to the air atmosphere plasma.
In an effort to understand the composition of each of the plasmas 4, a “Photon Control Silicon CCD Spectrometer, SPM-002-E” (from Blue Hill Optical Technologies, Westwood, Mass.) was used to collect the emission spectra for each of the plasmas 4.
Specifically, in reference to
Prior to the collection of any spectra created by each plasma 4, the atmosphere control device 35 was saturated with each gas for 30 seconds and a background spectrum was collected with 2 second exposure set in the software package. The plasma 4 was active for 10 minutes prior to any data collection. The primary spot from the laser 501 was aligned with the same point each time. Three separate spectra were collected for each run and then averaged. The results of each spectra are shown in
5b′
5c′
5e′
5f′
5g′
5h′
5b′
5c′
5e′
5f′
5g′
5h′
5b′
5c′
5e′
5f′
5g′
5h′
5b′
5c′
5e′
5f′
5g′
5h′
5b′
5c′
5e′
5f′
5g′
5h′
Atomic Absorption Spectroscopy (AAS) samples were prepared and measurement values were obtained, as discussed in Example 6. Table 14f shows the results. Note that Table 14f includes a column entitled “Electrode Configuration”. This column contains characters “1” and “0”. The “1's” represent an electrode configuration corresponding to Electrode Set #1 (i.e., a 1, 5 combination). The “0's” represent an electrode combination of 5, 5′. Thus, for example, “AT091” is represented by “10010000” which means an eight electrode set combination was used to make “AT091” and the combination corresponded to Set #1=1, 5; Set #2=5, 5; Set #3=5, 5; Set #4=1, 5 and Set #5-Set #8=5, 5.
Table 14f includes a column entitled “Measured Ag PPM”. This column corresponds to the silver content of each of the eight solutions. As shown, the measured ppm produced in each of the atmospheres of air, nitrogen, reducing and ozone were substantially similar. However, the atmosphere of helium (i.e., AT095) produced a much lower ppm level. Also, the size of particulate matter in the AT095 solution was significantly larger than the size of the particulate matter in each of the other four solutions. The particulate sizes were determined by dynamic light scattering methods, as discussed above herein.
It is clear from
In an effort to determine efficacy against an E. coli bacteria (discussed in greater detail earlier herein), each of the five solutions made according to this Example 9 were all diluted to the exact same ppm for silver in order to compare their relative efficacies in a normalized manner. Accordingly, for example, samples made according to Run AT091 were diluted from 44.0 ppm down to 4 ppm; whereas the samples associated with Run AT095 were diluted from 28.3 ppm, down to 4 ppm. These samples were then further diluted to permit Bioscreen measurements to be performed, as discussed above herein.
This Example utilizes essentially the same basic apparatus used to make the solutions of Examples 1-5, however, this Example uses two plasmas 4 formed by a DC-like Power Source (i.e., a diode bridge-rectified power source). Specifically, for Electrode Set #1 and Electrode Set #4, this Example uses a “1, 5” electrode configuration wherein the electrode 1 creates a plasma 4 in accordance with the power source shown in
Table 15 summarizes portions of electrode design, configuration, location and operating voltages. As shown in Table 15, the target voltages were set to a low of about 400 volts (Electrode Set #4) and a high of about 1,300 volts (Electrode Set #3).
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
5b′
5c′
5e′
5f′
5g′
5h′
Atomic Absorption Spectroscopy (AAS) samples were prepared and measurement values were obtained, as discussed in Example 6. Table 15a shows the results. Note that Table 15a includes a column entitled “Electrode Configuration”. This column contains characters “1*” and “0”. The “1*” represents an electrode configuration corresponding to Electrode Set #1 (i.e., a 1, 5 combination, wherein the electrode 1 is negatively biased and the electrode 5 is positively biased. The “0's” represent an electrode combination of 5, 5′.
Table 15a includes a column entitled “Measured Ag PPM”. This column corresponds to the silver content of the solution. As shown, the measured ppm was 51.2 ppm, which was substantially higher than any other samples made by the other eight electrode sets utilized in any other Example.
In an effort to determine efficacy against an E. coli bacteria (discussed in greater detail earlier herein), this solution AT096 was tested against each of the five solutions made according to Example 9 above herein. Specifically, all of the five solutions from Example 9 and AT096 were diluted to the exact same ppm for silver in order to compare their relative efficacies in a normalized manner as discussed in Example 9.
The atmosphere used for AT096 was air, and the corresponding spectra of the air plasma is shown in
Similarly,
This Example follows the teachings of Examples 2 [AT060], 3 [AT031-AT064] and 4 [BT006-BT012] to manufacture two different silver-based nanoparticle/nanoparticle solutions and one zinc-based nanoparticle/nanoparticle solution. Additionally, a new and different solution (i.e., PT001) based in part on the inventive process for making BT006 and BT012 was also produced. Once produced, three solutions were tested for efficacy and cytotoxicity.
Specifically, the solution made by the method of Example 2 (i.e., AT060) was tested for cytotoxicity against Murine Liver Epithelial Cells, as discussed above herein. The results are shown in
Mixtures of the materials (i.e., AT060, AT064 and BT012) were then made in order to form GR5 and GR8, in accordance with what is shown in Table 8 herein relating to the solutions GR5 and GR8. Specifically, AT064 and BT012 were mixed together to form GR5; and AT060 and BT012 were mixed together to form GR8 to result in the amounts of silver and zinc in each being the same as what is shown in Table 8.
Once the solutions of GR5 and GR8 were formed, the cytotoxicity for each was measured. Specifically, as shown in
In comparison,
The other inventive material in this Example 11, “PT001”, was made by the following process. Electrode Set #1 was a 1, 5 combination. Electrode Set #2 was also a 1, 5 combination. There were no electrode sets at positions 2-8. Accordingly, the designation for this electrode combination was a “11XXXXXX”. The composition of each of electrodes 1 and 5 in both Electrode Sets #1 and #2 were high-purity platinum (i.e., 99.999%). Table 16a sets forth the specific run conditions for PT001.
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
The solution PT001 was then treated as if it had an equivalent volume of zinc-based nanoparticles equivalent to those present in BT012 (i.e., 23 ppm zinc). In other words, a volume of about 150 ml of PT001 was added to about 50 ml of AT064 to produce GR5* and a volume of about 170 ml of PT001 was added to about 33 ml of AT060 to produce GR8*. Once mixed, these new material solutions (i.e., GR5* and GR8*) were allowed to sit for 24 hours prior to being tested for cytotoxicity.
Likewise,
Accordingly, the LD50 of each of GR5* and GR8* was higher than the corresponding LD50's of GR5 and GR8, respectively (i.e., with regard to the silver content in each of the mixes GR5 and GR8).
The biological efficacies against E. coli of each of GR5 and GR5* were then compared. Specifically,
Likewise, a comparison between the biological efficacy against E. coli was also performed for GR8 and GR8*. This comparison is shown in
Accordingly, this Example shows that cytotoxicity of solutions GR5 and GR8 can be lowered by utilizing the solution PT001 instead of BT012 in each of the mixes GR5 and GR8. Moreover, such cytotoxicity is lowered without sacrificing biological efficacy against E. coli, as shown in
However, it should be understood that other in vivo benefits can be obtained by the presence of, for example, the material corresponding to BT012 in the solutions GR5 and GR8.
The materials disclosed in Example 11, namely AT064 and AT060 and an equivalent to BT012 (i.e., BT013) were mixed together in varying proportions to determine if any differences in biological efficacy could be observed (e.g., similar to the studies shown in
Specifically,
Specifically,
However, the biological efficacy results are dramatically different in
Additional biological efficacy tests were run to determine if additional “hold time” had any further enhancing effects. Specifically, the data in
In an effort to clarify the differences in biological efficacy observed in
Specifically, two sets of DLS tests were performed. The first test mixed together AT064 and BT013 in proportion to produce GR5 (i.e., about 50 ml of AT064 and about 150 ml of BT013). The second test mixed together AT060 and BT013 in proportion to produce GR8 (i.e., about 33 ml of AT060 and about 170 ml of BT013).
The results of the DLS measurements as a function of time after mixing the aforementioned materials together are shown in
It is clear from the results shown in
Without wishing to be bound by any particular theory or explanation, it appears that particle size and biological performance (e.g., efficacy against E. coli) are related.
This Example utilizes essentially the same basic apparatus used to make the solutions of Examples 1-5, however, this Example uses three different temperatures of water input into the trough member 30.
Specifically: (1) water was chilled in a refrigerator unit until it reached a temperature of about 2° C. and was then pumped into the trough member 30, as in Examples 1-5; (2) water was allowed to adjust to ambient room temperature (i.e., 21° C.) and was then pumped into the trough member 30, as in Examples 1-5; and (3) water was heated in a metal container until it was about 68° C. (i.e., for Ag-based solution) and about 66° C. (i.e., for Zn-based solution), and was then pumped into the trough member 30, as in Examples 1-5.
The silver-based nanoparticle/nanoparticle solutions were all manufactured using a set-up where Electrode Set #1 and Electrode Set #4 both used a “1, 5” electrode configuration. All other Electrode Sets #2, #3 and #5-#8, used a “5, 5′” electrode configuration. These silver-based nanoparticle/nanoparticle solutions were made by utilizing 99.95% pure silver electrodes for each of electrodes 1 and/or 5 in each electrode set.
Also, the zinc-based nanoparticles/nanoparticle solutions were all manufactured with each of Electrode Sets #1-#8 each having a “1,5” electrode configuration. These zinc-based nanoparticles/nanoparticle solutions also were made by utilizing 99.95% pure zinc electrodes for the electrodes 1,5 in each electrode set.
Tables 17a-17f summarize electrode design, configuration, location and operating voltages. As shown in Tables 17a-17c, relating to silver-based nanoparticle/nanoparticle solutions, the target voltages were set to a low of about 620 volts and a high of about 2,300 volts; whereas with regard to zinc-based solution production, Tables 17d-17f show the target voltages were set to a low of about 500 volts and a high of about 1,900 volts.
Further, bar charts of the actual and target voltages for each electrode in each electrode set, are shown in
Once each of the silver-based nanoparticle/nanoparticle solutions AT110, AT109 and AT111, as well as the zinc-based nanoparticle/nanoparticle solutions BT015, BT014 and BT016 were manufactured, these six solutions were mixed together to make nine separate 50/50 volumetric mixtures. Reference is made to Table 17g which sets forth a variety of physical and biological characterization results for the six “raw materials” as well as the nine mixtures made therefrom.
Specifically, for example, in reference to the first mixture listed in Table 17g, that mixture is labeled as “Cold Ag/Cold Zn”. Similarly, the last of the mixtures referenced in Table 17g is labeled “Hot Ag/Hot Zn”. “Cold Ag” or “Cold Zn” refers to the input water temperature into the trough member 30 being about 2° C. “RT Ag” or “RT Zn” refers to the input water temperature being about 21° C. “Hot Ag” refers to refers to the input water temperature being about 68° C.; and “Hot Zn” refers to the input water temperature to the trough member 30 being about 66° C.
The physical parameters reported for the individual raw materials, as well as for the mixtures, include “PPM Ag” and “PPM Zn”. These ppm's (parts per million) were determined by the Atomic Absorption Spectroscopy techniques discussed above herein in Example 6. It is interesting to note that the measured PPM of the silver component in the silver-based nanoparticle/nanoparticle solutions was higher when the input temperature of the water into the trough member 30 was lower (i.e., Cold Ag (AT110) corresponds to an input water temperature of 2° C. and a measured PPM of silver of 49.4). In contrast, when the input temperature of the water used to make sample AT111 was increased to 68° C. (i.e., the “Hot Ag”), the measured amount of silver decreased to 31.1 ppm (i.e., a change of almost 20 ppm). Accordingly, when mixtures were made utilizing the raw material “Cold Ag” versus “Hot Ag”, the PPM levels of the silver in the resulting mixtures varied.
Each of the nine mixtures formulated were each approximately 50% by volume of the silver-based nanoparticle solution and 50% by volume of the zinc-based nanoparticle solution. Thus, whenever “Hot Ag” solution was utilized, the resulting PPM in the mixture would be roughly half of 31.1 ppm; whereas when the “Cold Ag” solution was utilized the silver PPM would be roughly half of 49.4 ppm.
The zinc-based nanoparticle/nanoparticle solutions behaved similarly to the silver-based nanoparticle/nanoparticle solutions in that the measured PPM of zinc decreased as a function of increasing water input temperature, however, the percent decrease was less. Accordingly, whenever “Cold Zn” was utilized as a 50 volume percent component in a mixture, the measured zinc ppm in the mixtures was larger than the measured zinc ppm when “Hot Zn” was utilized.
Table 17g includes a third column, entitled, “Zeta Potential (Avg)”. “Zeta potential” is known as a measure of the electro-kinetic potential in colloidal systems. Zeta potential is also referred to as surface charge on particles. Zeta potential is also known as the potential difference that exists between the stationary layer of fluid and the fluid within which the particle is dispersed. A zeta potential is often measured in millivolts (i.e., mV). The zeta potential value of approximately 25 mV is an arbitrary value that has been chosen to determine whether or not stability exists between a dispersed particle in a dispersion medium. Thus, when reference is made herein to “zeta potential”, it should be understood that the zeta potential referred to is a description or quantification of the magnitude of the electrical charge present at the double layer.
The zeta potential is calculated from the electrophoretic mobility by the Henry equation:
where z is the zeta potential, UE is the electrophoretic mobility, E is a dielectric constant, η is a viscosity, ƒ(ka) is Henry's function. For Smoluchowski approximation ƒ(ka)=1.5.
Electrophoretic mobility is obtained by measuring the velocity of the particles in an applied electric field using Laser Doppler Velocimetry (“LDV”). In LDV the incident laser beam is focused on a particle suspension inside a folded capillary cell and the light scattered from the particles is combined with the reference beam. This produces a fluctuating intensity signal where the rate of fluctuation is proportional to the speed of the particles (i.e. electrophoretic mobility).
In this Example, a Zeta-Sizer “Nano-ZS” produced by Malvern Instruments was utilized to determine zeta potential. For each measurement a 1 ml sample was filled into clear disposable zeta cell DTS1060C. Dispersion Technology Software, version 5.10 was used to run the Zeta-Sizer and to calculate the zeta potential. The following settings were used: dispersant—water, temperature—25° C., viscosity—0.8872 cP, refraction index—1.330, dielectric constant—78.5, approximation model—Smoluchowski. One run of hundred repetitions was performed for each sample.
Table 17g shows clearly that for the silver-based nanoparticle/nanoparticle solutions the zeta potential increased in negative value with a corresponding increasing input water temperature into the trough member 30. In contrast, the Zeta-Potential for the zinc-based nanoparticle/nanoparticle solutions was positive and decreased slightly in positive value as the input temperature of the water into the trough member 30 increased.
It is also interesting to note that the zeta potential for all nine of the mixtures made with the aforementioned silver-based nanoparticle/nanoparticle solutions and zinc-based nanoparticle/nanoparticle solutions raw materials were positive with different degrees of positive values being measured.
The fourth column in Table 17g reports the measured pH. The pH was measured for each of the raw material solutions, as well as for each of the mixtures. These pH measurements were made in accordance with the teachings for making pH measurements in Examples 1-5. It is interesting to note that the pH of the silver-based nanoparticle/nanoparticle solutions changed significantly as a function of the input water temperature into the trough member 30 starting with a low of 3.8 for the cold input water (i.e., 2° C.) and increasing to a value of 5.2 for the hot water input (i.e., 68° C.). In contrast, while the measured pH for each of three different zinc-based nanoparticle/nanoparticle solutions were, in general, significantly lower than any of the silver-based nanoparticle/nanoparticle solutions pH measurements, the pH did not vary as much in the zinc-based nanoparticle/nanoparticle solutions.
The pH values for each of the nine mixtures were much closer to the pH values of the zinc-based nanoparticle/nanoparticle solutions, namely, ranging from a low of about 3.0 to a high of about 3.4.
The fifth column in Table 17g reports “DLS % Transmission”. The “DLS” corresponds to Dynamic Light Scattering. Specifically, the DLS measurements were made according to the DLS measuring techniques discussed above herein (e.g., Example 7). The “% Transmission” is reported in Table 17g because it is important to note that lower numbers correspond to a lesser amount of laser intensity being required to report detected particle sizes (e.g., a reduced amount of laser light is required to interact with species when such species have a larger radius and/or when there are higher amounts of the species present). Accordingly, the DLS % Transmission values for the three silver-based nanoparticle/nanoparticle solutions were lower than all other % Transmission values. Moreover, a higher “% of Transmission” number (i.e., 100%) is indicative of very small nanoparticles and/or significant ionic character present in the solution (e.g., at least when the concentration levels or ppm's of both silver and zinc are as low as those reported herein).
The next column entitled, “Predominant DLS Mass Distribution Peak (Radius in nm)” reports numbers that correspond to the peak in the Gaussian curves obtained in each of the DLS measurements. For example, these reported peak values come from Gaussian curves similar to the ones reported in
The last two columns in Table 17g summarize detailed microbiological studies. In this regard, E. coli bacteria were tested in a Bioscreen apparatus. The procedures for testing were similar to those procedures discussed in Examples 1-5 herein. Specifically,
The column entitled “Relative Bioscreen Performance” is a merit ranking, wherein the higher numbers correspond to the highest performing raw materials and solutions relative to each other. In this regard, the numbers 11 and 11.7 corresponding to “RT Ag/Cold Zn” and “Hot Ag/Cold Zn”, respectively were the best performers, based on this ranking. However, in order to define the performances even more particularly, the column entitled, “Time (hours) to Bacteria Growth Beginning (1.0 McFarland)” shows that the “Cold Ag”, “RT Ag” and “Hot Ag” allow bacteria to begin to grow between 3 and 3.5 hours; the “Cold Zn”, “RT Zn” and “Hot Zn” did not inhibit bacterial growth at all (i.e., the bacterial growth curves substantially corresponded to control growth curves); and the nine different mixtures provided varying times when the bacteria begin to grow with the two worst performing mixtures being “Cold Ag/Cold Zn” (i.e., 5.25 hours) and “RT Ag/Hot Zn” (i.e., 5.00 hours); in contrast to the better performing mixtures showing growth times beginning around 16 and 17 hours.
Without wishing to be bound by any particular theory or explanation, it is clear that the input temperature of the liquid into the trough member 30 does have an effect on the inventive solutions made according to the teachings herein. Specifically, not only are amounts of components (e.g., ppm) affected by water input temperature, but physical properties and biological performance are also affected. Thus, control of water temperature, in combination with control of all of the other inventive parameters discussed herein, can permit a variety of particle sizes to be achieved, differing zeta potentials to be achieved, different pH's to be achieved and corresponding different performance (e.g., biological performances) to be achieved.
This Example utilizes essentially the same basic apparatus used to make the solutions of Examples 1-5, however, this Example use gold electrodes for the 8 electrode sets. In this regard, Tables 18a-18c set forth pertinent operating parameters associated with each of the 16 electrodes in the 8 electrode sets utilized to make gold-based nanoparticles/nanoparticle solutions.
Further,
Additionally, the following differences in manufacturing set-up were also utilized:
Table 18d summarizes the physical characteristics results for each of the three solutions GT032, GT031 and GT019. Full characterization of GT-019 was not completed, however, it is clear that under the processing conditions discussed herein, both processing enhancers (i.e., soda and salt) increase the measured ppm of gold in the solutions GT-031 and GT-019 relative to GT032.
This Example utilized a different apparatus from those used to make the solutions in Examples 1-5, however, this Example utilized similar technical concepts to those disclosed in the aforementioned Examples. In reference to
Once the solutions made in trough members 30a and 30b had been manufactured, these solutions were then processed in three different ways, namely:
(i) The Zn-based and Ag-based solutions were mixed together at the point 30d and flowed to the base portion 30o of the Y-shaped trough member 30 immediately after being formed in the upper portions, 30a and 30b, respectively. No further processing occurred in the base portion 30o;
(ii) The Zn-based and Ag-based solutions made in trough members 30a and 30b were mixed together after about 24 hours had passed after manufacturing each solution in each upper portion trough member 30a and 30b (i.e., the solutions were separately collected from each trough member 30a and 30b prior to being mixed together); and
(iii) The solutions made in trough members 30a and 30b were mixed together in the base portion 30o of the y-shaped trough member 30 substantially immediately after being formed in the upper portions 30a and 30b, and were thereafter substantially immediately processed in the base portion 30o of the trough member 30 by another four electrode set.
Table 19a summarizes the electrode design, configuration, location and operating voltages for each of trough members 30a and 30b (i.e., the upper portions of the trough member 30) discussed in this Example. Specifically, the operating parameters associated with trough member 30a were used to manufacture a zinc-based nanoparticle/nanoparticle solution; whereas the operating parameters associated with trough member 30b were used to manufacture a silver-based nanoparticle/nanoparticle solution. Once these silver-based and zinc-based solutions were manufactured, they were mixed together substantially immediately at the point 30d and flowed to the base portion 30o. No further processing occurred.
Table 19b summarizes the electrode design, configuration, location and operating voltages for each of trough members 30a and 30b (i.e., the upper portions of the trough member 30) discussed in this Example. Specifically, the operating parameters associated with trough member 30a were used to manufacture a zinc-based nanoparticle/nanoparticle solution; whereas the operating parameters associated with trough member 30b were used to manufacture a silver-based nanoparticle/nanoparticle solution. Once these silver-based and zinc-based solutions were manufactured, they were separately collected from each trough member 30a and 30b and were not mixed together until about 24 hours had passed. In this regard, each of the solutions made in 30a and 30b were collected at the outputs thereof and were not allowed to mix in the base portion 30o of the trough member 30, but were later mixed in another container.
Table 19c summarizes the electrode design, configuration, location and operating voltages for each of trough members 30a and 30b (i.e., the upper portions of the trough member 30) discussed in this Example. Specifically, the operating parameters associated with trough member 30a were used to manufacture a zinc-based nanoparticle/nanoparticle solution; whereas the operating parameters associated with trough member 30b were used to manufacture a silver-based nanoparticle/nanoparticle solution. Once these silver-based and zinc-based solutions were manufactured, they were mixed together substantially immediately at the point 30d and flowed to the base portion 30o and the mixture was subsequently processed in the base portion 30o of the trough member 30. In this regard, Table 19c shows the additional processing conditions associated with the base portion 30o of the trough member 30. Specifically, once again, electrode design, configuration, location and operating voltages are shown.
Table 19d shows a summary of the physical and biological characterization of the materials made in accordance with this Example 15.
This Example provides a spectrographic analysis of various adjustable plasmas 4, all of which were formed in air, according to the teachings of the inventive concepts disclosed herein. Example 9 herein utilized a single spectrometer (i.e., photon control silicon CCD Spectrometer 500) to analyze a variety of plasmas (i.e., collect spectral information in the 200-1090 nm range), including spectral information for plasmas made in different atmospheres. In this Example, three different spectrometers having greater sensitivities than the spectrometer used in Example 9 were used to collect similar spectral information. Further, spectrographic analysis was conducted on several plasmas, wherein the electrode member 1 comprised a variety of different metal compositions. Different species in the plasmas 4, as well as different intensities of some of the species, were observed. The presence/absence of such species can affect (e.g., positively and negatively) processing parameters and products made according to the teachings herein.
In this regard,
Specifically, the experimental setup for collecting plasma emission data (e.g., irradiance) is depicted in
The assembly 524 contained one UV collimator (LC-10U) with a refocusing assembly (LF-10U100) for the 170-2400 nm range. The assembly 524 also included an SMA female connector made by Multimode Fiber Optics, Inc. Each LC-10U and LF-10U100 had one UV fused silica lens associated therewith. Adjustable focusing was provided by LF-10U100 at about 100 mm from the vortex of the lens in LF-10U100 also contained in the assembly 524.
The collimator field of view at both ends of the adjustable plasma 4 was about 1.5 mm in diameter as determined by a 455 μm fiber core diameter comprising the solarization resistant UV optical fiber 523 (180-900 nm range and made by Mitsubishi). The UV optical fiber 523 was terminated at each end by an SMA male connector (sold by Ocean Optics; QP450-1-XSR).
The UV collimator-fiber system 523 and 524 provided 180-900 nm range of sensitivity for plasma irradiance coming from the 1.5 mm diameter plasma cylinder horizontally oriented in different locations in the adjustable plasma 4.
The X-Z stage 525 comprised two linear stages (PT1) made by Thorlabs Inc., that hold and control movement of the UV collimator 524 along the X and Z axes. It is thus possible to scan the adjustable plasma 4 horizontally and vertically, respectively.
Emission of plasma radiation collected by UV collimator-fiber system 523, 524 was delivered to either of three fiber coupled spectrometers 520, 521 or 522 made by StellarNet, Inc. (i.e., EPP2000-HR for 180-295 nm, 2400 g/mm grating, EPP2000-HR for 290-400 nm, 1800 g/mm grating, and EPP2000-HR for 395-505 nm, 1200 g/mm grating). Each spectrometer 520, 521 and 522 had a 7 μm entrance slit, 0.1 nm optical resolution and a 2048 pixel CCD detector. Measured instrumental spectral line broadening is 0.13 nm at 313.1 nm.
Spectral data acquisition was controlled by SpectraWiz software for Windows/XP made by StellarNet. All three EPP2000-HR spectrometers 520, 521 and 522 were interfaced with one personal computer 528 equipped with 4 USB ports. The integration times and number of averages for various spectral ranges and plasma discharges were set appropriately to provide unsaturated signal intensities with the best possible signal to noise ratios. Typically, spectral integration time was order of 1 second and number averaged spectra was in range 1 to 10. All recorded spectra were acquired with subtracted optical background. Optical background was acquired before the beginning of the acquisition of a corresponding set of measurements each with identical data acquisition parameters.
Each UV fiber-spectrometer system (i.e., 523/520, 523/521 and 523/522) was calibrated with an AvaLight-DH-CAL Irradiance Calibrated Light Source, made by Avantes (not shown). After the calibration, all acquired spectral intensities were expressed in (absolute) units of spectral irradiance (mW/m2/nm), as well as corrected for the nonlinear response of the UV-fiber-spectrometer. The relative error of the AvaLight-DH-CAL Irradiance Calibrated Light Source in 200-1100 nm range is not higher than 10%.
Alignment of the field of view of the UV collimator assembly 524 relative to the tip 9 of the metal electrode 1 was performed before each set of measurements. The center of the UV collimator assembly 524 field of view was placed at the tip 9 by the alignment of two linear stages and by sending a light through the UV collimator-fiber system 523, 524 to the center of each metal electrode 1.
The X-Z stage 525 was utilized to move the assembly 524 into roughly a horizontal, center portion of the adjustable plasma 4, while being able to move the assembly 524 vertically such that analysis of the spectral emissions occurring at different vertical heights in the adjustable plasma 4 could be made. In this regard, the assembly 524 was positioned at different heights, the first of which was located as close as possible of the tip 9 of the electrode 1, and thereafter moved away from the tip 9 in specific amounts. The emission spectroscopy of the plasma often did change as a function of interrogation position, as shown in
For example,
Table 20a shows specifically each of the spectral lines identified in the adjustable plasma 4 when a silver electrode 1 was utilized to create the plasma 4.
A variety of similar species associated with each metallic electrode composition plasma are identified in Tables 20a-20d. These species include, for example, the various metal(s) from the electrodes 1, as well as common species including, NO, OH, N2, etc. It is interesting to note that some species' existence and/or intensity (e.g., amount) is a function of location within the adjustable plasma. Accordingly, this suggests that various species can be caused to occur as a function of a variety of processing conditions (e.g., power, location, composition of electrode 1, etc.) of the invention.
Moreover, the plasma electron temperatures (see
AgI4d10(1S)5s2S1/2-4d10(1S)5p2P03/2
AgI4d10(1S)5s2S1/2-4d10(1S)5p2P01/2
AgI4d10(1S)5p2P01/2-4d10(1S)5d2D3/2
AgI4d10(1S)5p2P03/2-4d10(1S)5d2D5/2
Spectral line intensities used in all temperature measurements are given in units of spectral irradiance (mW/m2/nm) after the irradiance calibration of the spectrometers was performed.
The materials disclosed in Examples 11 and 12, namely, AT-060 and BT-06, were mixed together in varying proportions to form several different solutions to determine if any differences in zeta potential could be observed as a function of volumetric proportions in the various mixtures.
In this Example, a Zeta-Sizer “Nano-ZS” produced by Malvern Instruments was utilized to determine the zeta potential of each solution. For each measurement, a 1 ml sample was filled into clear disposable zeta cell DTS1060C. Dispersion Technology Software, version 5.10 was used to run the Zeta-Sizer and to calculate the zeta potential. The following settings were used: dispersant—water, temperature—25° C., viscosity—0.8872 cP, refraction index—1.330, dielectric constant—78.5, approximation model—Smoluchowski. One run of hundred repetitions was performed for each sample.
“Zeta potential” is known as a measure of the electro-kinetic potential in colloidal systems. Zeta potential is also referred to as surface charge on particles. Zeta potential is also known as the potential difference that exists between the stationary layer of fluid and the fluid within which the particle is dispersed. A zeta potential is often measured in millivolts (i.e., mV). The zeta potential value of approximately 25 mV is an arbitrary value that has been chosen to determine whether or not stability exists between a dispersed particle in a dispersion medium. Thus, when reference is made herein to “zeta potential”, it should be understood that the zeta potential referred to is a description or quantification of the magnitude of the electrical charge present at the double layer.
The zeta potential is calculated from the electrophoretic mobility by the Henry equation:
where z is the zeta potential, UE is the electrophoretic mobility, ε is a dielectric constant, η is a viscosity, ƒ(ka) is Henry's function. For Smoluchowski approximation ƒ(ka)=1.5.
Electrophoretic mobility is obtained by measuring the velocity of the particles in applied electric field using Laser Doppler Velocimetry (LDV). In LDV the incident laser beam is focused on a particle suspension inside a folded capillary cell and the light scattered from the particles is combined with the reference beam. This produces a fluctuating intensity signal where the rate of fluctuation is proportional to the speed of the particles, i.e. electrophoretic mobility.
As Table 21a below indicates, AT-060, BT-06 and DI water were mixed in different proportions and the zeta potential was measured right after mixing and one day after mixing. The results for zeta potential are shown in the table below. A clear trend exists for zeta potential of Ag:Zn 4:0 (−28.9) to Ag:Zn 0:4 (+22.7).
As a comparison, zinc sulfate heptahydrate (ZnSO47H2O) having a formula weight of 287.58 was added in varying quantities to the AT-060 solution to determine if a similar trend in zeta potential change could be observed for different amounts of zinc sulfate being added. The zinc sulfate heptahydrate was obtained from Fisher Scientific, had a Product # of Z68-500, a Cas # of 7446-20-0 and a Lot # of 082764. After mixing, the zeta potential of the AT-060/ZnSO47H2O mixture was measured. The data were very mixed and no clear trends in changes in zeta potential were evident.
The biological efficacy of seven different solutions made according to the inventive teachings herein, were tested for efficacy against a variety of bacteria and fungi.
The biological efficacy measurements made in Example 18 are different from those discussed earlier herein (e.g., the biological characterization discussed relative to Examples 1-5). Specifically, MIC/MID 50 levels were determined for each of the seven different solutions. The clinical and laboratory standards institute Broth Microdilution Methodology was employed, however, the growth medium used was an “RPMI” medium.
Additionally, the methods for dilution and antimicrobial susceptibility tests for bacteria that grow aerobically were also followed with the noted exception of testing with alternative media (CLSI document M7A7, CLSI, Wayne, Pa.).
The seven different solutions tested for efficacy were GR-05, GR-08, GR-21, GR-01, GR-24, GR-25 and GR-26. The solutions GR-05, GR-08 and GR-01 were previously discussed herein in conjunction with Examples 1-5. The solutions GR-24, GR-25 and GR-26 correspond to different mixtures of the same components used to form GR-05. In this regard, the volumetric proportions of GR-24 were 40% Ag/60% Zn; the volumetric proportions for GR-25 were 50% Ag/50% Zn; and the volumetric proportions for GR-26 were 60% Ag/40% Zn. Solution GR-21 corresponded to GR-08 for its Ag solution, but the Zn solution was replaced with an equivalent amount of solution PT001 made in accordance with the teachings in Example 11.
Table 22a shows results of the seven different solutions against a variety of bacteria. Under the column, “Isolate” identification beginning with either “GP” or “GN” occur. The “GP” corresponds to Gram-Positive bacteria and the “GN” corresponds to “Gram Negative” bacteria. Each of the organisms are specifically listed after the isolate identification. Table 22b shows the same testing results, but reported in a different way.
Staphylococcus aureus ATCC 29213 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 43300 - methicillin-resistant control strain
Staphylococcus aureus - methicillin-resistant clinical isolate
Staphylococcus aureus - multi-drug-resistant clinical isolate
Staphylococcus aureus - teicoplanin-intermediate clinical isolate
Staphylococcus epidermidis antibiotic susceptible clinical isolate
Staphylococcus epidermidis methicillin-resistant clinical isolate
Staphylococcus haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus saprophyticus - antibiotic susceptible clinical isolate
Enterococcus faecalis - ATCC 29212 antibiotic-susceptible control strain
Enterococcus faecalis vancomycin - susceptible clinical isolate
Enterococcus faecalis vancomycin - resistant (VanA) clinical isolate **
Enterococcus faecalis vancomycin - resistant (VanB) clinical isolate **
Enterococcus faecalis high-level gentamicin-resistant clinical isolate **
Enterococcus faecium vancomycin-susceptible clinical isolate
Enterococcus faecium vancomycin-resistant (VanA) clinical isolate
Enterococcus faecium vancomycin-resistant (VanB) clinical isolate
Enterococcus gallinarum vancomycin-resistant (VanC) clinical isolate
Streptococcus pneumoniae - ATCC 49619 antibiotic-susceptible control strain
Streptococcus pneumoniae - pencillin-susceptible clinical isolate
Streptococcus pneumoniae - penicillin-intermediate clinical isolate
Streptococcus pneumoniae - penicillin-resistant clinical isolate
Streptococcus pneumoniae - multi-drug resistant clinical isolate
Streptococcus pyogenes - antibiotic-susceptible clinical isolate @@
Streptococcus pyogenes - Macrolide (MLS) resistant clinical isolate
Streptococcus pyogenes - Macrolide (M-type) resistance clinical isolate::
Corynebacterium jeikeium - antibiotic-susceptible clinical isolate::
Corynebacterium jeikeium - multi-drug resistant clinical isolate::
Listeria monocytogenes - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - macrolide-resistant clinical isolate
Streptococcus mitis - antibiotic-susceptible clinical isolate::
Streptococcus mitis - macrolide-resistant clinical isolate
Streptococcus
constellatus - antibiotic-susceptible clinical isolate::
Streptococcus
constellatus - macrolide-resistant clinical isolate::
Streptococcus oralis - antibiotic-susceptible clinical isolate
Streptococcus oralis - macrolide-resistant clinical isolate
Streptococcus bovis - antibiotic-susceptible clinical isolate
Streptococcus bovis - macrolide-resistant clinical isolate
Streptococcus sanguis - antibiotic-susceptible clinical isolate
Streptococcus sanguis - macrolide-resistant clinical isolate
Clostridium perfringens - antibiotic-susceptible clinical isolate ##
Escherichia coli ATCC25922 - antibiotic-susceptible type strain
Escherichia coli ATCC35218 - β-lactamase positive type strain
Escherichia coli - multi-drug resistant clinical isolate
Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain
Klebsiella aerogenes - multi-drug resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Serratia marcescens - antibiotic-susceptible clinical isolate
Serratia marcescens - multi-drug resistant clinical isolate
Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain
Pseudomonas aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate
Stenotrophomonas maltophila - antibiotic-resistant clinical isolate
Burkholderia cepacia - antibiotic-susceptible clinical isolate
Proteus mirabilis - antibiotic-susceptible clinical isolate
Proteus mirabilis - multi-drug resistant clinical isolate
Salmonella sp - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Shigella sp - multi-drug resistant clinical isolate
Morganella morganii - antibiotic-susceptible clinical isolate ∧∧
Morganella morganii - multi-drug resistant clinical isolate **
Providencia stuartii - antibiotic-susceptible clinical isolate
Providencia stuartii - multi-drug resistant clinical isolate **
Neisseria meningitidis - antibiotic-susceptible clinical isolate susceptible &&
Haemophilus influenzae β- lactamase negative clinical isolate ++
Haemophilus influenzae β- lactamase positive clinical isolate ++
Haemophilus influenzae β- lactamase negative ampicillin-resistant clinical isolate ++
Moraxella catarrhalis β- lactamase positive clinical isolate
Moraxella catarrhalis β- lactamase positive clinical isolate
Acinetobacter baumanii - antibiotic-susceptible clinical isolate **
Acinetobacter baumanii - multi-drug resistant clinical isolate **
Bacteroides fragilis - antibiotic-susceptible clinical isolate ##
Citrobacter freundii - antibiotic-susceptible clinical isolate
Citrobacter freundii - resistant clinical isolate
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Staphylococcus aureus ATCC 29213 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 43300 - methicillin-resistant control strain
Staphylococcus aureus - methicillin-resistant clinical isolate
Staphylococcus aureus - multi-drug-resistant clinical isolate
Staphylococcus aureus - teicoplanin-intermediate clinical isolate
Staphylococcus epidermidis antibiotic susceptible clinical isolate
Staphylococcus epidermidis methicillin-resistant clinical isolate
Staphylococcus haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus saprophyticus - antibiotic susceptible clinical isolate
Enterococcus faecalis - ATCC 29212 antibiotic-susceptible control strain
Enterococcus faecalis vancomycin-susceptible clinical isolate
Enterococcus faecalis vancomycin-resistant (VanA) clinical isolate **
Enterococcus faecalis vancomycin-resistant (VanB) clinical isolate **
Enterococcus faecalis high-level gentamicin-resistant clinical isolate **
Enterococcus faecium vancomycin-susceptible clinical isolate
Enterococcus faecium vancomycin-resistant (VanA) clinical isolate
Enterococcus faecium vancomycin-resistant (VanB) clinical isolate
Enterococcus gallinarum vancomycin-resistant (VanC) clinical isolate
Streptococcus pneumoniae - ATCC 49619 antibiotic-susceptible control strain
Streptococcus pneumoniae - pencillin-susceptible clinical isolate
Streptococcus pneumoniae - penicillin-intermediate clinical isolate
Streptococcus pneumoniae - penicillin-resistant clinical isolate
Streptococcus pneumoniae - multi-drug resistant clinical isolate
Streptococcus pyogenes - antibiotic-susceptible clinical isolate @@
Streptococcus pyogenes - Macrolide (MLS) resistant clinical isolate
Streptococcus pyogenes - Macrolide (M-type) resistance clinical isolate::
Corynebacterium jeikeium - antibiotic-susceptible clinical isolate::
Corynebacterium jeikeium - multi-drug resistant clinical isolate::
Listeria monocytogenes - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - macrolide-resistant clinical isolate
Streptococcus mitis - antibiotic-susceptible clinical isolate::
Streptococcus mitis - macrolide-resistant clinical isolate
Streptococcus
constellatus - antibiotic-susceptible clinical isolate::
Streptococcus
constellatus - macrolide-resistant clinical isolate::
Streptococcus oralis - antibiotic-susceptible clinical isolate
Streptococcus oralis - macrolide-resistant clinical isolate
Streptococcus bovis - antibiotic-susceptible clinical isolate
Streptococcus bovis - macrolide-resistant clinical isolate
Streptococcus sanguis - antibiotic-susceptible clinical isolate
Streptococcus sanguis - macrolide-resistant clinical isolate
Clostridium perfringens - antibiotic-susceptible clinical isolate ##
Escherichia coli ATCC25922 - antibiotic-susceptible type strain
Escherichia coli ATCC35218 -β-lactamase positive type strain
Escherichia coli - multi-drug resistant clinical isolate
Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain
Klebsiella aerogenes - multi-drug resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Serratia marcescens - antibiotic-susceptible clinical isolate
Serratia marcescens - multi-drug resistant clinical isolate
Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain
Pseudomonas aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate
Stenotrophomonas maltophila - antibiotic-resistant clinical isolate
Burkholderia cepacia - antibiotic-susceptible clinical isolate
Proteus mirabilis - antibiotic-susceptible clinical isolate
Proteus mirabilis - multi-drug resistant clinical isolate
Salmonella sp - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Shigella sp - multi-drug resistant clinical isolate
Morganella morganii - antibiotic-susceptible clinical isolate ∧∧
Morganella morganii - multi-drug resistant clinical isolate **
Providencia stuartii - antibiotic-susceptible clinical isolate
Providencia stuartii - multi-drug resistant clinical isolate **
Neisseria meningitidis - antibiotic-susceptible clinical isolate susceptible &&
Haemophilus influenzae β-lactamase negative clinical isolate ++
Haemophilus influenzae β-lactamase positive clinical isolate ++
Haemophilus influenzae β-lactamase negative ampicillin-resistant clinical
Moraxella catarrhalis β-lactamase positive clinical isolate
Moraxella catarrhalis β-lactamase positive clinical isolate
Acinetobacter baumanii - antibiotic-susceptible clinical isolate **
Acinetobacter baumanii - multi-drug resistant clinical isolate **
Bacteroides fragilis - antibiotic-susceptible clinical isolate ##
Citrobacter freundii - antibiotic-susceptible clinical isolate
Citrobacter freundii - resistant clinical isolate
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Staphylococcus aureus ATCC 29213- antibiotic-susceptible control strain
Staphylococcus aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 43300 - methicillin-resistant control strain
Staphylococcus aureus - methicillin-resistant clinical isolate
Staphylococcus aureus - multi-drug-resistant clinical isolate
Staphylococcus aureus - teicoplanin-intermediate clinical isolate
Staphylococcus epidermidis antibiotic susceptible clinical isolate
Staphylococcus epidermidis methicillin-resistant clinical isolate
Staphylococcus haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus saprophyticus - antibiotic susceptible clinical isolate
Enterococcus faecalis - ATCC 29212 antibiotic-susceptible control strain
Enterococcus faecalis vancomycin-suscePtible clinical isolate
Enterococcus faecalis vancomycin-resistant (VanA) clinical isolate **
Enterococcus faecalis vancomycin-resistant (VanB) clinical isolate **
Enterococcus faecalis high-level gentamicin-resistant clinical isolate **
Enterococcus faecium vancomycin-susceptible clinical isolate
Enterococcus faecium vancomycin-resistant (VanA) clinical isolate
Enterococcus faecium vancomycin-resistant (VanB) clinical isolate
Enterococcus gallinarum vancomycin-resistant (VanC) clinical isolate
Streptococcus pneumoniae - ATCC 49619 antibiotic-susceptible control strain
Streptococcus pneumoniae - pencillin-susceptible clinical isolate
Streptococcus pneumoniae - penicillin-intermediate clinical isolate
Streptococcus pneumoniae - penicillin-resistant clinical isolate
Streptococcus pneumoniae - multi-drug resistant clinical isolate
Streptococcus pyogenes - antibiotic-susceptible clinical isolate @@
Streptococcus pyogenes - Macrolide (MLS) resistant clinical isolate
Streptococcus pyogenes - Macrolide (M-type) resistance clinical isolate::
Corynebacterium jeikeium - antibiotic-susceptible clinical isolate::
Corynebacterium jeikeium - multi-drug resistant clinical isolate::
Listeria monocytogenes - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - macrolide-resistant clinical isolate
Streptococcus mitis - antibiotic-susceptible clinical isolate::
Streptococcus mitis - macrolide-resistant clinical isolate
Streptococcus
constellatus - antibiotic-susceptible clinical isolate::
Streptococcus
constellatus - macrolide-resistant clinical isolate::
Streptococcus oralis - antibiotic-susceptible clinical isolate
Streptococcus oralis - macrolide-resistant clinical isolate
Streptococcus bpvis - antibiotic-susceptible clinical isolate
Streptococcus bovis - macrolide-resistant clinical isolate
Streptococcus sanguis - antibiotic-susceptible clinical isolate
Streptococcus sanguis - macrolide-resistant clinical isolate
Clostridium perfringens - antibiotic-susceptible clinical isolate ##
Escherichia coli ATCC25922 - antibiotic-susceptible type strain
Escherichia coli ATCC35218 -β-lactamase positive type strain
Escherichia coli - multi-drug resistant clinical isolate
Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain
Klebsiella aerogenes - multi-drug resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Serratia marcescens - antibiotic-susceptible clinical isolate
Serratia marcescens - multi-drug resistant clinical isolate
Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain
Pseudomonas aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate
Stenotrophomonas maltophila - antibiotic-resistant clinical isolate
Burkholderia cepacia - antibiotic-susceptible clinical isolate
Proteus mirabilis - antibiotic-susceptible clinical isolate
Proteus mirabilis - multi-drug resistant clinical isolate
Salmonella sp - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Shigella sp - multi-drug resistant clinical isolate
Morganella morganii - antibiotic-susceptible clinical isolate ∧∧
Morganella morganii - multi-drug resistant clinical isolate **
Providencia stuartii - antibiotic-susceptible clinical isolate
Providencia stuartii - multi-drug resistant clinical isolate **
Neisseria meningitidis - antibiotic-susceptible clinical isolate susceptible &&
Haemophilus influenzae β-lactamase negative clinical isolate ++
Haemophilus influenzae β-lactamase positive clinical isolate ++
Haemophilus influenzae β-lactamase negative ampicillin-resistant clinical
Moraxella catarrhalis β-lactamase positive clinical isolate
Moraxella catarrhalis β-lactamase positive clinical isolate
Acinetobacter baumanii - antibiotic-susceptible clinical isolate **
Acinetobacter baumanii - multi-drug resistant clinical isolate **
Bacteroides fragilis - antibiotic-susceptible clinical isolate ##
Citrobacter freundii - antibiotic-susceptible clinical isolate
Citrobacter freundii - resistant clinical isolate
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Staphylococcus aureus ATCC 29213 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus aureus ATCC 43300 - methicillin-resistant control strain
Staphylococcus aureus - methicillin-resistant clinical isolate
Staphylococcus aureus - multi-drug-resistant clinical isolate
Staphylococcus aureus - teicoplanin-intermediate clinical isolate
Staphylococcus epidermidis antibiotic susceptible clinical isolate
Staphylococcus epidermidis methicillin-resistant clinical isolate
Staphylococcus haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus saprophyticus - antibiotic susceptible clinical isolate
Enterococcus faecalis - ATCC 29212 antibiotic-susceptible control strain
Enterococcus faecalis vancomycin-susceptible clinical isolate
Enterococcus faecalis vancomycin-resistant (VanA) clinical isolate **
Enterococcus faecalis vancomycin-resistant (VanB) clinical isolate **
Enterococcus faecalis high-level gentamicin-resistant clinical isolate **
Enterococcus faecium vancomycin-susceptible clinical isolate
Enterococcus faecium vancomycin-resistant (VanA) clinical isolate
Enterococcus faecium vancomycin-resistant (VanB) clinical isolate
Enterococcus gallinarum vancomycin-resistant (VanC) clinical isolate
Streptococcus pneumoniae - ATCC 49619 antibiotic-susceptible control strain
Streptococcus pneumoniae - pencillin-susceptible clinical isolate
Streptococcus pneumoniae - penicillin-intermediate clinical isolate
Streptococcus pneumoniae - penicillin-resistant clinical isolate
Streptococcus pneumoniae - multi-drug resistant clinical isolate
Streptococcus pyogenes - antibiotic-susceptible clinical isolate @@
Streptococcus pyogenes - Macrolide (MLS) resistant clinical isolate
Streptococcus pyogenes - Macrolide (M-type) resistance clinical isolate::
Corynebacterium jeikeium - antibiotic-susceptible clinical isolate::
Corynebacterium jeikeium - multi-drug resistant clinical isolate::
Listeria monocytogenes - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - antibiotic-susceptible clinical isolate
Streptococcus agalactiae - macrolide-resistant clinical isolate
Streptococcus mitis - antibiotic-susceptible clinical isolate::
Streptococcus mitis - macrolide-resistant clinical isolate
Streptococcus
constellatus - antibiotic-susceptible clinical isolate::
Streptococcus
constellatus - macrolide-resistant clinical isolate::
Streptococcus oralis - antibiotic-susceptible clinical isolate
Streptococcus oralis - macrolide-resistant clinical isolate
Streptococcus bovis - antibiotic-susceptible clinical isolate
Streptococcus bovis - macrolide-resistant clinical isolate
Streptococcus sanguis - antibiotic-susceptible clinical isolate
Streptococcus sanguis - macrolide-resistant clinical isolate
Clostridium perfringens - antibiotic-susceptible clinical isolate ##
Escherichia coli ATCC25922 - antibiotic-susceptible type strain
Escherichia coli ATCC35218 -β-lactamase positive type strain
Escherichia coli - multi-drug resistant clinical isolate
Klebsiella aerogenes NCTC11228 - antibiotic-susceptible type strain
Klebsiella aerogenes - multi-drug resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Serratia marcescens - antibiotic-susceptible clinical isolate
Serratia marcescens - multi-drug resistant clinical isolate
Pseudomonas aeruginosa ATCC27853 - antibiotic-susceptible type strain
Pseudomonas aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas maltophilia - antibiotic-susceptible clinical isolate
Stenotrophomonas maltophila - antibiotic-resistant clinical isolate
Burkholderia cepacia - antibiotic-susceptible clinical isolate
Proteus mirabilis - antibiotic-susceptible clinical isolate
Proteus mirabilis - multi-drug resistant clinical isolate
Salmonella sp - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Shigella sp - multi-drug resistant clinical isolate
Morganella morganii - antibiotic-susceptible clinical isolate ∧∧
Morganella morganii - multi-drug resistant clinical isolate **
Providencia stuartii - antibiotic-susceptible clinical isolate
Providencia stuartii - multi-drug resistant clinical isolate **
Neisseria meningitidis - antibiotic-susceptible clinical isolate susceptible &&
Haemophilus influenzae β-lactamase negative clinical isolate ++
Haemophilus influenzae β-lactamase positive clinical isolate ++
Haemophilus influenzae βlactamase negative ampicillin-resistant clinical
Moraxella catarrhalis β-lactamase positive clinical isolate
Moraxella catarrhalis β-lactamase positive clinical isolate
Acinetobacter baumanii - antibiotic-susceptible clinical isolate **
Acinetobacter baumanii - multi-drug resistant clinical isolate **
Bacteroides fragilis - antibiotic-susceptible clinical isolate ##
Citrobacter freundii - antibiotic-susceptible clinical isolate
Citrobacter freundii - resistant clinical isolate
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
Clostridium difficile ==
∧∧ NG IN RPMI ALONE
Citrobacter
freundii - antibiotic-susceptible clinical isolate
Stenotrophomonas
maltophilia - antibiotic-susceptible clinical isolate
Acinetobacter
baumanii - multi-drug resistant clinical isolate ##
Burkholderia
cepacia - antibiotic-susceptible clinical isolate
Pseudomonas
aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas
maltophila - antibiotic-resistant clinical isolate
Acinetobacter
baumanii - antibiotic-susceptible clinical isolate ##
Moraxella
catarrhalis β-lactamase positive clinical isolate
Moraxella
catarrhalis β-lactamase positive clinical isolate
Shigella sp - multi-drug resistant clinical isolate ##
Enterococcus
faecium vancomycin-resistant (VanB) clinical isolate
Listeria
monocytogenes - antibiotic-susceptible clinical isolate
Citrobacter
freundii - resistant clinical isolate
Providencia
stuartii - multi-drug resistant clinical isolate
Staphylococcus
aureus - multi-drug-resistant clinical isolate
Staphylococcus
aureus - teicoplanin-intermediate clinical isolate
Enterococcus
faecalis - ATCC 29212 antibiotic-susceptible control strain ##
Pseudomonas
aeruginosa ATCC27853 - antibiotic-susceptible type strain ##
Streptococcus
bovis - antibiotic-susceptible clinical isolate
Streptococcus
bovis - macrolide-resistant clinical isolate
Staphylococcus
haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus
saprophyticus - antibiotic susceptible clinical isolate
Staphylococcus
aureus ATCC 43300 - methicillin-resistant control strain ##
Staphylococcus
epidermidis methicillin-resistant clinical isolate
Staphylococcus
aureus - methicillin-resistant clinical isolate
Staphylococcus
aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus
aureus ATCC 29213 - antibiotic-susceptible control strain
Staphylococcus
epidermidis antibiotic susceptible clinical isolate
Providencia
stuartii - antibiotic-susceptible clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Streptococcus
pyogenes - Macrolide (MLS) resistant clinical isolate
Enterococcus
faecalis vancomycin-susceptible clinical isolate
Morganella
morganii - multi-drug resistant clinical isolate
Streptococcus
agalactiae - antibiotic-susceptible clinical isolate
Streptococcus
agalactiae - macrolide-resistant clinical isolate
Streptococcus
mitis - macrolide-resistant clinical isolate
Streptococcus
pneumoniae - multi-drug resistant clinical isolate
Streptococcus
pneumoniae - ATCC 49619 antibiotic-susceptible control strain ##
Streptococcus
pneumoniae - pencillin-susceptible clinical isolate
Streptococcus
sanguis - macrolide-resistant clinical isolate
Streptococcus
pneumoniae - penicillin-intermediate clinical isolate
Streptococcus
pneumoniae - penicillin-resistant clinical isolate
Streptococcus
sanguis - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Proteus
mirabilis - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Klebsiella
aerogenes NCTC11228 - antibiotic-susceptible type strain
Escherichia
coli ATCC25922 - antibiotic-susceptible type strain
Serratia
marcescens - multi-drug resistant clinical isolate
Enterococcus
gallinarum vancomycin-resistant (VanC) clinical isolate
Proteus
mirabilis - multi-drug resistant clinical isolate
Klebsiella
aerogenes - multi-drug resistant clinical isolate
Streptococcus
oralis - antibiotic-susceptible clinical isolate
Streptococcus
oralis - macrolide-resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Serratia
marcescens - antibiotic-susceptible clinical isolate
Citrobacter
freundii - antibiotic-susceptible clinical isolate
Stenotrophomonas
maltophilia - antibiotic-susceptible clinical isolate
Acinetobacter
baumanii - multi-drug resistant clinical isolate ##
Burkholderia
cepacia - antibiotic-susceptible clinical isolate
Pseudomonas
aeruginosa - multi-drug resistant clinical isolate
Stenotrophomonas
maltophila - antibiotic-resistant clinical isolate
Acinetobacter
baumanii - antibiotic-susceptible clinical isolate ##
Moraxella
catarrhalis β-lactamase positive clinical isolate
Moraxella
catarrhalis β-lactamase positive clinical isolate
Shigella sp - multi-drug resistant clinical isolate ##
Enterococcus
faecium vancomycin-resistant (VanB) clinical isolate
Listeria
monocytogenes - antibiotic-susceptible clinical isolate
Citrobacter
freundii - resistant clinical isolate
Providencia
stuartii - multi-drug resistant clinical isolate
Staphylococcus
aureus - multi-drug-resistant clinical isolate
Staphylococcus
aureus - teicoplanin-intermediate clinical isolate
Enterococcus
faecalis - ATCC 29212 antibiotic-susceptible control strain ##
Pseudomonas
aeruginosa ATCC27853 - antibiotic-susceptible type strain ##
Streptococcus
bovis - antibiotic-susceptible clinical isolate
Streptococcus
bovis - macrolide-resistant clinical isolate
Staphylococcus
haemolyticus - antibiotic susceptible clinical isolate
Staphylococcus
saprophyticus - antibiotic susceptible clinical isolate
Staphylococcus
aureus ATCC 43300 - methicillin-resistant control strain ##
Staphylococcus
epidermidis methicillin-resistant clinical isolate
Staphylococcus
aureus - methicillin-resistant clinical isolate
Staphylococcus
aureus ATCC 25923 - antibiotic-susceptible control strain
Staphylococcus
aureus ATCC 29213 - antibiotic-susceptible control strain
Staphylococcus
epidermidis antibiotic susceptible clinical isolate
Providencia
stuartii - antibiotic-susceptible clinical isolate
Shigella sp - antibiotic-susceptible clinical isolate
Streptococcus
pyogenes - Macrolide (MLS) resistant clinical isolate
Enterococcus
faecalis vancomycin-susceptible clinical isolate
Morganella
morganii - multi-drug resistant clinical isolate
Streptococcus
agalactiae - antibiotic-susceptible clinical isolate
Streptococcus
agalactiae - macrolide-resistant clinical isolate
Streptococcus
mitis - macrolide-resistant clinical isolate
Streptococcus
pneumoniae - multi-drug resistant clinical isolate
Streptococcus
pneumoniae - ATCC 49619 antibiotic-susceptible control strain ##
Streptococcus
pneumoniae - pencillin-susceptible clinical isolate
Streptococcus
sanguis - macrolide-resistant clinical isolate
Streptococcus
pneumoniae - penicillin-intermediate clinical isolate
Streptococcus
pneumoniae - penicillin-resistant clinical isolate
Streptococcus
sanguis - antibiotic-susceptible clinical isolate
Enterobacter sp - multi-drug resistant clinical isolate
Proteus
mirabilis - antibiotic-susceptible clinical isolate
Salmonella sp - multi-drug resistant clinical isolate
Klebsiella
aerogenes NCTC11228 - antibiotic-susceptible type strain
Escherichia
coli ATCC25922 - antibiotic-susceptible type strain
Serratia
marcescens - multi-drug resistant clinical isolate
Enterococcus
gallinarum vancomycin-resistant (VanC) clinical isolate
Proteus
mirabilis - multi-drug resistant clinical isolate
Klebsiella
aerogenes - multi-drug resistant clinical isolate
Streptococcus
oralis - antibiotic-susceptible clinical isolate
Streptococcus
oralis - macrolide-resistant clinical isolate
Enterobacter sp - antibiotic-susceptible clinical isolate
Serratia
marcescens - antibiotic-susceptible clinical isolate
Specifically, Table 22a reports results in terms of dilution amounts to achieve an MID/MIC 50. Accordingly, for example, the number “ 1/64” under GR-05 for GP01 means that the original GR-05 solution was diluted to 1/64th its potency to achieve and MID/MIC50 for Staphylococcus aureus ATCC-29213. The numbers under the columns “Levofloxacin” correspond to the amount of antibiotic in μg/ml required to achieve a similar MID/MIC 50.
In contrast, the numbers reported in Table 22b are all reported in μg/ml. Additionally, the relative efficacy levels for three of the test solutions relative to Levofloxacin are also reported. Wherever the reported number is 1.0 or greater, it means that the test solution was as good as, or better, than the known antibiotic. Accordingly, a number of “1.5” means that when the ppm of the solution is converted to “μg/ml” and the number of μg/ml (i.e., from the converted ppm) is divided into the required μg/ml of the antibiotic needed to achieve an MIC/MID 50, 1.5 times as much antibiotic is needed to achieve the same effect. Thus, many of the test solutions significantly outperformed this antibiotic.
Table 22c uses a format similar to that used for Table 22a, however, the test solutions were tested against a variety of fungi. Again, the test solutions were significantly diluted to achieve MID/MIC 50 values (e.g., dilutions between ⅛th and 1/128th), showing that the test solutions also have significant efficacy against fungi.
Alternaria altemata 48 hrs §§
Aspergillus flavus 48 hrs §§
Aspergillus fumigatus 48 hrs §§
Aspergillus terreus 48 hrs §§
Candida glabrata - 24 hrs
Candida glabrata - 48 hrs
Candida lusitaniae - 24 hrs
Candida lusitaniae 48 hrs §§
Candida parapsilosis - 24 hrs
Candida parapsilosis 48 hrs §§
Epidermophyton floccosum -
Fusarium oxysporum - 48 hrs
Mucor circinelloides - 48 hrs
Penicillium chrysogenum 48 hrs §§
Rhizopus oligosporus - 24 hrs
Trychophyton interdigitale 72 hrs
Trichophyton mentagrophytes
Trichophyton rubrum 48 hrs §§
Trichophyton cutaneum 48 hrs §§
Alternaria altemata - 48 hrs §§
Aspergillus flavus - 48 hrs §§
Aspergiflus fumigatus - 48 hrs §§
Aspergillus terreus - 48 hrs §§
Candida glabrata - 24 hrs
Candida glabrata - 48 hrs
Candida lusitaniae - 24 hrs
Candida lusitaniae - 48 hrs §§
Candida parapsilosis - 24 hrs
Candida parapsilosis - 48 hrs §§
Epidermophyton floccosum -
Fusarium oxysporum - 48 hrs
Mucor circinelloides - 48 hrs
Penicillium chrysogenum 48 hrs §§
Rhizopus oligosporus - 24 hrs
Trychophyton interdigitale
Trichophyton mentagrophytes -
Trichophyton rubrum 48 hrs §§
Trichophyton cutaneum 48 hrs §§
The purpose of this Example was to evaluate the antiviral properties of two solutions, GR05 and GR-08 against duck Hepatitis B virus (i.e., as a surrogate virus for the human Hepatitis B virus) when exposed (in suspension) for the specified exposure period. The protocol utilized was a modification of the Standard Test Method for Efficacy of Virucidal Agents Intended for Special Applications (ASTM E1052).
The LeGarth strain of duck Hepatitis B virus (DHBV) used for this study was obtained commercially from Hepadnavirus Testing Inc., Palo Alto, Calif. and consisted of duck Hepatitis B virus serum obtained from congenitally infected ducklings. Virus aliquots were maintained at ≤−70° C. On the day of use, two aliquots were removed, thawed, combined and refrigerated or stored on ice until used in the assay.
A suspension of primary duck hepatocytes was achieved following an in situ perfusion of the duck liver. The hepatocytes were seeded into sterile disposable tissue culture labware, maintained at 36-38° C. in a humidified atmosphere of 5-7% CO2 and used at the appropriate density. Only ducklings verified to be free of test virus were utilized in the assay.
The test medium used in this study was Leibovitz L-15 medium supplemented with 0.1% glucose, 10 μM dexamethasone, 10 μg/mL insulin, 20 mM HEPES, 10 μg/mL gentamicin and 100 units/mL penicillin.
Table 23a lists the test and control groups, the dilutions assayed, and the number of cultures used.
A 4.5 mL aliquot of each of GR-05 and GR-08 was dispensed into separate sterile 15 mL conical tubes and mixed with a 0.5 mL aliquot of the stock virus suspension. The mixtures were vortex mixed for a minimum of 10 seconds and held for the remainder of the specified exposure times at 37.0° C. The exposure times assayed was six hours. Immediately following each exposure time, a 0.5 mL aliquot was removed from each tube and the mixtures were tittered by 10-fold serial dilutions (0.5 mL+4.5 mL test medium) and assayed for the presence of virus.
A 0.5 mL aliquot of stock virus suspension was exposed to a 4.5 mL aliquot of test medium in lieu of test substance and treated as previously described. Immediately following each exposure time, a 0.5 mL aliquot was removed from the tube and the mixture was titered by 10-fold serial dilutions (0.5 mL+4.5 mL test medium) and assayed for the presence of virus. All controls employed the FBS neutralizer as described in the Treatment of Virus Suspension section. A virus control was performed for each exposure time. The virus control titer was used as a baseline to compare the percent and log reductions of each test parameter following exposure to the test substances.
A 4.5 mL aliquot of each concentration of test substance was mixed with 0.5 mL aliquot of test medium in lieu of virus and treated as previously described. The cytotoxicity of the cell cultures was scored at the same time as virus-test substance and virus control cultures. Cytotoxicity was graded on the basis of cell viability as determined microscopically. Cellular alterations due to toxicity were graded and reported toxic (“T”) if greater than or equal to 50% of the monolayer was affected.
Each cytotoxicity control mixture (above) was challenged with low titer stock virus to determine the dilution(s) of test substance at which virucidal activity, if any was retained. Dilutions that showed virucidal activity were not considered in determining reduction of the virus by the test substance.
As previously described, 0.1 mL of each test and control parameter following the exposure period was added to fetal bovine serum (0.9 mL) followed immediately by 10-fold serial dilutions in test medium to stop the action of the test substance. To determine if the neutralizer chosen for the assay was effective in diminishing the virucidal activity of the test substance, low titer stock virus was added to each dilution of the test substance-neutralizer mixture. This mixture was assayed for the presence of the virus (neutralization control above).
Primary duck hepatocytes were used as the indicator cell line in the infectivity assays. Cells contained in cell culture labware were inoculated in quadruplicate with 1.0 mL of the dilutions prepared from the input virus control, virus control and test substances. The cytotoxicity and neutralization control dilutions were inoculated in duplicate. Uninfected indicator cell cultures (negative cell controls) were inoculated with test medium alone. A 2.0 mL aliquot of test medium was added to each cell culture well. The inoculum was allowed to adsorb overnight at 36-38° C. in a humidified atmosphere of 5-7% CO2. Following the adsorption period, a 3.0 mL aliquot of test medium was added to each cell culture well. The cultures were incubated at 36-38° C. in a humidified atmosphere of 5-7% CO2 for ten days. The test medium was aspirated from each test and control well and replaced with fresh medium as needed throughout the incubation period. On the final day of incubation, the cultures were scored microscopically for cytotoxicity and the cells were fixed with ethanol. An indirect immunofluorescence assay was then performed using a monoclonal antibody specific for the envelope protein of the DHBV.
Viral and cytotoxicity titers are expressed as −log10 of the 50 percent titration endpoint for infectivity (TCID50) or cytotoxicity (TCD50), respectively, as calculated by the method of Spearman Karber.
A valid test requires 1) that stock virus be recovered from the virus control, 2) that the cell controls be negative for virus, and 3) that negative cultures are viable.
Test substance cytotoxicity was not observed at any dilution assayed (≤1.5 log10). Under the conditions of this investigation, GR-05 and GR-08 demonstrated a ≥99.99% reduction in viral titer following a six hour exposure time to duck Hepatitis B virus. The log reduction in viral titer was ≥4.0 log10. Specifically, Table 23b sets forth the experimental results.
Table 23c sets forth the cytotoxicity and neutralization control results. As the date show, no cytotoxicity was measured for the GR-05 and GR-08 solutions.
Minimum Essential Medium (50 μl) supplemented according to Baltz et al. (1985) with 2-mercaptoethanol and 15% heat-inactivated horse serum was added to each well of a 96-well microtiter plate.
Serial drug dilutions were prepared covering a range from 90 to 0.123 μg/ml.
Then 104 bloodstream forms of Trypanosoma b. rhodesiense STIB 900 in 50 μl were added to each well and the plate incubated at 37° C. under a 5% CO2 atmosphere for 72 hours.
10 μl of Alamar Blue (12.5 mg resazurin dissolved in 100 mL distilled water) were then added to each well and incubation continued for a further 2-4 hours.
Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, Calif., USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm.
Data were analysed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, Calif., USA). Decrease of fluorescence (=inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations.
From the sigmoidal inhibition curves the IC50 values were calculated.
Cytotoxicity was assessed using the same assay and rat skeletal myoblasts (L-6 cells). The medium used for the L-6 cells was RPMI 1640 medium with 10% FBS and 2 mM L-glutamine.
Efficacy testing of 10 solutions against the Plasmodium falciparum (3D7 and Dd2 laboratory strains) occurred. The Anti-malarial activities of the ten solutions disclosed in Examples 1-5 (i.e., GR-01-GR-10) were investigated with the primary aim of identifying the most promising solution through in vitro efficacy testing. A second objective was to determine the anti-malarial activities of the same 10 solutions against two different strains of Plasmodium falciparum (3D7 and Dd2 laboratory strains) and to document any observable effect on the human erythrocytes used in the cultivation of the parasites.
The results show that all 10 solutions tested (i.e., GR-01-GR-10) had anti-malarial activity with the effects being dose dependent. GR 08 had the best anti-malarial activity as it had the lowest IC50 concentrating against both strains of parasites (i.e., 3.1 against 3D7; and 3.4 against Dd2) used in this study in comparison with the other solutions.
Two laboratory strains of malaria parasites, chloroquine sensitive (3D7) and chloroquine resistant (Dd2) were used for these in vitro studies. Parasites were cultivated using methods by Trager and Jensen (1976) with slight modifications. In brief, parasites were removed from liquid nitrogen and thawed in a water bath set at 37° C. and immediately centrifuged at 2000 rpm for 7 minutes and the supernatants were discarded. Equal volumes of thawing mix (3.5% NaCl in distilled water) were added and centrifuged as above and the supernatant discarded. The cells were then washed two times in parasite culture medium and the cells added to a culture flask containing 5 ml parasite culture medium (RPMI 1640, L-glutamine, Gentamycin and Albumax) and 200 μl of freshly washed human O+ red blood cells. The culture was then gassed for 30 seconds using a gas mixture containing Oxygen 2.0% Carbon dioxide 5.5% and the remainder Nitrogen. Cultures were maintained for at least two weeks continuously until a stable parasitaemia was obtained before being used for the efficacy assay.
Serial dilutions (2 fold) of each solution were prepared starting from 2 times dilution to 128 times dilution in parasite culture medium (RPMI 1640, L-glutamine, Gentamycin and Albumax). In other words, 100 μl of test solution was used per milliliter of culture mixture giving a start concentration of 100 μl test solution/ml of culture medium (100 μl/ml). They were prepared prior to the start of the assays and kept refrigerated until they were ready to be used.
The ten different solutions were investigated for their anti-malarial activities against two Plasmodium falciparum parasite strains (3D7 and Dd2). Briefly, parasites were prepared from in vitro cultivation as described above. Into each well of a 24-well culture plates was added 40 μl of O+ freshly washed RBC at 1.0% parasitaemia in 900 μl of complete parasite medium. Into each of the wells, 100 μl of the diluted test solutions (corresponding to 0.78 μl, 1.56 μl, 3.125 μl, 6.25 μl, 12.5 μl, 25 μl, 50 μl, and 100 μl of the undiluted solution), were added per ml of culture medium. Also included in each 24-well plate were wells containing 40 μl of uninfected RBC plus 100 μl of undiluted solution of each formulation and 40 μl of infected RBC (1.0%) without any of the ten test solutions. Assays were performed in triplicates. The plates were then placed in a modular incubator chamber (California, USA) and gassed for 10 minutes using a special gas mixture (Oxygen 2.0% Carbon dioxide 5.5% and Nitrogen 92.5%). The chamber containing the plates was incubated at 37° C. for 48 hours. At approximately 48 hours cultures were removed and thin blood films prepared from each well on double frosted microscope slides. The slides were air-dried, fixed in methanol and stained with 10% giemsa in phosphate buffer.
The anti-malarial activities of all 10 solutions evaluated are shown in
Each of the 10 solutions also inhibited the growth of chloroquine sensitive strain of P. falciparum (3D7) parasites. The highest concentration (100 μl test solution/ml) of the test solutions recorded a maximum inhibition ranging between 71% and 85% (
The growth inhibition characteristics of the ten solutions were similar to that observed for chloroquine (
The concentration that inhibited the growth of each strain of P. falciparum (3D7 and Dd2) by 50% (IC50) are presented in Table 25a. The IC50 values for the test solutions against chloroquine sensitive P. falciparum (3D7) parasites ranged from 3.1 μl/ml-6.2 μl/ml. For chloroquine sensitive P. falciparum (Dd2) parasites the IC50 ranged from 3.4 μl/ml-7.9 μl/ml. GR-08 recorded the lowest IC50 against both chloroquine sensitive and chloroquine resistant strains of the Plasmodium parasites.
Plasmodium falciparum (3D7- chloroquine-sensitive strain
There were anti-malarial activities for all 10 test solutions. The anti-malarial effects were dose dependent. The 10 test solutions did not show observable adverse effects on infected and uninfected RBCs. GR-08 had the lowest IC50 against both chloroquine-resistant and chloroquine sensitive strains of Plasmodium parasites.
This Example 22 demonstrates how the silver-based constituents in GR-05 bind to a lipid bilayer membrane. Briefly, large unilamellar vesicles were used as a membrane mimetic. Different amounts of vesicle solution were added to the GR-05 solution. After incubation of the mixture for about one hour, the vesicles were centrifugally spun down to a pellet, leaving unbound silver constituents in the supernatant. Next, the silver concentration (i.e., Ag ppm) in the supernatant was measured by the atomic absorbance spectrometer techniques discussed above herein. The measured concentration in the supernatant was compared to the silver concentration in the control solution, where no vesicles were added, to determine the amount of silver constituents from GR-05 that bound to the vesicles. Finally, the bound fraction of silver constituents was plotted against lipid concentration to determine the binding (equilibrium) constant.
Large unilamellar vesicles were prepared in the following manor: 50 mol % BrPC, 40 mol % POPC, 10 mol % POPG lipids, in original stock solution in chloroform, were mixed together and were dried under a flowing nitrogen stream. Lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.) and were used without further purification. POPC lipids (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine) are the most commonly used lipids for vesicle preparation. BrPC lipids (1,2-Dibromostearoyl-sn-Glycero-3-Phosphocholine) were used to make the vesicle bilayers more dense for easy centrifugal separation (i.e., spinning down). Negatively charged POPG lipids (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt)) were used to mimic the negative charged bilayer membranes of bacteria. After the lipids were mixed together, they were rehydrated in deionized water to achieve a 5 mM total lipid concentration; and were extruded multiple times through a 0.1 μm pore membrane (extruder and membranes were purchased from Avanti Polar Lipids, Inc., Alabaster, Ala.) thus forming large unilamellar vesicles.
The binding of lipids to silver constituents in GR-05 can be described in a first approximation with the following relationship:
where “α” is the number of lipids “L” that bind to a silver constituent in GR-05, thus forming a lipid-silver complex “LαAg”.
The binding constant, or equilibrium constant, K is given as:
where [LαAg] is a concentration of bound silver constant, [L] is the concentration of lipids, and [Ag] is the concentration of unbound silver constituent. Total silver concentration [TAg] equals [LαAg] plus [Ag] and fraction of bound silver constituent ƒB is given as:
While GR-05 also contains Zn-based constituents, as a first approximation, these were ignored for the purposes of this Example.
The present application is a divisional of U.S. application Ser. No. 14/010,721, filed on Aug. 27, 2013 (now U.S. Pat. No. 9,387,452), which is a divisional of U.S. application Ser. No. 12/686,815, filed on Jan. 13, 2010 (now U.S. Pat. No. 8,540,942). The aforementioned present application claims priority to U.S. Provisional Patent Application No. 61/144,625, filed on Jan. 14, 2009. All of the applications mentioned above are hereby expressly incorporated by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5478533 | Inculet | Dec 1995 | A |
| 5876663 | Laroussi | Mar 1999 | A |
| 5965994 | Seo et al. | Oct 1999 | A |
| 6093422 | Denkewicz et al. | Jul 2000 | A |
| 6214299 | Holladay et al. | Apr 2001 | B1 |
| 6358398 | Halldorson et al. | Mar 2002 | B1 |
| 6743348 | Holladay et al. | Jun 2004 | B2 |
| 6749759 | Denes et al. | Jun 2004 | B2 |
| 6802981 | Ryazanova et al. | Oct 2004 | B2 |
| 7033415 | Mirkin | Apr 2006 | B2 |
| 7118852 | Purdum | Oct 2006 | B2 |
| 7135054 | Jin | Nov 2006 | B2 |
| 7135055 | Mirkin et al. | Nov 2006 | B2 |
| 7135195 | Holladay et al. | Nov 2006 | B2 |
| 7276283 | Denes et al. | Oct 2007 | B2 |
| 7348882 | Goodwin et al. | Oct 2008 | B2 |
| 8088193 | Zeng et al. | Jan 2012 | B2 |
| 8540942 | Pierce et al. | Sep 2013 | B2 |
| 8617360 | Pierce et al. | Dec 2013 | B2 |
| 9067263 | Pierce et al. | Jun 2015 | B2 |
| 20020014400 | Zadiraka et al. | Feb 2002 | A1 |
| 20040022702 | Christensen | Feb 2004 | A1 |
| 20040131524 | Josephson et al. | Jul 2004 | A1 |
| 20060037177 | Blum et al. | Feb 2006 | A1 |
| 20060060464 | Chang | Mar 2006 | A1 |
| 20060068026 | Hu et al. | Mar 2006 | A1 |
| 20060249705 | Wang et al. | Nov 2006 | A1 |
| 20070080054 | Parkansky et al. | Apr 2007 | A1 |
| 20070108056 | Nyberg et al. | May 2007 | A1 |
| 20070267289 | Jabs et al. | Nov 2007 | A1 |
| 20080169182 | Denes | Jul 2008 | A1 |
| 20080277272 | Pierce et al. | Nov 2008 | A1 |
| 20090178933 | Zeng | Jul 2009 | A1 |
| 20130259903 | Mortenson et al. | Oct 2013 | A1 |
| 20140081725 | Blackhurst et al. | Mar 2014 | A1 |
| 20150167189 | Pierce et al. | Jun 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| PCTAU9600768 | Jun 1997 | AU |
| 12908 | May 1910 | EP |
| 432101 | Jul 1935 | EP |
| 0857695 | Apr 2001 | EP |
| PCTUS0547699 | Jul 2006 | WO |
| Entry |
|---|
| Wu, C.; Zeng, T., Size-Tunable Synthesis of Metallic Nanoparticles in a Continuous and Steady-Flow Reactor, Chem. Mater. 2007, 112-125, vol. 19, No. 2. |
| Ultra Professional Instructions for Making Premium AC Colloidal Silver with your HVAC Ultra Professional System. |
| HVAC ARC Silver Solutions, System Tech Engineering, http://web.archive.org/web/20021125133241/http://www.hvacsilver.com; Nov. 12, 2008. |
| Plasma (physics); Wikipedia; http://en.wikipedia.org/wiki/plasma_(physics); 2010. |
| Taylor cone; Wikipeia, http://en.wikipedia.org/wiki/Taylor_cone; 2010. |
| Manolache, S; Shamamian, V.; Denes F., Dense Medium Plasma-Plasma-Enhanced Decontamination of Water of Aromatic Compounds, J. of Environ Eng. Jan. 2004, 17-25. |
| Riegel, E.R.; Osthoff, R.C., Flach, D.O. Bredig Sols: A Lecture Demonstration, J. Chem. Educ, 1949, p. 519, vol. 26, No. 10. |
| Weiser, Harry Boyer, Inorganic Colloid Chemistry, 1933, p. 1, 8-17, 45-46, 116-117, 124-125, 132-135, John Wiley & Sons, Inc., New York. |
| Kraemer, E.O., Svedberg, T., Formation of Colloid Solutions by Electrical Pulverization in the High-Frequency Alternating Current Arc, J. Am. Chem Soc. 1924, 46(9) p. 1980-1991. |
| Rodriguez-Sanchez, L., Blanco, M.C., Lopez-Quintela, M.A., Electrochemical Synthesis of Silver Nanoparticles, J. Phys. Chem. B., 2000, 104, p. 9683-9688. |
| Liu, Yu-Chuan, et al., Active Catalysts of Electrochemically Prepared Gold Nanoparticles for the Decomposition of Aldehyde in Alcohol Solutions. Electrochemistry Communications, 2006, 1163-1167, 8. |
| Hickling, A., et al. Contact Glow-Discharge Electrolysis. Trans Faraday Soc., 1964, 783-793, 60. |
| Toriyabe, Yu, et al. Controlled Formation of Metallic Nanoballs During Plasma Electrolysis, Applied Physics Letters, 2007, 041501-1-041501-3, 91. |
| Wuthrich, Rolf, et al., Electrochemical Discharges—Discovery and Early Applications, Eletrochimica Acta, 2009, 4031-4035, 54. |
| Locke, B. R., et al., Electrohydraulic Discharge and Nonthermal Plasma for Water Treatment, Ind. Eng. Chem. Res., 2006, 882-905, 45. |
| Meiss, Sebastian A., Employing Plasmas as Gaseous Electrodes at the Free Surfacte of Ionic Liquids: Deposition of Nanocrystalline Silver Particles. ChemPhsyChem, 2007, 50-53, 8. |
| Hickling, A., et al., Glow-Discharge Electrolysis, J. Electroanal.Chem, 1964, 65-81, 8. |
| Chaffin, John H., et al., Hydrogen Production by Plasma Electrolysis. Journal of Energy Engineering, 2006, 104-108, 132:3. |
| Aqil, A., et al.; Preparation of Stable Suspensions of Gold Nanoparticles in Water by Sonoelectrochemistry. Ultrasonics Sonochemistry, 2008, 1055-1061, 15. |
| Liu, Yu-Chen, et al. Size-Controlled Synthesis of Gold Nanoparticles from Bulk Gold Substrates by Sonoelectrochemical Methods. J. Phys. Chem. B, 2004, 19237-19240, 108. |
| Torimoto, Tsukasa, et al. Sputter deposition onto ionic liquids: Simple and clean synthesis of highly dispersed ultrafine metal nanoparticles. Applied Physics Letters, 2006, 243117-1-243117-3, 89. |
| Nagata, Yoshio, et al. Sonochemical Formation of Gold Particles in Aqueous Solution. Radiation Research, 1996, 333-338, 146. |
| Saez, Veronica, et al. Sonoelectrochemical Synthesis of Nanoparticles. Molecules, 2009, 4284-4299, 14. |
| Fujimoto, Taku, et al. Sonolytical Preparation of Various Types of Metal Nanoparticles in Aqueous Solution. Scripta mater., 2001, 2183-2186, 44. |
| Vasudevamurthy, Gokul, et al. “Effect of System Parameters on Size Distribution of 304 Stainless Steel Particles Produced by Electrical Discharge Mechanism”, Materials Letters, 2007, p. 4872-4874, vol. 61. |
| Staack, David, et al. Nanoscale Corona Discharge in Liquids, Enabling Nanosecond Optical Emission Spectroscopy. Angew.Chem.Int.Ed., 2008, 8020-8024, 47. |
| Xu, Z.P.; Zeng, Q.H.; Lu, G.Q.; Yu, A.B.; Inorganic Nanoparticles as Carriers for Efficient Cellular Delivery, Chemical Engineering Science. Nov. 3, 2005. vol. 61. pp. 1027-1040. |
| Burda, et al., Chemistry and Properties of Nanocrystals of Different Shapes; Chem. Rev. 2005, 1025-1102. |
| Cushing, et al., Recent Advances in the Liquid-Phase Synthesis of Inorganic Nanoparticles; Chem. Rev. 2004, 104, 3893-3946. |
| Wiley, et al., Shape-Controlled Synthesis of Metal Nanostructures: The Case of Silver, Chem. Eur. J. 2005, 11, 454-463. |
| Tas, M.A., Van Hardeveld, R., Van Veldhuizen, E.M.; Reactions of NO in a Positive Streamer Corona Plasma; Plasma Chem and Plasma Proc; 1997, p. 371-391, vol. 17, No. 4. |
| Yan, K., Corona Plasma Generation, 2001, Technische Universiteit Eindhoven. |
| Hoeben, W.F.L.M., Pulsed Corona-Induced Degradation of Organic Materials in Water, 2000, Technische Universiteit Eindhoven. |
| Lofton, C., Sigmund, W.; Mechanisms Controlling Crystal Habits of Gold and Silver Colloids, Adv. Funct. Mater. 2005, p. 1197-1208, 15. |
| Martinez, S.S., Gallegos, A.A., Martinez, E.; Electrolytically Generated Silver and Copper Ions to Treat Cooling Water: An Environmentally Friendly Novel Alternative, International Journal of Hydrogen Energy. 2004, p. 921-932, 29. |
| Bhattacharyya, S., et al.; Localized Synthesis of Metal Nanoparticles, Using Nanoscale Corona Discharge in Aqueous Solutions, Adv. Mater, 2009, p. 4039-4044, 21. |
| Mucalo, M.R., Bullen, C.R.; Electric Arc Generated (Bredig) Palladium Nanoparticles. Surface Analysis by X-Ray Photoelectron Spectroscopy for Samples Prepared at Different pH, J. Mat. Sci. Let., 2001, p. 1853-1856, 20. |
| Lange, H., et al., Nanocarbon Production by Arc Discharge in Water, Carbon, 2003, pp. 1617-1623, 41. |
| Ma, H; Yin, B; Wang, S; Jiao, Y; Pan, W; Huang, S; Chen, S; Meng, F; Synthesis of Silver and Gold Nanoparticles by a Novel Electrochemical Method, ChemPhysChem, 2004, 5, 68-75. |
| Powell, J., Our Mightiest Germ Fighter, Science Digest, 1978, p. 57-59. |
| Svedberg, T., The Formation of Colloids, 1921, p. 22-41, D. Van Nostrand Company, New York. |
| Zsigmondy, R., The Chemistry of Colloids, 1917, p. 11, 19, 30, 68, 86-95, 114-119, 122-123, 127-128, John Wiley & Sons, Inc., New York. |
| Bechhold, H., Colloids in Biology and Medicine. 1919, p. 89-127, D. Van Nostrand Company, New York. |
| Federal Register/vol. 64, No. 158/Aug. 17, 1999/Rules and Regulations, p. 44653-44658. |
| Gibbs, R. J., Silver Coolids, Do They Work?, 1999, Ronald J. Gibbs, Newark, DE. |
| Grier, Silver and Its Compounds. Disinfection, Sterilization, and Preservation, 2001, p. 403-407, Lippincott Williams & Wilkins, Philadelphia. |
| Pacheco, G., Studies on the Action of Metallic Colloids on Immunisation. Mem. Inst. Oswaldo Cruz. 1925, vol. 18, n. 1, pp. 119-149. |
| DeVries, C.A.M.; De Hoog, F.J.; Schram, D.C.; Ionic Species in a Negative Corona in Humid Air, 1983, p. 317-321, Eindhoven University of Technology, The Netherlands. |
| Liu, Yu-Chuan, et al., New Pathway for the Synthesis of Ultrafine Silver Nanoparticles From Bulk Silver Substrates in Aqueous Solutions by Sonoelectrochemical Methods. Electrochemistry Communications, 2004, 1163-1168, 6. |
| Bruggeman, Peter, et al., Non-Thermal Plasmas in and in Contact With Liquids; J.Phys.D.:Appl.Phys,2009. |
| Kravchenko, A.V., et al.; On the Change in Properties of Water Subjected to Low-Temperature Plasma Electrolysis. High Energy Chemistry, 2004, 333-337. 38-5. |
| Koo, Il Gyo, et al.; Platinum Nanoparticles Prepared by a Plasma-Chemical Reduction Method. J.Mater.Chem., 2005, 4125-4128, 15. |
| Lelievre, J., et al; Electrolysis Processes in D.C. Corona Discharges in Humid Air. J. Phys. III France 5 (1995) 447-457. |
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| 20160375058 A1 | Dec 2016 | US |
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| 61144625 | Jan 2009 | US |
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| Parent | 14010721 | Aug 2013 | US |
| Child | 15199009 | US | |
| Parent | 12686815 | Jan 2010 | US |
| Child | 14010721 | US |
