The invention generally relates to methods and apparatus for manipulation of charged molecules in solution. More particularly, the invention provides nanofluidic CCD arrays, and related methods, that are capable of manipulate one, or a group of molecules or nanoparticle(s) on an individual bases such that they undergo controlled physical and/or chemical movements and/or transformations.
Currently, microfluidic devices strive for the control of small numbers of molecules, or ideally single molecules, throughout various microfluidic compartments and channels with micrometer or submicrometer precision. To date, the two most widely used forms of actuation in microfluidic devices are electrokinetic (electrophoresis and electro-osmosis) and pressure driven flow.
In electrokinetic actuation, an electrical connection is made with the microfluidic channels through the use of electrodes in fluidic reservoirs located at the ends of the microfluidic channels. A voltage difference between electrodes results in electrokinetic forces in the device. However, if an array of channels, or any network of channels, is present, it is extremely difficult to establish an electric field in one channel without affecting other channels. Not only is it currently impractical to electrically isolate single channels, if more than one molecule is present within a single channel, the molecules may not be controlled or manipulated independently.
Pressure driven flow may be considered a blunt method for moving molecules within a network of microfluidic channels. Typically, pressure fittings are fixed at interfaces between the microfluidic channel network and the microscopic world, and pressure is applied at the channel endpoints. In its most complex form to date, a microfluidic pressure based device is made from an elastomeric material, and microfluidic pumps and valves are integrated at various points within the fluidic network. In these devices, channels may be isolated from one another; however, control over fluidic elements is limited in resolution by the minimum size and density of the pumps and valves. Bulky connections to macroscale pumps and valves must be made, further increasing the complexity and size of the overall device, and further limiting the density of elastomeric microfluidic pumps and valves.
Similarly, DNA molecules in conventional nanofluidic devices are moved in bulk via electrophoresis, electroosmotic flow, capillary action, or pressure-driven flow. All molecules have an equal chance of interacting with a specific region of the device. For example, a typical nanofluidic device may contain a restriction digestion activity in one region, but all molecules have an equal probability of being digested. Thus, with existing devices it is not difficult to select one or a few molecules and divert them to a particular region of the device to undergo a preselected transformation (e.g., to a digestion zone), without the use of complicated pumps and/or valves.
Precise localization of biochemical activities in nanofluidic devices remains challenging. Nanofluidic fabrication methods often involve high temperatures, chemical etchings, vacuum, etc., and are often incompatible with maintaining biological activities of enzymes or nucleic acids integrated into the device. Furthermore, if enzymes are introduced to the device after fabrication, the bulk flow of the enzyme into the device may preclude localizing the enzyme to a specific area. Finally, even if the enzyme can be localized to a specific region of the device, the necessary bulk flow of analytes by pressure or electrophoretic flow may dislodge the enzyme rendering the biochemical transformation in effective or incomplete.
Thus, unmet needs remain for novel nanofluidic apparatus and methods that are capable of individualized movement and controlled manipulation of a molecule or group of molecules in solution environment, particularly for large biological molecules such as DNA molecules.
The invention is based in part on the unexpected discovery of novel nanofluidic apparatus and methods that are capable of individualized movement and controlled manipulation of a molecule or group of molecules in solution environment. A key feature of the nanofluidic CCD array technology disclosed herein is that molecules or nanoparticles, in particular DNA and other large biomolecules, can be individually manipulated and moved to spatially distinctive parts (e.g., reaction stations) of the nanofluidic device, without the use of electrophoresis or pressure. For example, a nanofluidic CCD array can be constructed such that different regions of the array have different biochemical activities (e.g., a restriction enzyme to digest the DNA, or a polymerase to incorporate labeled nucleotides). An exceptional aspect of the present invention is that biomolecules such as DNA molecules can be readily moved between reaction stations by a combination of diffusion and nanofluidic charge coupling, rather than by bulk flow. These DNA molecules can be made to stay at a reaction station to under biophysical or biochemical transformations for preselected and controllable time periods. Thus, different DNA molecules in a single nanofluidic device can be addressed and treated individually, all without the use of valves.
In one aspect, the invention generally relates to an apparatus for manipulation of a molecule or group of molecules in a solution. The apparatus includes: (1) a nanofluidic cavity, a bottom of which is made of a dielectric layer, wherein the depth of the nanofluidic cavity is on the order of or less than the ionic screening length of the solution filling the nanofluidic cavity; (2) a plurality of electrodes arranged on a surface of the dielectric layer opposite a surface of the dielectric layer having the nanofluidic cavity, wherein a spacing between individual electrodes in the array of electrodes is not significantly greater than the ionic screening length of the solution filling the nanofluidic cavity; and (3) a plurality of vias individually connected to the individual electrodes for connection to an external electronic device.
In another aspect, the invention generally relates to an apparatus for manipulation of a molecule or group of molecules in a solution. The apparatus includes: (1) a nanofluidic cavity, wherein a depth of the nanofluidic cavity is on the order of or less than the ionic screening length of the solution filling the nanofluidic cavity; (2) an array of electrodes arranged on a surface the nanofluidic cavity, wherein a spacing between individual electrodes in the array of electrodes is not significantly greater than the ionic screening length of the solution filling the nanofluidic cavity; and (3) an array of vias individually connected to the individual electrodes in the array of electrodes for connecting the array of electrodes to an external electronic device. An applied voltage applied to the individual electrodes in the array is less than an overpotential required to transfer an electron from the metal electrode to any chemical species in solution.
In the following detailed description of embodiments of the invention, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be apparent to one of ordinary skill in the art that the invention may be practiced without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the description.
In general, embodiments of the invention relate to an apparatus for the manipulation of a molecule, or group of molecules, or nanoparticle(s) in a solution. More specifically, embodiments of the invention relate to an apparatus for the independent actuation of single molecules or groups of molecules or nanoparticle(s) in a nanofluidic environment. Single molecule studies in nanofluidic environments are currently being explored; see for example, U.S. Patent Publication No. 20110227558, the contents of which are hereby incorporated by reference in its entirety for all purposes.
Additionally, embodiments of the invention relate to a method for the manipulation of a molecule, or group of molecules, or nanoparticle(s) in a solution. More particularly, embodiments of the invention provide nanofluidic CCD arrays-based methods that are capable of manipulating one or a group of molecules or nanoparticle(s) on an individual bases such that they undergo controlled physical and/or chemical movements and/or transformations.
Analogous to how a charge coupled device (CCD) array shuffles charges through an imaging chip, embodiments of the invention allow for the discrete movement of single molecules, or groups of molecules, dispersed in a solution within a cavity. For the purposes of this invention, a cavity refers to slit, channel, or similar space that contains the solution. A cavity may also refer to wide channel or chamber definable by a shallow depth in the vicinity of a buried gate electrode. In some embodiments, the cavity may be referred to as a nanocavity. Also, for the purposes of this invention, the term nanofluidic cavity refers to a fluid filled cavity with at least one dimension on the order of, or smaller than, one hundred nanometers. The phrase “on the order of” as used herein indicates that one quantity is of the same order of magnitude as another quantity. In this document, two quantities are of the same order of magnitude as long as the two quantities do not differ from one another by more than a factor of one hundred.
One or more embodiments of the invention include an array of independently addressable buried gate electrodes in close proximity to the cavity. The array of independently addressable buried electrodes may or may not be separated from the solution of molecules in the cavity by a dielectric. The depth of the cavity that contains the solution of molecule forces some or all of the molecules to reside within a distance to the electrodes that is on the order of or less than the ionic screening length of the solution.
Therefore, in one or more embodiments of the invention, a voltage applied to a single buried electrode may not be fully shielded by buffer ions in solution except at distances that are large compared with the depth of the channel. As such, the electrostatic potential through substantially the entire depth of the channel above the electrode pixel may be perturbed. Embodiments of the invention may enable discrete, independent, and programmable actuation of charged molecules, macromolecules, or packets of charged molecules in a nanofluidic environment. The motion of the molecules induced by the array of buried gate electrodes is distinct from traditional electrophoretic motion of charged species in microfluidic or nanofluidic environments because no electron transfer actually occurs at the electrode and solution interface. Thus, there may be no direct current flowing through the device.
The substrate 112, dielectric layer 104, and ceiling 106 are electrically insulating. Examples of materials of the substrate 112 include, but are not limited to, silicon, or fused silica. Examples of the material of the dielectric layer 104 and ceiling 106 include, but are not limited to, silicon dioxide, silicon nitride, or aluminum oxide. The electrode 108 and conductive via 110 may be constructed of metal, polysilicon, or other conductive materials.
The depth of the cavity 102 in the z direction in
In one or more embodiments of the invention, the dielectric layer 104 is of sufficient thickness and quality such that electrical breakdown does not occur within the range of voltages applied to the electrode 108 through the conducting via 110. In one or more embodiments of the invention the dielectric layer 104 is made of a material that prevents any electrochemical reactions from occurring at the electrode 108.
In one or more embodiments of the invention, the dielectric layer 104 may be omitted. In these embodiments, the applied potential is controlled to compensate for the lack of the dielectric layer. In these embodiments, the bare electrode may cause current to flow into the solution containing the molecule, or groups of molecules, and may allow molecules to be moved by electrophoresis, as is traditionally done in microfluidic and nanofluidic devices, if the applied voltage was too high. However, one of ordinary skill in the art would recognize, in view of the present disclosure, that molecule manipulation in the nanofluidic CCD mode may be achieved without the dielectric layer 104, if the voltage applied to the electrodes in the array is less than an overpotential required to transfer an electron from the electrode to any chemical species in the solution.
The cavity 102 may be deep enough to allow the molecules of interest to enter, but shallow enough such that the perturbation caused by the potential on the electrode 108 on the fluid is not screened by mobile charge carriers over substantially the entire depth of the cavity 102, or at least over some noticeable fraction of the cavity. In other words, the depth of the cavity may be on the order of, or less than, the ionic screening length of the solution. Further, the shallower the cavity is in relation to the ionic screening length, the stronger the electric coupling between the buried electrode and the molecules of interest may be throughout the depth of the cavity. Additionally, as mentioned previously, the screening length may be an adjustable parameter because the screening length depends on the ionic strength of the buffer solution.
It should furthermore be noted that in the case where the ionic screening length is only a fraction of the channel depth (5-10% for example) it may still be possible to move molecules across the array in the CCD mode of operation. For example, if a long DNA molecule spanned a part of the channel over a number of buried electrodes, but the channel depth was ten times the ionic screening length the device may still be used in the CCD mode of operation. Under these conditions, if a large negative potential is applied to one of the electrodes, the segment of DNA above the electrode may be excluded from the bottom fraction of the channel (the bottom tenth of the channel volume). The large negative potential may effectively cause the molecule to be more tightly confined in the vertical direction. Such confinement may result in a horizontal stretching of the molecule, as shown by molecule 516-3 illustrated in
Non-uniform confinement along the length of the molecule may result in entropic forces in the horizontal plane of the nanocavity, which may be used to move the molecule across the array. Such confinement-induced entropic forces, acting on DNA molecules in nanofluidic environments, are well understood in the field of nanofluidics. (See, e.g., “Conformational Analysis of Single DNA Molecules Under going Entropically Induced Motion in Nanochannels” by Mannion, et. al. 2006 Biophysical Journal Vol. 90, p4538). An electrostatic force may be used to push the molecule vertically upwards in the cavity, pressing the molecule partly against the ceiling. The vertical squeezing may result in an entropic force that acts in the horizontal direction, driving the molecule out of the space above the negative electrode, toward the space above an adjacent electrode. Embodiments of the invention include one and two-dimensional arrays of the basic component 100 shown in
In one or more embodiments of the invention, each of the electrodes 208-1 . . . 208-6 may be independently addressable through the conductive vias 210-1 . . . 210-6 by the external or on-chip electronics. Alternatively, two or more of the electrodes 208-1 . . . 208-6 may be addressable as a group, depending on the relative size of the molecule, or molecules, and the electrodes 208-1 . . . 208-6.
For example, in
In one or more embodiments of the invention, the area of the electrode may be designed relative to the size of the molecule or molecules of interest. If the molecules of interest are smaller than the area covered by the electrode, then the space between adjacent electrodes should be approximately equal to or less than the ionic screening length, and consequently the depth of the nanochannel. Therefore, the molecule may not become trapped in a “dead zones” in between the electrodes. If the molecules of interest cover a hydrodynamic area larger than the area of the electrodes, then the array pitch may be less than or equal to the hydrodynamic radius of the molecules. In one or more embodiments of the invention, both of the previous conditions may be met. As the size and spacing between adjacent electrodes decrease, the spatial resolution of the apparatus may be increased.
In one or more embodiments of the invention, molecules are moved during the brief transition period after the electrode voltages have been changed, and before a steady state rearrangement of mobile charge carriers is reached in solution. For example, movement of the molecules is achieved by the charging and discharging closely spaced fluidic electrodes. Because rearrangement happens quickly, to move the molecule over an appreciable distance of the array, the voltages on the array electrodes may be continually switched, to shuffle the charges from one adjacent electrode to the next. This is a distinction in the device operation as compared with the standard “electrode in reservoir” apparatus and, advantageously, may open new realms of possibilities for nanofluidic sample handling.
In one or more embodiments of the invention, certain analyte molecules may respond to the change in voltage of the buried electrode over a timescale longer than that required for the majority of electrolyte ions to respond. For example, a large DNA molecule may initially occupy the space above multiple neutral electrodes, and one electrode potential may be suddenly set to a high negative voltage. The DNA molecule may eventually shift its position so that it resides above the neutral electrode. However, the speed at which the molecule shifts position depends, in part, on the relationship between the depth of the channel and the ionic screening length. If the ionic screening length is longer that the depth of the nanocavity, the DNA molecule may experience a strong repulsive force in the vertical direction, and may not only move upwards in the cavity, away from the negative electrode, but may also be excluded from the entire volume above the negative electrode as a result of the electric fields present in that space. If the ionic screening length is relatively short however, for example a tenth of the channel depth, the DNA molecule may only be excluded from the bottom portion of the volume of fluid above the negative electrode (the bottom tenth of that volume). The DNA may still reside in the top 90% of that volume, without experiencing a strong electrical repulsion. However, this slight squeezing in the vertical direction by electric fields may lead to a confinement-induced entropic force acting in the horizontal direction. Thus, even after the electrolyte ions rearrange in response to the negative applied voltage, the confinement-induced entropic forces may continue to act on a macromolecule such as a long DNA molecule. In both cases, the DNA molecule may be moved long distances over the electrode array through a sequence of toggled voltages that shuffle the molecule from electrode to electrode. However, in the second case, the electrode voltages may need to be toggled at a slower clock speed, such that the DNA molecule may not fall behind pace of the changing electrode voltages. Similarly, if a charged molecule being moved has a low electrophoretic mobility, the molecule may move slower than the amount of time required to set new voltages on electrodes. Thus, a sufficiently long delay period may be provided during the toggling of the voltages.
In one or more embodiments of the invention, a network of nanofluidic channels may allow for molecules to be sorted in a serial fashion without being dumped into a macrofluidic reservoir, where they may be lost in the large fluidic volume, or where they may directly contact the electrode surface.
For example, a collection of chromosome fragments 620-1 . . . 620-3 may be loaded into the cavity area, either from above (e.g., through access in the ceiling) or may be supplied from a microchannel (not shown) on the right side of
In one or more embodiments of the invention, the nanofluidic array apparatus may be combined with pressure-driven flow to isolate highly charged molecules (e.g., DNA, RNA) from lysed cells from other cellular components. In one or more embodiments of the invention, the nanofluidic array apparatus may be coupled to a microfluidic channel to enable discrete sampling of the microfluidic solution.
As described previously, embodiments of the claimed invention may include independently addressable electrodes through electrical contact to the vias on the backside of the apparatus. The vias may be connected to external electronics through a circuit board. In other words, the external electronics referred to herein may include additional circuit boards or components to facilitate connections to the vias.
In one or more embodiments of the invention, as shown in
In addition, further components may be incorporated in the apparatus in accordance with one or more embodiments of the invention. For example,
In certain embodiments of the invention, certain regions of the nanofluidic array are coated with gold, to which proteins with free cysteine residues can bind via a gold-thiolate bond. (Ulman, et al. 2011 J Nanobiotech. 9:26 “Highly active engineered-enzyme oriented monolayers: formation, characterization and sensing applications”.)
The gold surface can be created in a specific region by etching the dielectric away to expose a gold electrode (
In other embodiments, protein enzymes can bind to antibodies bound to the modified surface. If the dielectric layer over certain electrodes is removed (
Devices and methods of the invention can be adopted in a microfluidic or nanofluidic device together with other functions, such as a nanofluidic “bump array” for fluid exchange, cell lysing, or particle sorting, or nanofluidic channels for polymer sizing. For example, a combined lab-on-chip device can be constructed which lyses individual bacterial cells, pass the genome of the cells to restriction digestion stations (this invention) and size the resulting fragments in nanofluidic channels. (Morton, et al. 2008 Lab Chip. 9:1448-53. “Crossing microfluidic streamlines to lyse, label and wash cells”, E. pub. 2008, Jul. 23); Mannion, et al. 2007 Biopolymers 85(2): 131-43, “Nanofluidic structures for single biomolecule fluorescent detection”).
In certain embodiments, a polymerase may be bound to the surface of the chip to incorporate labeled nucleotides at breaks or nicks in the DNA. In other embodiments, antibodies to DNA modifications such as methylation can be used, to retain methylated DNAs in one region of the device. Alternatively, DNA aptamers can provide the enzymatic activity, either as bound entitites or as nucleic acids localized to an electrode via the nanofluidic CCD array technology. For example, devices of the invention provides a nanofluidic device that has enzymatic activities bound to certain regions the molecule sensing and actuation ability provided by the nanofluidic CCD array.
In one aspect, the invention generally relates to an apparatus for manipulation of a molecule or group of molecules in a solution. The apparatus includes: (1) a nanofluidic cavity, a bottom of which is made of a dielectric layer, wherein the depth of the nanofluidic cavity is on the order of or less than the ionic screening length of the solution filling the nanofluidic cavity; (2) a plurality of electrodes arranged on a surface of the dielectric layer opposite a surface of the dielectric layer having the nanofluidic cavity, wherein a spacing between individual electrodes in the array of electrodes is not significantly greater than the ionic screening length of the solution filling the nanofluidic cavity; and (3) a plurality of vias individually connected to the individual electrodes for connection to an external electronic device.
In certain embodiments of the invention, the array of the electrodes is a two dimensional array. In certain preferred embodiments, one or more electrodes in the array of electrodes are independently addressable, through the array of vias, by the external electronic device. The electric potential applied to the one or more electrodes is chosen such that negligible electric current flows between the electrode and the solution in the cavity.
In certain preferred embodiments, the dielectric layer is of sufficient thickness, quality, and material composition to limit electron transfer during operation while also allowing for strong electrostatic coupling between the gate electrode and molecules in the fluidic cavity. For example, the depth of the nanofluidic cavity may be greater than about 1 nm and less than about 1,000 nanometers (e.g., from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 100 nm to about 1,000 nm, from about 100 nm to about 1,000 nm, from about 100 nm to about 1,000 nm, from about 100 nm to about 900 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 500 nm, from about 100 nm to about 400 nm, from about 100 nm to about 300 nm, from about 100 nm to about 200 nm). In some preferred embodiments, the depth of the nanofluidic cavity is less than approximately 150 nanometers and greater than approximately 1 nm.
In certain embodiments, the apparatus further includes a hole in the ceiling to enable diffusion of the molecule through the hole into the fluid-filled cavity.
In certain embodiments, the apparatus further includes a second array of electrodes disposed on a top surface of the dielectric layer of the ceiling; and a second array of vias individually connected to the individual electrodes in the second array of electrodes for connecting the second array of electrodes to the external electronic device. The one or more electrodes of the second array of electrode are independently addressable, through the second array of vias, by the external electronic device.
In certain embodiments, the external electronic device includes integrated on chip electronics.
In certain embodiments, the apparatus further includes one or more physical barriers partitioning the nanofluidic cavity into two or more passages or compartments. In some embodiments, at least one of the passages or compartments allows therein a physical or biochemical manipulation of the one or more analyte molecules or particles in the sample, without affecting molecules in the other passages or compartments. In some embodiments, one or more of the passages or compartments is connected to fluidic channels allowing fluid exchange between said passages or compartments, without exchanging fluid in the entire cavity. In some embodiments, a portion of the cavity is modified to expose selected molecules to biochemical or physical modification. In some embodiments, the modification of the cavity comprises immobilization of one or more protein or DNA molecules at a specific region of the cavity.
In certain embodiments, the thickness of the dielectric layer is less than about 30 nanometers (e.g., less than about 25 nm, less than about 20 nm, less than about 15 nm, less than about 10 nm, less than about 5 nm). In some preferred embodiments the thickness of the dielectric layer is less than approximately 15 nanometers.
In certain embodiments, the biochemical manipulation is selected from an oxidation-reduction reaction, an enzymatic reaction, a nuclease digestion, a fluorescent labeling reaction, affinity-based binding, a phosphorylation or dephosphorylation reaction, or covalent modification.
In certain embodiments, the biochemical manipulation is selected from an oxidation-reduction reaction, an enzymatic reaction, a nuclease digestion, a fluorescent labeling reaction, affinity-based binding, a phosphorylation or dephosphorylation reaction, or covalent modification.
In another aspect, the invention generally relates to an apparatus for manipulation of a molecule or group of molecules in a solution. The apparatus includes: (1) a nanofluidic cavity, wherein a depth of the nanofluidic cavity is on the order of or less than the ionic screening length of the solution filling the nanofluidic cavity; (2) an array of electrodes arranged on a surface the nanofluidic cavity, wherein a spacing between individual electrodes in the array of electrodes is not significantly greater than the ionic screening length of the solution filling the nanofluidic cavity; and (3) an array of vias individually connected to the individual electrodes in the array of electrodes for connecting the array of electrodes to an external electronic device. An applied voltage applied to the individual electrodes in the array is less than an overpotential required to transfer an electron from the metal electrode to any chemical species in solution.
In yet another aspect, the invention generally relates to an apparatus for manipulating one or more analyte molecules or nanoparticles in a solution. The apparatus includes: a nanofluidic cavity defined by a dielectric ceiling and a floor substrate, having a depth from about 10 nm to about 1,000 nm; a plurality of electrodes disposed on a surface of the floor substrate opposite a surface of the dielectric ceiling defining the nanofluidic cavity; and a plurality of vias, each being individually connected to an electrode of the plurality of electrodes for connection to an external electronic device. The apparatus may further include one or more barriers or gates partitioning the nanofluidic cavity into two or more passages or compartments, wherein each of the passages or compartments may be designed to allow therein a physical or biochemical manipulation of the one or more analyte molecules or particles in the sample. The apparatus may additionally include one or more apertures in the dielectric ceiling allowing diffusion into and/or out of the nanofluidic cavity.
In yet another aspect, the invention generally relates to a method for manipulating of one or more analyte molecules or particles. The method includes: providing a nanofluidic cavity, defined by a dielectric ceiling and a floor substrate, having a depth from about 10 nm to about 1,000 nm; providing a plurality of electrodes disposed on a surface of the floor substrate opposite a surface of the dielectric ceiling defining the nanofluidic cavity; providing a plurality of vias, each being individually connected to an electrode in the plurality of electrodes, whereby each electrode is connected to and individually addressable by an external electronic control unit; depositing inside the nanofluidic cavity a solution having the one or more analyte molecules or particles to be manipulated; and asserting one or more electrical voltages or signals to one or more of the electrodes in the plurality of electrodes to induce a spatial movement of the one or more analyte molecules or particles in the sample inside the nanofluidic cavity.
Specific embodiments of the invention will now be described in detail with reference to the accompanying figures. Like elements in the various figures are denoted by like reference numerals for consistency. Further, the use of “Fig.” in the drawings is equivalent to the use of the term “Figure” in the description.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference, unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.
While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims.
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.
The representative examples disclosed herein are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
Number | Date | Country | Kind |
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61580952 | Dec 2011 | US | national |
This application is the national phase of PCT/US12/71004, filed on Dec. 20, 2012, which claims the benefit of priority from U.S. Provisional Application Ser. No. 61/580,952, filed on Dec. 28, 2011, the entire content of each of which is incorporated herein by reference in its entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US2012/071004 | 12/20/2012 | WO | 00 |