Gradient elution isotachophoretic apparatus, and systems for performing gradient elution isotachophoresis to separate, purify, concentrate, quantify, and/or extract charged analytes from a sample. The isotachophoretic apparatus include an electrophoretic assembly, a sampling assembly connected to the electrophoretic assembly, and/or a support structure connected to the electrophoretic assembly and/or to the sampling assembly. The system includes an isotachophoretic apparatus, and a controller communicatively coupled to the isotachophoretic apparatus. The controller includes a storage medium and a processor for executing computer readable and executable instructions.

TECHNICAL FIELD

The present disclosure generally rates to electrophoretic apparatus, and more particularly, relates to gradient elution isotachophoretic apparatus for separating, purifying, concentrating, quantifying, and/or extracting charged analytes, to systems for performing gradient elution isotachophoresis, and to methods for separating, purifying, concentrating, quantifying, and/or extracting charged analytes from samples.

BACKGROUND

The separation, purification, concentration, quantification, and/or extraction of charged analytes, such as, e.g., biomolecules and/or deoxyribonucleic acid (i.e., DNA), from crude samples remains a technical and practical challenge. For example, crude samples may contain environmental contaminants, such as, e.g., soil, blood, bacteria, particulate material, cell detritus, and/or ionic species, and/or biomolecule inhibitors, such as, e.g., qPCR inhibitors such as polymerase inhibitors, which complicate analysis thereof. Additionally, conventional apparatus and/or techniques for analyzing crude samples are generally labor intensive, time consuming, and/or require access to a laboratory, skilled technicians, and/or specialized equipment. Moreover, such apparatus and/or techniques typically deliver the purified analytes, such as, e.g., DNA, in small fluid volumes, such as, e.g., about 50 μL, which limits further analysis thereof. Further, conventional apparatus and/or methods for separation of charged analytes from crude samples may require pre-separation and/or post-separation sample preparation steps, such as, e.g., filtration, centrifugation, and/or precipitation. Such further sample preparation steps may reduce the quantity of charged analytes delivered from the sample and may also lower the final concentration of charged analytes. The reduction of both quantity and concentration of delivered charged analytes can negatively impact the likelihood of further post-separation analyses, such as, e.g., in the case of DNA, short tandem repeat (i.e., STR) analysis for human identification. Accordingly, additional embodiments of apparatus for separating, purifying, concentrating, quantifying, and/or extracting charged analytes and methods thereof are desired.

SUMMARY OF THE INVENTION

In one embodiment, an apparatus for performing gradient elution isotachophoresis (GEITP) to separate charged analytes in a sample is disclosed. The apparatus may include a moveable electrophoretic assembly including: a separation unit, a detection unit operably connected to the separation unit, wherein the detection unit includes: a support structure, a conductivity detection device accommodated by the support structure, a light source accommodated by the support structure, and a light source detection device accommodated by the support structure, and at least one moveable support structure connected to at least one of the separation unit or the detection unit; a sampling assembly operably connected to the moveable electrophoretic assembly; and a support structure connected to at least one of the moveable electrophoretic assembly or the sampling assembly.

In another embodiment, a system for performing gradient elution isotachophoresis (GEITP) to separate charged analytes in a sample is disclosed. The system includes: an apparatus for performing GEITP including: a moveable electrophoretic assembly including: a separation unit including at least one separation channel in a vertical orientation, a leading electrolyte (LE) reservoir in open fluidic communication with the at least one separation channel, a voltage supply device communicatively coupled to the at least one separation channel, and a pressure control device connected to the LE reservoir, a detection unit operably connected to the separation unit, wherein the detection unit includes a support structure, a conductivity detection device accommodated by the support structure, a light source accommodated by the support structure, and a light source detection device accommodated by the support structure, at least one moveable support structure connected to at least one of the separation unit or the detection unit; a sampling assembly operably connected to the moveable electrophoretic assembly, wherein the sampling assembly includes a trailing electrolyte (TE) reservoir and a delivery reservoir, wherein the TE reservoir includes the sample and TE fluid; a support structure connected to at least one of the moveable electrophoretic assembly or the sampling assembly; and a controller communicatively coupled to the moveable electrophoretic assembly and the sampling assembly, wherein the controller includes a storage medium including computer readable and executable instructions and a processor for executing the computer readable and executable instructions, wherein the processor executes the computer readable and executable instructions to: (1) optionally pre-treat the at least one separation channel, (2) insert LE fluid and sensor molecules from the LE reservoir into the at least one separation channel, (3) contact the at least one separation channel with the sample and TE fluid in the TE reservoir of the sampling assembly, (4) separate the charged analytes via GEITP by: (a) producing a pressure-driven counterflow of the LE fluid through the at least one separation channel with the pressure control device and/or the voltage supply device, (b) applying a voltage to the at least one separation channel with the voltage supply device to produce an electric field, thereby driving electrophoretic migration of charged analytes in the TE reservoir of the sampling assembly toward the at least one separation channel, and (c) varying with respect to time the pressure-driven counterflow through the at least one separation channel with the pressure control device to control focusing of the charged analytes via initiation of a pressure ramp, thereby focusing and separating the charged analytes, (5) direct light through the at least one separation channel to excite fluorescence in any of the charged analytes contacted with the sensor molecules, (6) detect the charged analytes in the at least one separation channel via conductivity detection with the conductivity detection device and/or fluorescence detection with the light source detection device, and (7) deliver the charged analytes to a delivery reservoir in the sampling assembly.

Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements, as well as conventional parts removed, to help to improve understanding of the various embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used in the present application:

As used herein, the term “communicatively coupled” refers to the electrical, signal, wireless, wired, and/or optical interconnectivity of various components of the gradient elution isotachophoretic apparatus which are connected, such as, e.g., through wires, through optical fibers, and/or wirelessly, such that electrical, optical, and/or electromagnetic signals may be exchanged therebetween.

As used herein, the term “crude sample” refers to a specimen suspected of containing charged analytes of interest, such as, e.g., positively charged analytes and/or negatively charged analytes, and/or particulates, which has not undergone procedures for the separation, concentration, purification, and/or extraction thereof. In further embodiments, the crude sample refers to a specimen which has also not undergone sample preparation procedures, including but not limited to pre-separation and/or post-separation sample preparation procedures, such as, e.g., filtration, centrifugation, and/or precipitation (excluding simple dilution and lysis in some embodiments). In particular embodiments, crude sample refers to a specimen suspected of containing negatively charged small inorganic species, such as, e.g., chloride, bromide iodide, chlorate, perchlorate, iodate, and/or periodate, which has not undergone procedures for the separation, concentration, purification, and/or extraction thereof. In other particular embodiments, crude sample refers to a specimen suspected of containing negatively charged organic species, such as, e.g., acetate, formate, lactate, and/or biomolecules such as, e.g., DNA and/or RNA, which has not undergone procedures for the separation, concentration, purification, and/or extraction thereof. Where the charged analytes of interest include biomolecules, such as, e.g., DNA and/or RNA, cells in the crude sample may be lysed. Additionally, where the charged analytes of interest include biomolecules, by way of example, a buccal swab and/or a soiled buccal swab of cheek cells may be a crude sample. In still other particular embodiments, crude sample refers to a specimen suspected of containing positively charged species, such as, e.g., copper, which has not undergone procedures for the separation, concentration, purification, and/or extraction thereof. For example, crude samples may contain environmental contaminants, such as, e.g., soil, blood, bacteria, particulates, cell detritus, solid cell lystae, plant material detritus, and/or ionic species, and/or biomolecule inhibitors, such as, e.g., qPCR inhibitors such as polymerase inhibitors.

As used herein, the term “gradient elution isotachophoresis” refers to a fluid-phase electroseparation technique that involves concentration of charged analytes via isotachophoresis (i.e., ITP) and controlled focusing of such charged analytes via pressure-driven counterflow. In some embodiments, the pressure-driven counterflow improves control and/or selectivity of the charged analytes during focusing thereof and/or excludes contaminants from focusing with the charged analytes. In further embodiments, the pressure-driven counterflow excludes contaminants, such as, e.g., particulates, inhibitors, and/or other contaminant molecules, from being introduced into a separation channel in which the gradient elution isotachophoresis (i.e., GEITP) occurs. GEITP is further described in U.S. Pat. No. 8,080,144, the contents of which are hereby incorporated by reference in their entirety,

As used herein, the term “isotachophoresis” refers to the separation and/or concentration of charged analytes in a sample which is introduced in between a zone of leading electrolyte (i.e., LE) fluid, such as, e.g., LE solution, and a zone of trailing electrolyte (i.e., TE) fluid, such as, e.g., TE solution, upon application of an electric field thereto. The LE fluid includes electrophoretically fast ions and the TE fluid includes electrophoretically slow ions (relative to the electrophoretic mobility of the charged analytes). The charged analytes have an electrophoretic mobility which is intermediate to the electrophoretically fast ions of the LE fluid and the electrophoretically slow ions of the TE fluid. Upon application of the electric field, the charged analytes focus at the interface of the LE fluid and the TE fluid.

As used herein, the term “selective fluidic communication” refers to the controlled flow and/or lack of flow of a fluid from a first position to a second position. When a fluid flows and/or is capable of flowing from a first position to a second position, the fluidic communication is open therebetween. When a fluid does not flow and/or is incapable of flowing from a first position to a second position, the fluidic communication is closed therebetween.

Embodiments of the present disclosure are directed toward gradient elution isotachophoretic apparatus for performing GEITP. More specifically, embodiments of the present disclosure are directed toward gradient elution isotachophoretic apparatus for separating, purifying, concentrating, quantifying, and/or extracting charged analytes (hereinafter, “isotachophoretic apparatus”) in and/or from a sample via GEITP. In further embodiments, the present disclosure is directed toward isotachophoretic apparatus for performing automated GEITP.

Embodiments of the isotachophoretic apparatus will now be described in detail with reference toFIGS. 1-16. Thereafter, embodiments of isotachophoretic systems will be described with reference toFIG. 23. Finally, methods for separating, purifying, concentrating, quantifying, and/or extracting charged analytes will be described with reference toFIG. 17.

ReferencingFIGS. 1-4, in one or more embodiments, an isotachophoretic apparatus100includes an electrophoretic assembly110, a sampling assembly350operably connected to the electrophoretic assembly110, and/or a support structure510connected to the electrophoretic assembly110and/or to the sampling assembly350.

Referring toFIGS. 1-5, in one or more embodiments, the isotachophoretic apparatus100includes an electrophoretic assembly110. In some embodiments, the electrophoretic assembly110is moveable. In embodiments, the electrophoretic assembly110includes a separation unit130, a detection unit230operably connected and/or communicatively coupled to the separation unit130, and a first and second moveable support structure280,300attached to, connected to, and/or attachable to the separation unit130and/or to the detection unit230. In one or more embodiments, the separation unit130includes at least one separation channel140, a leading electrolyte (i.e., run buffer) reservoir160in fluidic communication with the at least one separation channel140, a voltage supply device180electrically connected and/or communicatively coupled to the separation channel140, and/or a pressure control device200connected to the leading electrolyte reservoir160.

Referring toFIGS. 5-8, in one or more embodiments, the separation unit130includes at least one separation channel140. In some embodiments, the separation channel140includes an elongate body which defines a channel (not shown) therethrough. In some embodiments, the channel (not shown) extends from an inlet144to an outlet (not shown). In further embodiments, the channel (not shown) extends from the inlet144to the outlet, along a length L of the elongate body. In still further embodiments, the elongate body includes a length L of from about 5 cm to about 100 cm, or from about 6 cm to about 50 cm, or from about 7 cm to about 20 cm, or about 10 cm. In one particular embodiment, the elongate body includes a length L of about 8.4 cm.

In embodiments, the elongate body has a substantially circular, ovular, oblong, and/or square cross-sectional shape. In some embodiments, the body has an inner diameter (not shown) of from about 1 μm to about 200 μm, or from about 5 μm to about 150 μm, or from about 15 μm to about 100 μm, or about 75 μm. In particular embodiments, the separation channel140is a capillary tube and/or a microfluidic channel. Additional suitable separation channels140are known to those of ordinary skill in the art.

In one or more embodiments, the inlet144functions as an outlet and/or the outlet functions as an inlet. In further embodiments, the inlet144functions to allow LE fluid, such as, e.g., LE solution, to enter and/or exit the separation channel140. Similarly, in further embodiments, the outlet functions to allow TE fluid, such as, e.g., TE solution, sample, and/or DNA sensor molecules to enter and exit the separation channel.

Referring specifically toFIGS. 6 and 8, in some embodiments, the separation unit130includes a plurality of separation channels140. In embodiments, the plurality of separation channels140form a bundle of separation channels150. In particular embodiments, the separation unit130includes from about 2 to about 200 separation channels140, or from about 5 to about 100 separation channels140, or from about 10 to about 50 separation channels140, or about 20 separation channels140. In one specific embodiment, the separation unit130includes 19 separation channels140. In further embodiments, the separation unit130includes 19 separation channels140having respective lengths L of from about 1 cm to about 20 cm, or from about 5 cm to about 15 cm, or about 10 cm. In one or more embodiments, the separation channels140are arranged, such as, e.g., in a bundle, such that the lengths L of each of the separation channels140are substantially parallel to one another and/or such that the inlets144and the outlets of each of the separation channels140are substantially aligned.

Referring now toFIGS. 2, 5, and 7-8, in one or more embodiments, the separation unit130includes a leading electrolyte reservoir160in fluidic communication with the at least one separation channel140. In some embodiments, the leading electrolyte reservoir160is in continuous, open fluidic communication with the at least one separation channel140. In embodiments, the leading electrolyte reservoir160includes a lower housing162, a cavity (i.e., hole)164defined by the lower housing162, and an upper housing166releasably and/or movably attached to, connected to, and/or attachable to the lower housing162for enclosing the cavity164. In some embodiments, the upper housing166is releasably and/or movably attached to or connected to the lower housing162such that the cavity164defined therein may be accessed, such as for, e.g., inserting LE solution therein. In some embodiments, the lower housing162includes an upper surface168, side surfaces170, and a lower surface172. In further embodiments, the upper surface168defines the cavity164. In one or more embodiments, the cavity164extends from the upper surface168to the lower surface172of the lower housing162, such that, e.g., a LE solution may flow through the leading electrolyte reservoir160into a separation channel140and/or bundle of separation channels releasably attached or connected thereto. In embodiments, the cavity164includes an upper surface diameter at the upper surface168of the lower housing162and a lower surface at the lower surface172of the lower housing162. The upper surface diameter may be equal to the lower surface diameter. In embodiments, the diameter of the upper surface and the lower surface may be equal and may be from about 0.5 to about 2 cm, or from about 1 to about 1.5 cm, or about 1 cm. In further embodiments, the cavity164may be substantially cylindrical. In specific embodiments, the upper housing166is formed from a poly(sulfone).

As shown inFIGS. 5-8, the separation channel140and/or bundle of separation channels150is releasably attached to, connected to, and/or attachable to the lower housing162. More specifically, the separation channel140and/or bundle of separation channels150is releasably attached to, connected to, and/or attachable to the cavity164defined by the lower housing162via an adapter148, such as, e.g., an adapter nut and/or a one-piece fitting, commercially available from LabSmith (Livermore, Calif.), connected to a port connector (not shown) within the cavity164. In embodiments, the separation channel140and/or bundle of separation channels are releasably attached to and/or connected to the lower housing162such that it may be readily interchanged, such as, e.g., in between GEITP experiments. In some embodiments, the port connector is adhered, such as, e.g., via glue and/or epoxy, to the cavity164of the lower housing162. In embodiments, the adapter148further includes a flangeless ferrule154. In some embodiments, the lower surface diameter (not shown) of the cavity164is shaped and/or sized to accommodate the adapter148, port connector, and/or flangeless ferrule154.

In one or more embodiments wherein the separation channel140and/or the bundle of separation channels150is releasably connected to the lower housing162via the adapter148, the port connector, and/or the flangeless ferrule154, the at least one separation channel140and/or the bundle of separation channels150is positioned in a vertical orientation relative to the lower surface172of the lower housing162. More specifically, in such vertical orientation, the length L of the separation channel140or the lengths L of the bundle of separation channels150may be positioned substantially perpendicular to or perpendicular to the lower surface172of the lower housing162(and/or to a horizontal plane parallel to the lower surface172of the lower housing162). For example, in some embodiments, the length L of the separation channel140or the lengths L of the bundle of separation channels150is positioned substantially parallel to the Z axis (as indicated by the X, Y, and Z coordinates inFIG. 1). Such vertical positioning and/or orientation of the separation channel140and/or bundle of separation channels150is operable to prevent contamination thereof during GEITP, to cleanly deliver charged analytes, such as, e.g., DNA, to a delivery reservoir in the sampling assembly350(as described in greater detail below), wherein a horizontal orientation would not provide for clean delivery out of the separation channel as fluid may be re-introduced into a horizontal separation channel during delivery, and to provide automated GEITP.

In further embodiments, the separation channel140or bundle of separation channels150is releasably connected to the lower housing162such that at least a portion of the separation channel140or the bundle of separation channels150extends into the cavity164such that the separation channel140or bundle of separation channels150is in selective fluidic communication with the leading electrolyte reservoir160. More specifically, in some embodiments, the inlet144of the separation channel140or the inlets144of the bundle of separation channels150extend into the cavity164such that the separation channel140or bundle of separation channels150is in selective fluidic communication with the leading electrolyte reservoir160. In this way, LE fluid may flow from the leading electrolyte reservoir160through the inlet(s)144of the separation channel140or bundle of separation channels150for the GEITP when the separation channel140or bundle of separation channels150is in open fluidic communication with the leading electrolyte reservoir160.

Referring specifically toFIGS. 1-2, 7, 9, and 15, in some embodiments, the separation unit130includes a voltage supply device180electrically connected and/or communicatively coupled to the separation channel140or the bundle of separation channels150and electrically connected and/or communicatively coupled to a power source (not shown). In further embodiments, the voltage supply device180is electrically connected and/or communicatively coupled to the separation channel140or the bundle of separation channels150via LE fluid in the leading electrolyte reservoir160and via fluid, such as, e.g., LE solution, in the separation channel140or bundle of separation channels150. In some embodiments, the voltage supply device180is a high voltage supply device180configured to provide a high voltage for GEITP. Referring toFIGS. 7 and 9, in some embodiments, the voltage supply device180includes a high voltage electrode182electrically connected and/or communicatively coupled to the leading electrolyte reservoir160and/or a ground electrode184electrically connected and/or communicatively coupled to sample372(described in greater detail below).

In some embodiments, the high voltage electrode182is configured to contact leading electrolyte fluid in the leading electrolyte reservoir160, in order to provide a voltage thereto. In this way, the high voltage electrode182may be electrically connected and/or communicatively coupled to the separation channel140or bundle of separation channels150via LE fluid in the leading electrolyte reservoir160and via fluid, such as, e.g., LE solution, in the separation channel140or bundle of separation channels150. In some embodiments, the ground electrode184is configured to contact specimen fluid in the sample372.

Additionally, the high voltage electrode182may provide a polarity, such as, e.g., positive or negative, dependent upon the polarity of the charged analytes for GEITP. Thus, the polarity of the high voltage electrode182may be selected based upon the polarity of the charged analytes. For example, in some embodiments, where the charged analytes are negative, the high voltage electrode182may be positive. Similarly, where the charged analytes are positive, the high voltage electrode182may be negative. In this way, the voltage supply device180is configured to control the direction of the flow of the charged molecules during GEITP. Suitable voltage supply devices180are known to those of ordinary skill in the art.

Referring toFIG. 7, in some embodiments, the voltage supply device180includes a resistor186. In some embodiments, the resistor186is electrically connected and/or communicatively coupled to the high voltage electrode182and/or the ground electrode184. In embodiments, the resistor186is in series with the at least one separation channel140or the bundle of separation channels150.

Referring toFIGS. 7 and 23, in some embodiments, the high voltage electrode182and the ground electrode184are configured to detect current of fluid, such as, e.g., LE fluid, present in the separation channel140and/or in the bundle of separation channels150via use of a current detection device320, such as, e.g., a data acquisition device320. More specifically, the high voltage electrode182and the ground electrode184is configured to detect current of fluid present in the separation channel140and/or in the bundle of separation channels150during GEITP. In some particular embodiments, the current in the fluid in the separation channel140and/or bundle of separation channels150is detected via measuring and/or monitoring with a current detection device320, such as, e.g., a data acquisition device320, a voltage drop across the resistor186. In some embodiments, the current data acquisition device320includes an input channel and an analog-to-digital converter. However, the current in the fluid in the separation channel140and/or in the bundle of separation channels150may be measured, monitored, and/or detected via any methods known to those of ordinary skill in the art. For example, the current may be measured via suitable current detection devices320known to those of ordinary skill in the art. In embodiments, the current detection device320is communicatively coupled to the voltage detection device.

Referring toFIGS. 1-2 and 7, in some embodiments, the separation unit130includes at least one pressure control device200connected to the leading electrolyte reservoir160. In some embodiments, the pressure control device200is fluidly connected to the leading electrolyte reservoir160, in order to provide pressure control thereto. In further embodiments, the pressure control device200is connected to headspace, such as, e.g., above the cavity164, of the leading electrolyte reservoir160, in order to provide pressure control thereto. The pressure control device200is configured to create a variable pressure-driven counterflow for GEITP. In some embodiments, the pressure control device200is configured to create a pressure differential across the inlet144and the outlet of the separation channel140and/or bundle of separation channels150. In embodiments, the pressure differential across the inlet144and the outlet of the separation channel140varies with time throughout the GEITP such that charged analytes of interest may be selectively and/or sequentially separated, purified, concentrated, quantified, and/or extracted from a sample and/or such that contaminants may be excluded therefrom. In particular embodiments, the pressure control device200creates a pressure differential of from about −20,000 Pa to about 20,000 Pa, or from about −10,000 Pa to about 10,000 Pa, or from about −7,000 Pa to about 7,000 Pa.

Suitable pressure control devices200include, but should not be limited to, pumping devices, such as, e.g., a syringe pump. However, additional suitable pressure control devices200are known to those of ordinary skill in the art. In some embodiments, the pumping device is accommodated by and/or within the support structure510(described in greater detail below). In some embodiments, the pressure control device200further includes a pressure detection device (not shown), such as, e.g., a bidirectional pressure gauge (not shown) communicatively coupled to the leading electrolyte reservoir160. In some embodiments, the bidirectional pressure gauge is accommodated by and/or within the support structure510. Suitable pressure detection devices are known to those of ordinary skill in the art. Suitable bidirectional pressure gauges are commercially available from Omega Engineering (Stamford, Conn.).

Referring toFIGS. 1-4, 7, 9, and 11, in one or more embodiments, the electrophoretic assembly110includes a detection unit230communicatively coupled to the separation unit130. In embodiments, the detection unit230includes a support structure250, a conductivity detection device270attached to, connected to, attachable to and/or accommodated by the support structure250and communicatively coupled to the separation channel140, at least one light source290attached to, connected to, attachable to, and/or accommodated by the support structure250, and/or a light source detection device310attached to, connected to, attachable to, and/or accommodated by the support structure250.

Referring toFIGS. 11-14, in some embodiments, the detection unit230includes a support structure250. In some embodiments, the support structure250includes an upper portion252and a lower portion262. In some embodiments, the upper portion252includes an upper surface256, side surfaces258, and a lower surface (not shown). In embodiments, the side surfaces258include a first side surface, a second side surface adjacent to the first side surface, a third side surface adjacent to the second side surface, and a fourth side surface adjacent to the third side surface and the first side surface. In embodiments, the first side surface and the third side surface are substantially parallel, and the second side surface and the fourth side surface are substantially parallel. In further embodiments, the second side surface and the fourth side surface are substantially normal to the first side surface and the third side surface. In one or more embodiments, the lower portion262includes an upper surface (not shown), a lower surface266, and side surfaces268. In one or more embodiments, the support structure250provides an interface for substantial alignment and/or alignment of the conductivity detection device270, the light source290, the light source detection device310, and/or the separation channel140(described in greater detail below). In this way, in operation, the conductivity detection device270may detect charged analytes present in the at least one separation channel, the light source290may direct and/or emit light through the at least one separation channel140(as described in greater detail below), exciting fluorescence in charged analytes contacted with sensor molecules present therein (as described in greater detail below), and the light source detection device310may detect the fluorescence excited in the at least one separation channel140(as described in greater detail below). In some embodiments, the support structure250is formed from a polyoxymethylene, i.e., Delrin.

Referring toFIGS. 1-3, 5-6, and 9-11, in some embodiments, the detection unit230includes a conductivity detection device270attached to, connected to, and/or attachable to the support structure250and communicatively coupled to the separation channel140and/or bundle of separation channels150. More specifically, in one or more embodiments, the conductivity detection device270is attached to, connected to, attachable to and/or accommodated by the upper portion252of the support structure250and the lower portion262of the support structure250. In further embodiments, the conductivity detection device270is attached to, connected to, attachable to, and/or accommodated by the lower surface (not shown) of the upper portion252of the support structure and to the upper surface (not shown) of the lower portion262of the support structure250.

In some embodiments, the conductivity detection device270includes ring electrodes. In one or more embodiments, the conductivity detection device270defines a cavity (not shown) for accommodating the separation channel140and/or bundle of separation channels150. In some embodiments, the conductivity detection device270is communicatively coupled to and/or interfaced with the separation channel140and/or bundle of separation channels150, in order to detect conductivity therethrough. In further embodiments, the conductivity detection device270is configured to detect conductivity of fluid present in the separation channel140and/or bundle of separation channels150during GEITP. Suitable conductivity detection devices270include contactless conductivity detectors. However, additional suitable conductivity detection devices270are known to those of ordinary skill in the art.

Referring toFIGS. 1 and 5-6, in some embodiments, the detection unit230includes a light source290. In one or more embodiments, the light source290is accommodated by the support structure250. In some embodiments, the light source290includes a light source input292, which is accommodated the support structure250. More specifically, in some embodiments, the light source290is attached to, connected to, attachable to, and/or accommodated by the upper portion252of the support structure250(described in greater detail below).

In some embodiments, the light source290directs and/or emits light through the separation channel140and/or bundle of separation channels150. In one or more embodiments, the light source290is configured to direct and/or emit light through fluid in the separation channel140and/or bundle of separation channels150during GEITP to excite fluorescence therein. For example, in embodiments wherein the charged analytes are DNA and wherein DNA sensor molecules are in contact with and/or intercalated within DNA in the separation channel140and/or bundle of separation channels150(described in greater detail below), the light source290is configured to direct and/or emit light through such fluid during GEITP to excite and/or induce fluorescence therein. In some specific embodiments, the light source290is a laser. In some embodiments wherein the light source290is a laser, the light source input292is a laser-induced fluorescence input292. However, additional suitable light sources290are known to those of ordinary skill in the art.

Referring toFIGS. 1-2, 5-6, and 9-14, in embodiments, the detection unit230includes a light source detection device310. In one or more embodiments, the light source detection device310is attached to, connected to, attachable to, and/or accommodated by the support structure250. More specifically, in some embodiments, the light source detection device310(which may include a photomultiplier tube (i.e., PMT) output312as described in greater detail below) is accommodated by the upper portion252of the support structure250(described in greater detail below).

In some embodiments, the light source detection device310is configured to detect light and/or fluorescence excited and/or emitted through and/or by fluid in the separation channel140and/or bundle of separation channels150during GEITP, as previously described with regard to the light source290. In one or more embodiments, the light source detection device310is communicatively coupled to and/or interfaced with the light source290and/or the separation channel140and/or bundle of separation channels150, in order to detect light and/or fluorescence excited and/or emitted from the separation channel140and/or bundle of separation channels150, as previously described with regard to the light source290. In one or more specific embodiments, the light source detection device310is photomultiplier tubes, accommodated by the support structure250. In some embodiments, the light source detection device310includes a photomultiplier tube (i.e., PMT) output312. However, additional suitable light source detection devices310are known to those of ordinary skill in the art.

Referring toFIGS. 5-6 and 10-14, in some embodiments, the support structure250provides an interface for substantial alignment and/or alignment of the conductivity detection device270, the light source290, the light source detection device310, the separation channel140, the bundle of separation channels150, the laser-induced fluorescence input292, the PMT output312, and/or the ground electrode184. For example, the support structure250defines a separation channel cavity260,264, for accommodating the at least one separation channel140or bundle of separation channels150therein. Referring specifically toFIGS. 5 and 12-13, each of the upper portion252and the lower portion262of the support structure250provides a cavity (i.e., hole)260,264, respectively, for accommodating the separation channel140and/or bundle of separation channels150therethrough. More specifically, each of the upper portion252, the lower portions262, and the conductivity detection device270provides corresponding cavities through which the separation channel140and/or bundle of separation channels150may extend therethrough and be accommodated. In this way, at least a portion of the separation channel140and/or bundle of separation channels150may extend into the cavities260,264of the upper portion252and the lower portion262and the cavity of the conductivity detection device270such that light may be directed and/or emitted therethrough and such that resulting emitted and/or excited fluorescence may be detected therefrom during GEITP.

In embodiments, the support structure250defines a light source cavity334for accommodating the light source290therein and also defines a light source detection cavity336for accommodating the light source detection device310therein. As another example, referring specifically toFIG. 10, the support structure250provides a cavity334for accommodating the input292, e.g., the laser-induced fluorescence input292, and a recess and/or cavity336for accommodating the PMT output312. More specifically, adjacent side surfaces258of the upper portion252of the support structure250respectively provide the cavity334and the recess and/or cavity336. In some embodiments, as shown inFIGS. 13-14, the support structure250provides a cavity342for accommodating the ground electrode184. The cavities334,336,342, and/or the cavity of the conductivity detection device270may respectively correspond substantially in size and/or shape to the laser-induced fluorescence input292, the PMT output312, and/or the ground electrode184.

In embodiments, the light source290is accommodated by the support structure250at a fluorescence excitation angle α. More specifically, the support structure250may define the light source cavity334at a fluorescence excitation angle α of from about 30° to about 150°. In embodiments, the fluorescence excitation angle α is about 90°. The fluorescence excitation angle α is formed by intersection of an axis of the light source290and a plane bisecting the separation channel cavity260,264and being parallel to the side surfaces258of the support structure250(and/or to a plane parallel to a vertical axis). In embodiments, the light source detection device310is accommodated by the support structure250at a fluorescence detection angle β. More specifically, the support structure250may define the light source detection cavity336at a fluorescence detection angle β of from about 30° to about 150°. In embodiments, the fluorescence detection angle β is about 45°. The fluorescence detection angle β is formed by intersection of an axis of the light source detection device310and a plane bisecting the separation channel cavity260,264and being parallel to the side surfaces258of the support structure250(and/or to a plane parallel to a vertical axis). In this way, the support structure250provides an interface such that fluorescence in the separation channel140and/or bundle of separation channels150may be excited and/or detected.

Referring toFIGS. 1-3, in some embodiments, the electrophoretic assembly110is capable of movement. In some embodiments, the electrophoretic assembly110is attached to, connected to, and/or attachable to a first moveable support structure280. The first moveable support structure280is configured to provide movement of the electrophoretic assembly110in the Z direction (as indicated by the X, Y, and Z coordinates inFIG. 1) via movement thereof. For example, in one or more embodiments, the electrophoretic assembly110is capable of upward and/or downward movement via movement of the first moveable support structure280, relative to a central resting position thereof.

In some embodiments, the first moveable support structure280is attached to, connected to, and/or attachable to a second moveable support structure300. The second moveable support structure300is configured to provide movement of the electrophoretic assembly110in the X direction (as indicated by the X, Y, and Z coordinates inFIG. 1) via movement thereof. For example, in one or more embodiments, the electrophoretic assembly110is capable of movement from side to side, relative to a central resting position thereof.

Referring toFIG. 1, in further embodiments, the first and second moveable support structures280,300are capable of automated movement in the respective the X and Z directions. In some embodiments, each of the first and second moveable support structures280,300is movably attached to a respective support surface284,302. The first and second moveable support structures280,300are capable of automated movement in the respective X and Z directions relative to the respective support surfaces284,302. In this way, the moveable support structure280and the electrophoretic assembly110provided thereon are capable of movement in the X and Z directions such that samples372(described in greater detail below) may be placed in contact with the outlet of the separation channel140and/or outlets of the bundle of separation channels150.

In some embodiments, the voltage supply device180, the data acquisition device320, the pressure control device200, the pressure detection device (not shown), the conductivity detection device270, the at least one light source290, the light source detection device310, the first, second, and third (described in greater detail below) moveable support structures280,300,380, and/or the first, second, and third (described in greater detail below) motors,410,430,440, are communicatively coupled to a data receiver (not shown) and/or a controller (as described in greater detail below with regard to GEITP systems). It is to be understood that any structures and/or devices previously described herein with regard to the isotachophoretic apparatus100may be communicatively coupled to a data receiver and/or the controller, such that the GEITP methods (described in greater detail below) may be fully automated.

Referring toFIGS. 1-3 and 15-16, in one or more embodiments, the isotachophoretic apparatus100includes a sampling assembly350. In further embodiments, the sampling assembly350is operably connected to the electrophoretic assembly110. In embodiments, the sampling assembly350is operably connected to the electrophoretic assembly110such that samples372may be placed in contact with the outlet of the separation channel140and/or outlets of the bundle of separation channels150. In some embodiments, the length L of the separation channel140and/or lengths L bundle of separation channels150are oriented substantially perpendicular to the horizontal plane of a sampling platform360of the sampling assembly350, such that the samples372may be placed in contact with the outlet of the separation channel140and/or outlets of the bundle of separation channels150. In some embodiments, the sampling assembly350includes a sampling platform360attached to, connected to, and/or attachable to a moveable support structure380.

Referring toFIGS. 1-2 and 15-16, in one or more embodiments, the sampling assembly350includes a sampling platform360. In embodiments, the sampling assembly350is attached to, connected to, and/or attachable to a third moveable support structure380. In some embodiments, the sampling assembly includes an upper surface362, side surfaces364, and a lower surface366. In further embodiments, the upper surface362defines a plurality of cavities (i.e., holes)368extending therethrough. The cavities368may be configured such that samples372, such as samples372provided in sample reservoirs, such as, e.g., Eppendorf, microcentrifuge, and/or PCR tubes, may be accommodated therein. Examples of suitable samples372which may be accommodated by the cavities368include, but should not be limited to, sample reservoirs including specimens of interest, pre-treatment reagent reservoirs, buffer reservoirs, and/or delivery reservoirs for delivery of the separated, purified, concentrated, and/or quantified charged analytes. In some embodiments, the cavities368have a substantially circular, ovular, oblong, and/or square cross-sectional shape. However, the cavities368may have any cross-sectional shape and/or size such that the cavities368may accommodate samples372therein. In one or more embodiments, as shown inFIG. 16, the sampling assembly defines a space370in between the upper surface362and the lower surface366and the side surfaces364, such that samples372may extend at least partially therethrough and/or be securely accommodated therein.

Referring toFIGS. 1-4, in one or more embodiments, the sampling assembly350includes a third moveable support structure380, i.e., a sampling moveable support structure. The third moveable support structure380is configured to provide movement of the sampling platform360in the Y direction (as indicated by the X, Y, and Z coordinates inFIG. 1) via movement thereof. For example, in one or more embodiments, the sampling platform360is capable of forward and/or backward movement via movement of the third moveable support structure380, relative to a central resting position thereof.

In further embodiments, the third moveable support structure380(as well as the first moveable support in the Z direction and the second moveable support in the Y direction) is capable of automated movement in the Y direction. The third moveable support structure380may include a moveable surface382movably attached to a support surface384. In some embodiments, the sampling platform360is attached to, connected to, and/or attachable to the moveable surface382of the third moveable support structure380. In this way, the third moveable support structure380and the sampling platform360provided thereon are capable of moving in the Y direction such that samples372may be placed in contact with the outlet of the separation channel140and/or outlets of the bundle of separation channels150during GEITP.

Referring toFIGS. 1-4, in some embodiments, the isotachophoretic apparatus100includes a first, second, and third motor,410,430,440configured to provide motion of the first, second, and/or third moveable support structures280,300,380. In some embodiments, the first, second, and/or third motor410,430,440, are respectively electrically connected and/or communicatively coupled to the first, second, and/or third moveable support structures280,300,380, in order to provide motion thereto. In embodiments, the first, second, and third moveable support structures280,300,380, and the first, second, and third motors410,430,440, are configured to provide movement, such as, e.g., translation, of the electrophoretic assembly110and the sampling assembly350. Suitable examples of motors for providing motion to the first, second, and/or third moveable support structures280,300,380include electric and/or hydraulic motors. In some embodiments, the first, second, and/or third motors410,430,440, are stepper motors. However, additional motors for providing movement of the first, second, and third moveable support structures280,300,380are known to those of ordinary skill in the art.

Referring toFIGS. 1-4, in one or more embodiments, the isotachophoretic apparatus100includes a support structure510connected to the electrophoretic assembly110and/or to the sampling assembly350. In some embodiments, the support structure510accommodates the at least one pumping device, such as, e.g., a syringe pump and at least one pressure detection device (not shown).

Embodiments of isotachophoretic apparatus100have been described in detail. Embodiments of systems for performing GEITP to separate charged analytes from samples will be described with reference toFIG. 23. Thereafter, methods for separating, purifying, concentrating, quantifying, and/or extracting charged analytes will be described with reference toFIG. 17.

In one or more embodiments, a system600for performing GEITP to separate, purify, concentrate, and/or extract charged analytes is disclosed. In embodiments, the system600includes an apparatus100for performing GEITP, as previously described herein. For example, the apparatus100may include a moveable electrophoretic assembly110, a sampling assembly350operably connected to the moveable electrophoretic assembly110, and a support structure510connected to at least one of the moveable electrophoretic assembly110or the sampling assembly510.

Referring toFIG. 23, in embodiments, the system600includes a data receiver and/or controller610communicatively coupled (as shown via double headed arrows) to the moveable electrophoretic assembly110, the sampling assembly310, and/or the support structure510. As previously described, the voltage supply device180, the data acquisition device320of the voltage supply devices180, the pressure control device200, the pressure detection device (not shown), the conductivity detection device270, the at least one light source290, the light source detection device310, the first, second, and third (described in greater detail below) moveable support structures280,300,380, and/or the first, second, and third (described in greater detail below) motors,410,430,440, may be communicatively coupled to a data receiver (not shown) and/or a controller610.

In some embodiments, the controller includes a processor630and a storage medium650containing computer readable and executable instructions which, when executed by the processor, cause the controller to automatically execute a series of steps to control and/or adjust the voltage applied during GEITP and/or the pressure applied during GEITP, and/or to control and/or adjust the positioning of the moveable support structures280,300,380. In this way, the focusing of the charged analytes may be controlled. The processor630may be communicatively coupled to the storage medium650.

In some embodiments, the computer readable and executable instructions execute a series of steps, such as, e.g., steps (1)-(7), as described in greater detail below with regard to methods for separating, purifying, concentrating, quantifying, and/or extracting charged analytes utilizing the isotachophoretic apparatus100.

In one or more embodiments, methods for separating, purifying, concentrating, quantifying, and/or extracting charged analytes utilizing the isotachophoretic apparatus100and/or the isotachophoretic system600as previously described herein are disclosed. In one or more embodiments, such methods relate to separating, purifying, concentrating, quantifying, and/or extracting biomolecules, such as, e.g., DNA and/or RNA, from samples, such as, e.g., crude samples, utilizing the isotachophoretic apparatus100and/or the isotachophoretic system600as previously described herein. In some embodiments, the methods generally include:

(1) optionally pre-treating the separation channel140and/or bundle of separation channels150(hereinafter, collectively referred to as the separation channel140);

(2) inserting LE fluid into the separation channel140;

(3) contacting the separation channel140with a sample and TE fluid;

(4) separating, purifying, concentrating, and/or quantifying charged analytes in and/or from the sample via GEITP by:(a) producing a pressure-driven counterflow of fluid through the separation channel140;(b) applying a voltage to the separation channel to produce an electric field to drive electrophoretic migration of charged analytes toward the separation channel140; and(c) varying with respect to time the pressure-driven counterflow through the separation channel to control focusing and/or separation of the charged analytes via initiation of a pressure ramp (such as is shown inFIG. 17);

(5) optionally directing light through the separation channel to excite fluorescence in the charged analytes;

(7) optionally delivering the charged analytes to a delivery reservoir (such as is shown inFIG. 17).

Performance of steps (1)-(7) may be referred to as a run of GEITP. Additionally, the positioning of the sampling assembly350may be adjusted in the Y direction as needed in order to perform any of steps (1)-(7) via movement of the third moveable support structure380. In some embodiments, the positioning of the sampling assembly350may be adjusted prior to, during, and/or after performance of any of steps (1)-(7).

With regard to pre-treating the separation channel140, in one or more embodiments, such pre-treatment involves rinsing the separation channel140with pre-treatment reagents, such as, e.g., distilled water, negatively-charged and/or positively-charged capillary coatings, and/or salmon sperm DNA. In some embodiments, the separation channel140is rinsed with pre-treatment reagents by: (1) placing a pre-treatment reagent in a pre-treatment reagent reservoir on the sampling platform360in contact with the outlet of the separation channel140via movement of the first and/or second moveable support structures280,300; and (2) varying the pressure of the headspace in the LE reservoir160to achieve insertion of the pre-treatment reagents within the channel (not shown). In embodiments, the separation channel140is rinsed with pre-treatment reagents only one time each day. Stated another way, in embodiments, the separation channel140is not rinsed with pre-treatment reagents prior to every run of GEITP performed; rather, it may be rinsed with pre-treatment reagents periodically, such as, e.g., one time before each series of GEITP runs performed.

Similarly, varying the pressure of the headspace in the LE reservoir160also achieves extraction of the pre-treatment reagents therefrom. Specifically, the pressure may be adjusted, such as, e.g., decreased, to from about −100 Pa about −1200 Pa to achieve insertion of the pre-treatment reagents within the channel (not shown), and may be adjusted, such as, e.g., increased, to from about −200 Pa to about −1000 Pa to achieve extraction of the pre-treatment reagents therefrom. For example, during the pre-treating and/or washing of the separation channel140, the pressure may initially be about −200 Pa. Once the separation channel140has been contacted with the appropriate pre-treatment reagent, the pressure may be decreased to about −1200 Pa for from about 60 seconds to about 300 seconds. In particular embodiments, the pressure was decreased to about −1200 Pa for approximately 300 seconds. In embodiments, the separation channel140is then moved to a waste reservoir and the pressure is increased to about −1000 Pa. At the end of the pre-treating and/or washing, the pressure may be adjusted to from about 0 Pa to about −700 Pa. In this pre-treatment step, the separation channel140and the electrophoretic assembly110may move in the X and/or Z (i.e., in the horizontal and/or vertical) direction via movement of the first and/or second moveable support structures280,300. In this way, the separation channel140and the electrophoretic assembly110may contact varying reservoirs, such as, e.g., pre-treatment reagent reservoirs and waste reservoirs, in the sampling assembly350.

With regard to inserting LE fluid, such as, e.g., LE solution, into the separation channel140, in one or more embodiments, such insertion involves varying the pressure to achieve insertion thereof. More specifically, the pressure may be adjusted to from about −700 Pa to about −200 Pa to achieve insertion of the LE fluid from the LE reservoir160through the inlet144of the separation channel140and into the channel thereof (not shown). In some embodiments, following insertion, the pressure may be adjusted, such as, e.g., increased, to a slight positive value and may be regulated at a slight positive value to drive a small flow of the LE fluid into the separation channel140. Suitable LE fluids include, but should not be limited to, Tris buffers. The LE fluids, such as, e.g., LE buffers, should be selected such that the electrophoretic mobility of the LE fluid is higher than that of the charged analytes. Moreover, where biomolecules, such as, e.g., DNA, RNA, proteins, and/or carbohydrates, are the charged analyte of interest, the LE fluid may include biomolecule sensor molecules, such as, e.g., DNA sensor molecules, RNA sensor molecules, protein sensor molecules, and/or carbohydrate sensor molecules, such as affinity-labeled molecules, which are capable of fluorescing upon contact with biomolecules and excitation thereof. Specific examples of such biomolecule sensor molecules which are capable of fluorescing upon contact with biomolecules are known to those of ordinary skill in the art. Thus, in embodiments wherein the detection unit230includes at least one light source290and a light source detection device310, the LE fluid may include biomolecule sensor molecules such that direction and/or emission of light through the separation channel140by the light source290will excite and/or emit fluorescence of biomolecules present therein which have contacted and/or interacted with biomolecule sensor molecules. As a result of such fluorescence excitation and/or emission, biomolecule analytes may then be detected via fluorescence detection with the light source detection device310.

With regard to contacting the separation channel140with sample and TE fluid, such as, e.g., TE solution, in one or more embodiments, such contacting is achieved by: (1) placing a sample of interest prepared in a TE fluid in a TE reservoir on the sampling platform360in contact with the outlet of the separation channel140via movement of the first and/or second moveable support structures280,300; and (2) varying the pressure of the headspace in the LE reservoir160to prevent insertion of the sample and TE fluid within the channel (not shown). In some embodiments, the sample may be treated with lysing agents and/or may be diluted prior to insertion into the separation channel140. In other embodiments, the sample of interest is prepared in the TE fluid via dissolution and/or slurrying therein. In embodiments, the pressure is adjusted to about −700 Pa to prevent insertion of the sample and TE fluid within the channel. In further embodiments, the sample is not filtered, centrifuged, and/or precipitated prior to insertion into the separation channel140. Suitable TE fluids include, but should not be limited to, Tris HEPES. The TE fluids, such as, e.g., TE buffers, should be selected such that the electrophoretic mobility of the TE fluid is lower than that of the charged analytes. In this contacting step, the separation channel140and the electrophoretic assembly110may move in the X and/or Z direction via movement of the first and/or second moveable support structures280,300. In this way, the separation channel140and the electrophoretic assembly110may contact varying reservoirs, such as, e.g., the TE reservoir, in the sampling assembly350. However, contact between the separation channel140and the TE reservoir in the sampling assembly350should be maintained continuously from steps (3)-(6) (discussed in greater detail below).

With regard to separating, purifying, concentrating, and/or quantifying charged analytes in the sample via GEITP, as described in U.S. Pat. No. 8,080,144, the contents of which are hereby incorporated by reference in their entirety, GEITP is a fluid-phase electroseparation technique which combines the ability of isotachophoresis (i.e., ITP) to concentrate, such as, e.g., to focus, charged analytes with a controlled, variable pressure-driven counterflow for improved control and selectivity thereof. GEITP allows for high-resolution separations in short separation channel140lengths, does not require a discrete injection method, and is fast relative to other electrophoretic methods. During GEITP, electrophoretic mobilities of the LE and TE fluids and/or ions thereof, bracket that of the charged analytes in the sample. In some embodiments, GEITP is performed by: (a) producing a pressure-driven counterflow of fluid through the separation channel140, (b) applying a voltage to the separation channel140to produce an electric field to drive electrophoretic migration of charged analytes toward the separation channel140, and/or (c) varying with respect to time the pressure-driven counterflow of the LE fluid through the separation channel140.

With regard to producing a pressure-driven counterflow of fluid through the separation channel, such as, e.g., the LE fluid, the pressure-driven counterflow includes electroosmotic flow (i.e., EOF) and/or controlled, variable pressure-driven flow, i.e., hydrodynamic flow. Thus, the pressure-driven counterflow of the LE fluid may be produced by application of a voltage (such as is described with regard to step (4)(b)), and/or via application of a pressure differential across the inlet144and the outlet of the separation channel140(such as was previously described). In embodiments, the pressure differential is adjusted to from about −10,000 Pa to about 10,000 Pa, or to from about −5,000 Pa to about 5,000 Pa, or from about −1,000 Pa to about 1,000 Pa, or from about −750 Pa, to about 750 Pa. In particular embodiments, the pressure differential is adjusted to about −700 Pa. In embodiments, the pressure-driven counterflow (i.e., bulk flow) is initially produced such that the charged analytes are prevented from being inserted into the outlet of the separation channel140and/or such that LE fluid is introduced into the TE reservoir.

The pressure-driven counterflow may be driven in a direction from the inlet144to the outlet of the separation channel140, or it may be driven in a direction from the outlet to the inlet144of the separation channel140. Typically in GEITP, the pressure-driven counterflow is driven in a direction opposing the net migration of charged analytes through the separation channel140. Thus, in embodiments, the pressure-driven counterflow is driven in a direction from the inlet144to the outlet of the separation channel140, In embodiments, the pressure-driven counterflow of LE fluid is produced by application of a voltage and application of a pressure differential.

With regard to applying a voltage to the separation channel to drive electrophoretic migration of charged analytes toward the separation channel140, the polarity of the voltage is dependent upon the direction of the pressure-driven counterflow and the sign of the charged analytes. For example, if the charged analytes are negative in fluid and the pressure-driven counterflow is from the inlet144to the outlet of the separation channel140, the voltage applied to the inlet144of the separation channel140may be positive relative to the voltage applied to the outlet of the separation channel140. In embodiments, a constant voltage of about 100 V to about 5,000 V or from about 500 V to about 2,000 V, or about 2000 V is applied to the separation channel140to drive electrophoretic migration of the charged analytes. Such constant voltage may be applied to drive electrophoretic migration of the charged analytes toward the junction of the TE reservoir in the sampling assembly350and the outlet of the separation channel140.

With regard to varying with respect to time the pressure-driven counterflow of the fluid through the separation channel140, such as, e.g., the LE fluid, such variation may be effective to control focusing of the charged analytes in the TE reservoir prior to introduction into the separation channel140and/or to control focusing of the charged analytes in the separation channel140prior to detection thereof. In this manner, charged analytes and TE fluid may be driven into an ionic interface, forming enriched ITP layers in the TE reservoir. Thus, controlled focusing of the charged analytes may be achieved outside of the separation channel140in the TE reservoir. In some embodiments, controlled focusing of the charged analytes may be achieved within the separation channel140prior to encountering the conductivity detection device270and/or the light source detection device310(i.e., upstream of the detection device270and/or the light source detection device310).

In embodiments, controlled focusing of the charged analytes is achieved by ensuring that the current (which may serve a measure of occlusion of the separation channel140) of fluid in the separation channel140does not exceed a threshold value. Thus, in embodiments, the current of fluid in the separation channel140is determined, detected, and/or monitored, such as, e.g., with a data acquisition device320communicatively coupled to the at least one separation channel and/or to the voltage supply device, to ensure that it does not exceed a threshold value. The threshold value for the current of fluid in the separation channel140may be from about 10 μA to about 500 μA. In embodiments wherein a single separation channel140is employed, the threshold value for the current of fluid in the separation channel140is from about 20 μA to about 30 μA. In one particular embodiment, the threshold value for the current of fluid in the separation channel140is about 23 μA. In embodiments wherein a bundle of separation channels150is employed, the threshold value for the current of fluid in the separation channel140is from about 310 μA to about 500 μA.

Generally, the pressure may be varied to ensure that the current of fluid in the separation channel140does not exceed the threshold value. In some embodiments, the pressure is varied when the current of the solution is determined to be about 90% of the threshold value. In embodiments, in view of the above-noted guidelines, the pressure is varied and/or adjusted to afford a constant current for focusing the charged analytes, such as, e.g., to afford a constant current that is about 80% of the threshold value. In some embodiments, the pressure is held at from about −700 Pa to about −200 Pa, or at from about −600 Pa to about −300 Pa, or at about −500 Pa. In general, however, the pressure is initially adjusted until a desired, constant current value is maintained. In this way, the TE fluid may be prevented from moving into the separation channel140and/or the prevented from encountering the detection device270and/or the light source detection device310. Thus, the charged analytes may focus in between the LE solution and the TE solution in the TE reservoir and/or within a portion of the separation channel140upstream of the detection device270and/or the light source detection device310. Such focusing of the charged analytes may be accomplished in from about 1 minute to about 10 minutes. Thus, the pressure may be varied: (i) to achieve controlled focusing of the charged analytes; (ii) to introduce the charged analytes into the separation channel140(and/or to a region thereof wherein the conductivity detection device270and/or the light source detection device310are located); and/or (iii) to deliver the charged analytes to a delivery reservoir.

With regard to introduction of the charged analytes into the separation channel140via the outlet thereof and/or migration of the charged analytes within the separation channel140to encounter the detection device270and/or the light source detection device310, a pressure ramp is initiated. In general, the pressure may be adjusted such that it is decreased. More specifically, the pressure is generally adjusted, such as, e.g., decreased, to about −700 Pa to about −1,500 Pa, or to about −600 Pa to about −1,300 Pa, or to about −500 Pa to about −1,200 Pa. In particular embodiments, the pressure is adjusted at a rate of from about −10 Pa/s to about −25 Pa/s, or at a rate of about −20 Pa/s. Such may be completed in from about 125 to about 500 s. Generally, the pressure ramp may be terminated after the interface is detected via conductivity of the fluid in the separation channel140and/or the fluorescence emitted and/or excited by the charged analytes in the separation channel140, as previously described and as described below. In embodiments, the pressure ramp is terminated once the interface of the charged analytes has passed and/or is downstream of the detection device270and/or the light source detection device310. As shown inFIG. 17, once the current (μA) is low, such as, e.g., is reduced to approximately less than about 10 μA, the charged analytes have been incorporated into the separation channel140and/or have encountered the detection device270and/or the light source detection device310. At this point, delivery of the charged analytes to a delivery reservoir may be initiated.

With regard to detecting separated, purified, concentrated, and/or quantified charged analytes in the sample via conductivity detection and/or fluorescence detection, in one or more embodiments, such is achieved via detecting and/or monitoring the conductivity of the fluid in the separation channel140and/or the fluorescence emitted and/or excited by the charged analytes in the separation channel140(wherein peaks may be determined). Such detection methods are known to those of ordinary skill in the art. In embodiments, the conductivity detection device270detects movement of the interface of the charged analytes (bracketed by the TE solution and the LE solution) thereby. After the conductivity detection device270detects movement of the interface thereby, the voltage is turned off and the pressure is adjusted to 0 Pa (thereby stopping flow of the solution in the separation channel140). Similarly, in embodiments, the light source detection device310detects fluorescence of biomolecule sensor molecules, such as, e.g., DNA sensor molecules, in the LE fluid interacting with biomolecule analytes, such as, e.g., DNA. In some embodiments, the quantity of charged analytes in the sample may be determined via calculating the area under the curve provided via the fluorescence detection and comparing such calculation to a standard curve (such as is described in greater detail in the Example below).

With regard to delivery of the charged analytes to a delivery reservoir, in embodiments, the delivery of the charged analytes to the delivery reservoir includes: (1) placing a rinsing reagent, such as, e.g., TE fluid, in a rinsing reagent reservoir on the sampling platform360in contact with the outlet of the separation channel140via movement of the first and/or second moveable support structures280,300; (2) placing the outlet of the separation channel140within and/or above the delivery reservoir (which is a clean reservoir) via movement of the first and/or second moveable support structures280,300; and (3) extracting the charged analytes from the separation channel140into the delivery reservoir.

With regard to placing a rinsing reagent in contact with the outlet of the separation channel140, in embodiments, the separation channel140is contacted with the rinsing reagent such that the rinsing reagent is not inserted within the separation channel140. Such contacting is effective to remove environmental contaminants and/or biomolecule inhibitors from an outside surface of the separation channel140. In embodiments, contacting the separation channel140with the rinsing reagent is performed in about 1 s.

With regard to placing the outlet of the separation channel140within and/or above the delivery reservoir, in embodiments, the delivery reservoir includes a delivery buffer, such as, e.g., Tris EDTA−4. In further embodiments, the outlet of the separation channel140is contacted with the delivery buffer in the delivery reservoir. Such contact with the delivery buffer functions to aid in the extraction of the charged analytes from the separation channel140. In alternative embodiments, the delivery reservoir does not include a delivery buffer and/or is empty prior to extraction of the charged analytes therein. Placing the outlet of the separation channel140within and/or above a delivery reservoir may be effected via movement in the X and/or Z (i.e., in the horizontal and/or vertical) direction of the first and/or second moveable support structures280,300.

With regard to extracting the charged analytes from the separation channel140into the delivery reservoir, in embodiments, varying the pressure of the headspace in the LE reservoir160achieves extraction of the charged analytes from the separation channel140into the delivery reservoir. In embodiments, such variation of the pressure of the headspace to achieve extraction of the charged analytes involves adjusting, such as, e.g., increasing, the pressure to from about −1,500 Pa to about 2,000 Pa, or to from about −1,400 Pa to about 1,500 Pa, or to from about −1,200 Pa to about 1,000 Pa. In particular embodiments, the pressure is adjusted to about 2,000 Pa. Such delivery may be completed in from about 30 s to about 300 s. In this delivery step, the separation channel140and the electrophoretic assembly110may move in the X and/or Z (i.e., in the horizontal and/or vertical) direction via movement of the first and/or second moveable support structures280,300. In this way, the separation channel140and the electrophoretic assembly110may contact varying reservoirs, such as, e.g., rinsing reagent reservoirs and delivery reservoirs, in the sampling assembly350.

In embodiments, the separation channel140is contacted with a storage reagent in a storage reagent reservoir on the sampling platform36following the delivery step. In embodiments, the storage reagent reservoir has a storage reagent, such as, e.g., distilled water, inserted therein. Such contact is effected via movement in the X and/or Z (i.e., in the horizontal and/or vertical) direction of the first and/or second moveable support structures280,300. In embodiments, the storage reagent is not inserted within the separation channel140. In embodiments, the pressure is adjusted to about −700 Pa. In further embodiments, the separation channel140is contacted with the storage reagent in the storage reagent reservoir until an additional run of GEITP is performed and/or until the separation channel140is rinsed in between runs of GEITP. Such contacting may be effective to prevent evaporation of LE buffer and/or formation of crystals within the separation channel140.

In embodiments wherein DNA is the charged analyte of interest, DNA may be detected separated, purified, concentrated, and/or quantified via GEITP as described herein in less than 5 minutes.

EXAMPLES

The following non-limiting examples illustrate use of an isotachophoretic apparatus.

Example 1: DNA Separation, Purification, Concentration, Quantification, and Delivery from a Crude Sample Using Gradient Elution Isotachophoresis

1. Materials and Methods

DNA purification, concentration, and delivery required several buffer solutions. LE solution had a measured pH of 8.2 and consisted of 50 mmol/L Tris (Sigma), 25 mmol/L HCl (Fluka), 0.1% w/w PVP (40 000 amu, Sigma), 0.1% w/w Triton-X 100 (Sigma), and 1×SYBR Green I (Molecular Probes). TE solution was 25 mmol/L Tris, 25 mM HEPES (Sigma), 0.1% w/w PVP, and 0.1% w/w Triton-X 100 with a measured pH of 8.0. Triton-X 100 was included in the LE and TE solutions, because this surfactant resulted in the degradation of cellular membranes during cell lysis of the buccal swab samples. PVP coated the capillary surfaces dynamically, with the intended purpose of aiding in eliminating the carry-over of DNA between samples. The fluorescent dye SYBR Green I allowed on-line DNA quantification using LIF during gradient elution isotachophoresis (i.e., GEITP). After purification and concentration, DNA molecules were delivered into a buffer solution that consisted of 10 mmol/L Tris and 0.1 mmol/L EDTA (Sigma).

A schematic of the experimental isotachophoretic apparatus is shown inFIG. 18. The upper end of a separation channel, i.e., a 9 cm long fused silica capillary with nominal outer and inner diameters of 360 and 75 μm, respectively, was affixed to the bottom of a polyetherimide reservoir containing 1 mL of LE solution using a miniature compression fitting from LabSmith (Livermore, Calif.) and Bondit B45TH epoxy from McMaster-Carr (Cleveland, Ohio). A custom autosampling stage controlled the placement of the lower end of the capillary into various solutions during analysis, as described below. The headspace pressure in the LE solution reservoir was regulated using a 20 mL, air-filled, disposable syringe driven by a custom syringe pump. Platinum electrodes were used to apply a high voltage of +2000 V dc from EMCO High Voltage (Sutter Creek, Calif.) at the LE solution reservoir and ground the solution electrically at the lower end of the capillary. The capillary passed through a custom LIF detector and a custom capacitively coupled contactless conductivity C4D detector from eDAQ Inc (Colorado Springs, Colo.). The laser-induced fluorescence (i.e., LIF) and conductivity detection points were approximately 58 and 44 mm from the lower end of the capillary, respectively. The apparatus was controlled and the data recorded using custom LabVIEW software from National Instruments (Austin, Tex.).

For method characterization, Plexor HY Promega Genomic DNA Standard (Madison, Wis.), comprised of a mixture of male individuals, was used as a control sample. The DNA concentration was determined to be 48.5±1.7 ng/μL (mean±SD) through qPCR using the Quantifiler Human Quantification Kit from Life Technologies (Grand Island, N.Y.). The control DNA solution was vortexed and diluted in TE solution to concentrations of 0.05, 0.17, 0.50, 1.5, and 5 ng/μL. The resulting samples were vortexed, and 100 μL of each were placed into 200 μL PCR tubes for analysis using the isotachophoretic apparatus.

To demonstrate the GEITP method for DNA purification, concentration, quantification, and delivery from crude samples for human identification, buccal swabs were collected from six anonymous human donors. A cotton-tipped swab containing a buccal sample was rehydrated with 120 μL of TE solution and placed into a 1.5 mL microcentrifuge tube containing 485 μL TE solution and 15 μL proteinase K solution (20 ng/μL) from Ambion® (Grand Island, N.Y.) for cell lysis. The cotton swab tip was submerged, and the swab stick was cut to allow the tube to close. The tube was then inverted by hand gently for approximately 30 s and placed into a hot water bath at 56° C. for 15 min for cell lysis. After vortexing briefly, fluid from the tube containing the swab was placed into 200 μL microcentrifuge tubes to create 1×, ⅓×, and 1/10× concentration samples with a final volume of 100 μL, diluted using TE solution. These samples were vortexed briefly just prior to analysis using the GEITP method.

Soiled buccal swabs were prepared to mimic crude environmental samples. Approximately 85-100 mg of soil collected for previous work was placed into 1.5 mL microcentrifuge tubes. A buccal swab containing a human buccal sample was rehydrated with 120 μL of TE solution and then placed into the tube containing soil, the stick was cut to allow the tube to close securely, and the tube was agitated by hand to coat the swab with soil thoroughly. The soiled swab was then prepared for analysis similarly to a clean swab. No attempt was made to avoid pipetting soil and other particulates into a 200 μL microcentrifuge tube for analysis using the GEITP method.

To determine the amount of DNA delivered from each sample, qPCR was performed using a 7500 Real-Time PCR System from Life Technologies (Grand Island, N.Y.) and Quantifiler Human DNA Quantification Kit from Life Technologies (Grand Island, N.Y.). The analysis used 8.2 μL Quantifiler Human Primer Mix, 9.8 μL Quantifiler Human PCR Reaction Mix, and 2 μL of the delivered DNA solution, for a total reaction volume of 20 μL. Amplification proceeded according to the manufacturer's recommended program of 95° C. for 10 min, followed by 40 cycles of 15 s at 95° C. and 1 min at 60° C. A standard curve was constructed for each plate using NIST Standard Reference Material 2372 Human DNA Quantitation Standard Component A at concentrations ranging from 52.44 to 0.07 ng/μL. An automatic threshold was applied to each plate using Applied Biosystems 7500 System SDS software version 1.2.3 (Grand Island, N.Y.). Each sample was measured in duplicate wells on the same plate.

STR analysis was performed to demonstrate human identification from DNA delivered from a crude sample using the GEITP method. STR analysis used the Promega PowerPlex 16 HS STR Amplification Kit. The 16 locus multiplex PCR kit contained primers to type the 13 core STR markers, the sex-typing marker Amelogenin, and the two additional STR markers Penta E and Penta D. The multiplex PCR reaction used a 12.5 μL total reaction volume containing 2.5 μL PowerPlex HS 5× Master Mix, 1.25 μL PowerPlex 16 HS Primer Set, 5 μL of the delivered DNA solution with a target DNA amount of 0.5 ng, and 3.75 μL deionized water. Thermal cycling was performed in a GeneAmp PCR System 9700 from Life Technologies (Grand Island, N.Y.) operating in the 9600 emulation mode with the following cycling parameters: 2 min incubation at 96° C.; 10 cycles of ramp 100% to 94° C. for 30 s, ramp 29% to 60° C. for 30 s, and ramp 23% to 70° C. for 45 s; 20 cycles of ramp 100% to 90° C. for 30 s, ramp 29% to 60° C. for 30 s, and ramp 23% to 70° C. for 45 s; and a 30 min incubation at 60° C. The temperature was subsequently held at 4° C. until the samples were removed.

Following multiplex PCR, 1 μL of amplified product was diluted in 10 μL Hi-Di formamide from Life Technologies (Grand Island, N.Y.) and 1 μL Internal Lane Standard 600 from Promega (Madison, Wis.) and analyzed with an ABI PRISM 3130 xl Genetic Analyzer using Data Collection v 3.0 software from Life Technologies (Grand Island, N.Y.), POP-4 polymer from Life Technologies (Grand Island, N.Y.), and a 36 cm capillary array. All genotyping was performed with GeneMapper ID v 3.2 software from Life Technologies (Grand Island, N.Y.) using allelic ladders, bins, and panels provided by the manufacturer and a peak detection threshold of 50 relative fluorescent units.

The steps used for DNA purification, concentration, quantification, and delivery are shown schematically inFIG. 18, while the voltage, pressure, current, and C4D detector signal are plotted versus time inFIG. 19. Buccal swabs were first incubated in lysis buffer (seeFIGS. 18(A)and18), after which a portion of the sample was taken for analysis using the GEITP method. At the beginning of each analysis, the pressure applied to the LE solution reservoir was regulated at a value that ensured a slight flow of LE solution through the capillary to prevent TE solution or the crude sample from entering the lower end of the capillary. This pressure was slightly negative (−700 Pa) relative to the ambient pressure due to the height difference between the upper and lower ends of the capillary. The lower end of the capillary was then dipped into the sample in the TE solution reservoir, and 2 kV was applied (seeFIG. 18(B)). At the same time, the pressure was increased to 0 Pa at the LE solution reservoir to prevent the ITP interface from migrating into the capillary. The pressure was then decreased at −20 Pa/s, and the focusing interface entered the capillary (seeFIG. 18(C)). During this pressure ramp, the electrical current through the capillary was monitored. A typical current at the beginning of the analysis was approximately 23 μA. As the focusing interface entered the capillary, a decreasing current indicated that the capillary was filled partially with TE solution. The pressure ramp was stopped when the current reached approximately 18 μA, indicating that the focusing interface was inside the capillary near the capillary entrance. For the next 60 s, the pressure was adjusted to maintain a constant current. This held the interface at an approximately constant position to purify and concentrate DNA. The pressure was subsequently reduced at −20 Pa/s to transport the interface and the focused DNA past the C4D and LIF detectors (seeFIGS. 18(D)and (E)). The interface was detected with the C4D detector, which triggered an automated DNA delivery sequence. Eight seconds after the interface passed the C4D detector, the high voltage was turned off and the applied pressure was set to −940 Pa, leaving the focused DNA stationary in the capillary. The lower end of the capillary was then rinsed in TE solution and placed into a clean 200 μL PCR tube containing 8.8 μL delivery solution. A pressure of 2 kPa was applied for 30 s to drive the purified and concentrated DNA past the LIF detector again for quantification and delivery into the clean tube (seeFIG. 18(F)). The lower end of the capillary was then placed into 18M Ω cm water and the pressure at the LE reservoir returned to −700 Pa between analyses.

The LIF signal was used for on-line DNA quantification during the delivery step. Representative fluorescence measurements for a 0.5 ng/μL control DNA sample, clean buccal swab, and soiled buccal swab show comparable performance between the samples (seeFIGS. 21(A)-(C)). DNA passed the LIF detector twice during analysis, which resulted in two peaks. The earlier of these tended to have a variable shape, possibly due to the complex behavior of macromolecular polyelectrolytes focusing in the strong electric field gradient at the interface between the LE and TE solutions. Consequently, the later peak, which was measured after the electric field was removed and had a more regular shape, was used to quantify the amount of DNA delivered by calculating the area under the peak. This area was determined, with a baseline subtraction, using measurements from 11 s before (FIGS. 18(D) and 21(D)) to 29 s after triggering the delivery sequence. The baseline was calculated by a linear fit to the LIF signal for only the first and last 6 s of the data in this range.

The LIF signal was calibrated against qPCR measurements to allow on-line DNA quantification in real time during analysis. Representative qPCR measurements of DNA delivered from a 0.5 ng/μL control DNA sample, clean buccal swab, and soiled buccal swab gave threshold cycles consistent with clean samples free of PCR inhibitors (seeFIGS. 21(D)-(F)). As shown inFIG. 21, ReferencingFIG. 20, the LIF measurements plotted versus the qPCR measurements are shown. Data for the control DNA samples and clean buccal swab samples show similar trends, indicating that the on-line LIF measurement can be used for DNA quantification for the clean buccal swab samples. However, LIF measurements for the soiled buccal swab samples were generally larger than expected from the trend measured with the control DNA samples. This is consistent with nonhuman DNA in the soil focusing along with human DNA in the sample and increasing the total amount of DNA measured using LIF. In contrast, the qPCR measurement used primers specific to human DNA only. To confirm this, the GEITP method was used to deliver DNA from a blank soiled swab sample with no human buccal cells. The result is plotted as the open circle inFIG. 20and shows a significant amount of DNA measured using LIF, while the qPCR measurement detected no human DNA. As shown inFIG. 20, soiled buccal swab samples generally resulted in larger LIF measurements than clean buccal swab samples, for a given qPCR value, likely due to the presence of nonhuman DNA in the soil. Additionally, the green open circle inFIG. 20represents a measurement of a blank soiled swab sample without human buccal cells. By calibrating the LIF measurements against qPCR measurements in this way, the amount of DNA delivered can be estimated during analysis in real time.

Reliable human identification using STR analysis requires that a specific amount of human DNA, between approximately 0.5 and 1.5 ng, be used for multiplex PCR. As implemented here, the GEITP method quantifies all DNA focused from the sample. For samples containing significant amounts of nonhuman DNA, the LIF measurement would ideally incorporate specificity for human DNA for subsequent human identification. The results inFIG. 20indicate that the quantification provided by the on-line LIF measurement compares favorably with qPCR measurement and may be adequate, with specificity for human DNA, to eliminate the need for the additional quantification step prior to multiplex PCR.

DNA delivered using the GEITP method was suitable for human identification using STR analysis (seeFIG. 21). ReferencingFIG. 21, the average threshold cycles of 30.7, 29.8, and 30.2 for (D), (E), and (F), respectively, gave no evidence for significant inhibition due to contaminants present in the soiled buccal swab sample (seeFIGS. 21(C)and (F)). Representative STR profiles from the same individual obtained from DNA delivered from clean and soiled buccal swab samples show comparable data that successfully locate all 16 STR loci. Clean and soiled buccal swab samples were also diluted in TE solution to ⅓× and 1/10× concentrations, and STR analysis was performed using the delivered DNA (see Table 1 below). GEITP delivered an appropriate amount of DNA for STR analysis for the undiluted samples, giving full STR profiles for all six clean and six soiled buccal swab samples. These clean and soiled buccal swab samples were in agreement for each individual, with average peak height ratios for heterozygous loci >80%. The fluorescence signal intensity was similar for these samples, with no DNA degradation apparent in the soiled buccal swab samples. However, diluting the DNA concentration in the sample prior to GEITP analysis resulted in a significant decrease in the amount of delivered DNA and, consequently, a loss of genetic information as STR profiles had an increasing number of alleles lost or loci dropped from the profiles. As implemented here, the GEITP method consistently delivered approximately 1% of the DNA in a sample, which is significantly less than the approximately 16-33% typical of more conventional techniques. Despite this drawback, the results shown here indicate that the GEITP method is adequate for DNA delivery from crude samples containing a relative abundance of DNA, as demonstrated using the soiled buccal swab samples. Further optimization of the GEITP method is expected to increase the efficiency of DNA purification and concentration for delivery.

The method presented here addresses several of the challenges associated with conventional techniques for DNA purification, concentration, and quantification from crude samples for human identification. First, GEITP requires no sample preparation, aside from suspension in a buffer solution for cell lysis; if this lysis solution also serves as the TE solution, DNA is purified and concentrated directly from the lysate. Additionally, no analysis steps are required to further purify or concentrate the DNA after delivery prior to STR analysis. Second, on-line DNA quantification can potentially eliminate the need for quantification by qPCR after DNA delivery. For samples containing significant amounts of nonhuman DNA, additional specificity for human DNA should be incorporated into the on-line quantitation. Third, nanogram amounts of DNA can be delivered into a small volume ≤10 μL, which is well matched to the volume requirements for input into the multiplex PCR reaction for STR analysis. Fourth, GEITP occurs in free solution, imposing no thresholds for low or high molecular weight DNA, as for gel-based methods, so that all of the DNA in the sample may be delivered. This capability is important for DNA delivery from aged or otherwise degraded samples. Finally, the GEITP method implemented here delivers DNA rapidly from crude samples in under 5 min after cell lysis, which is approximately 3-4 times faster than typical magnetic bead-based methods (Qiagen, EZ1R_DNA Investigator Handbook, 4th ed. 2009, www.qiagen.com/Knowledge-and-Support/Resource-Center/?catno=104640.) and an order of magnitude faster than the SCODA method. Because any DNA that may have carried over in the GEITP apparatus between deliveries was below the LOD, no additional time was required to change buffer solutions, rinse the capillary, or otherwise clean the apparatus between analyses.

The method described here based on GEITP enabled DNA purification, concentration, quantification, and delivery from crude samples with minimal sample preparation for human identification using STR analysis. In addition to the automated stand-alone format demonstrated here, GEITP is suitable for incorporation as a sample preparation step at the front end of a more complete DNA analysis system.

All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present disclosure.

It is to be further understood that where descriptions of various embodiments use the term “comprising,” and/or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”