Patent Application
20160146755
There is a significant interest in the development of paper point-of-care (POC) devices that are cheap, user friendly, robust, sensitive, and portable. Such devices pose an effective solution to the existing economic and healthcare accessibility problems in underdeveloped countries, as well as the growing trend in more affluent societies to become better informed in terms of its health. Although commercial paper-based sensors have been around for about 25 years (e.g., pregnancy test and glucose test strips), few paper POC devices have been successfully commercialized. Such failure to produce trustworthy paper POC devices is a combination of many factors, including poor limits of detection (LOD), high non-specific adsorption (NSA), unstable reagents, long analysis time, complex user-technology interface, detection method, and poor sensitivity.
Described herein are devices that can be used to perform electrophoretic separations and/or the isotachophoretic concentration of samples. The devices can comprise a plurality of planar segments with each planar segment comprising a fluid permeable region defined by a fluid impermeable boundary. The plurality of planar segments can be stacked (e.g., to form a stack) such that the plurality of planar segments are parallel and aligned. When stacked, the fluid permeable regions of the plurality of planar segments together can form a fluid permeable column within the stack of segments extending from a first end to a second end. The device can further comprise a first electrode in electrical contact with the first end, a second electrode in electrical contact with the second end, or a combination thereof.
In some embodiments, the device can further comprise a first reservoir in fluid contact with the first end, a second reservoir in fluid contact with the second end, or a combination thereof. The device can further comprise a first separator in fluid contact with the first reservoir and the first end, a second separator in fluid contact with the second reservoir and the second end, or a combination thereof. In some embodiments, the first separator can be located between the first reservoir and the first end, the second separator can be located between the second reservoir and the second end, or a combination thereof.
In some embodiments, the device can further comprise a slip layer. The slip layer can comprise a fluid permeable region defined by a fluid impermeable boundary. The slip layer can be translocated from a retracted position to a deployed position, wherein in the retracted position the fluid permeable region of the slip layer is fluidly independent from the fluid permeable column, and wherein in the deployed position, the fluid permeable region of the slip layer is in fluid contact with the fluid permeable column. The slip layer can serve as a loading layer to introduce a sample into the fluid permeable column. The slip layer can also serve as a collection layer on which an analyte can be collected.
In some embodiments, the plurality of segments are joined together in a sheet. When joined in a sheet, the plurality of segments can optionally be coplanar. For example, the plurality of segments can be joined end to end to form an elongate strip. The stack can be formed by folding the sheet so as to align the segments in a stack. In some embodiments, folding the sheet can comprise accordion folding the sheet.
The devices herein can be fabricated from any suitable material or combination of materials. In some embodiments, the devices can be paper based.
In some embodiments, the devices disclosed herein can comprise two or more fluid permeable columns.
Also disclosed herein are methods of use of the devices disclosed herein. In some embodiments, the method can comprise introducing a sample to the fluid permeable column of the device and applying a potential to the fluid permeable column. In some embodiments, the method can comprise electrophoresis (e.g., the device can be configured to electrophoretically localize and/or separate the sample). In some embodiments, the sample can comprise an analyte. In some embodiments, the potential can be 40 volts (V) or less.
In some embodiments, introducing the sample to the fluid permeable column can comprise translocating the slip layer to the deployed position, wherein the sample is initially located in the fluid permeable region of the slip layer.
In some embodiments, the method can further comprise separating the analyte from the sample (e.g., the method can comprise electrophoretically separating the analyte from the sample). In some embodiments, the method can further comprise accumulating the sample, the analyte, or a combination thereof in a section of the fluid permeable column. The section can comprise one or more of the planar segments, the slip layer, or a combination thereof. In some embodiments, the method can further comprise removing the section of the fluid permeable column to isolate the sample, the analyte, or a combination thereof. In some embodiments, the method can further comprise analyzing the sample, analyte, or a combination thereof to determine a property of the sample, the analyte, or a combination thereof.
Also disclosed herein are methods of use of the devices comprising a first fluid permeable column and a second permeable column. For example, the method can comprise a multichannel analysis of one or more sample. In some embodiments, the method can comprise a multi-step analysis, where a sample is loaded into the first fluid permeable column, partially separated such that a first analyte is collected in a section of the first fluid permeable column comprising a slip layer, then translocating the slip layer to introduce the first analyte to the second fluid permeable column and perform another analysis step.
The devices and methods described herein are inexpensive, user friendly, sensitive, portable, robust, efficient, thin (e.g., column is ˜2 mm in length), rapid (completion of analysis in ˜5 min), and use low voltage (e.g., 10-20 V). As such, the device and methods are well suited for use in numerous applications including point-of-care (POC) diagnostics.
The methods and devices described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter, figures and the examples included therein.
Before the present devices and methods are disclosed and described, it is to be understood that the aspects described below are not intended to be scope by the specific devices and methods described herein, which are intended as illustrations. Various modifications of the devices and methods in addition to those shown and described herein are intended to fall within the scope of that described herein. Further, while only certain representative devices and method steps disclosed herein are specifically described, other combinations of the devices and method steps also are intended to fall within the scope of that described herein, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various examples, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific examples of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.
As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms.
Throughout the specification, the terms “planar” and “parallel” are used to describe segments and the relative arrangement of segments. It will be understood that such terms allow for some variation (e.g., segments need not be absolutely planar or parallel but merely substantially planar or parallel) provided that device function is not compromised (e.g., provided that the fluid impermeable boundary that defines the fluid permeable region and by extension to the electrophoretic column remains sufficiently continuous to allow for a device to function).
Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
Devices
Disclosed herein are devices 100 that can comprise a plurality of planar segments 104. Referring now to
Referring now to
In some embodiments, the plurality of planar segments 104 can be stacked such that the bottom surface 134 of a first planar segment 104 and the top surface 132 of a second segment 104 are in intimate contact at a juncture 140.
The fluid permeable regions 116 can form a fluid permeable column 106 within the stacked plurality of segments 104 (e.g., within the stack 102) extending from a first end 108 to a second end 110, wherein the first end 108 can comprise the fluid permeable region 116 at the top surface 132 of the first planar segment 104, and wherein the second end 110 can comprise the fluid permeable region 116 at the bottom surface 134 of the last planar segment 104.
In some embodiments, the fluid permeable column 106 can be 10 mm or less in length (e.g., 9.5 mm or less, 9 mm or less, 8.5 mm or less, 8 mm or less, 7.5 mm or less, 7 mm or less, 6.5 mm or less, 6 mm or less, 5.5 mm or less, 5 mm or less, 4.5 mm or less, 4 mm or less, 3.5 mm or less, 3 mm or less, 2.5 mm or less, 2.4 mm or less, 2.3 mm or less, 2.2 mm or less, 2.1 mm or less, 2 mm or less, 1.9 mm or less, 1.8 mm or less, 1.7 mm or less, 1.6 mm or less, 1.5 mm or less, 1.4 mm or less, 1.3 mm or less, 1.2 mm or less, 1.1 mm or less, 1 mm or less, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, or 0.5 mm or less), wherein the length is the distance from the first end 108 to the second end 110.
The device 100 can further comprise a first electrode 112 in electrical contact with the first end 108, a second electrode 114 in electrical contact with the second end 110, or a combination thereof.
Referring now to
The device 100 can further comprise a first separator 124 in fluid contact with the first reservoir 120 and the first end 108, a second separator 126 in fluid contact with the second reservoir 122 and the second end 110, or a combination thereof. The first separator 124, the second separator 126, or a combination thereof can, for example, separate the fluid permeable region 116 of the device 100 from the volume of solution in the first reservoir 120, the second reservoir 122, or a combination thereof, to prevent the fluid permeable region 116 from being damaged by long-term exposure to solution, undesirable pH changes, the effects of pressure-driven flow, and the like. In some examples, the first separator 124, the second separator 126, or a combination thereof can comprise a housing, a separation material, or a combination thereof. In some examples, the separation material can comprise a hydrogel (e.g., an agar gel).
In some embodiments, the first separator 124 can be located between the first reservoir 120 and the first end 108, the second separator 126 can be located between the second reservoir 122 and the second end 110, or a combination thereof.
Referring now to
In some embodiments, the plurality of segments 104 can be independent (i.e., non-attached) planar segments that can be stacked to form the devices described herein. In other embodiments, the plurality of segments 104 can be joined together in a sheet. When joined in a sheet, the plurality of segments can optionally be coplanar. In some embodiments, the stack can be formed by folding the sheet, for example as shown in
The devices herein can be fabricated from any suitable material or combination of materials. In some embodiments, the devices 100 can be paper based, meaning that the fluid permeable regions 116 can be formed from a porous, cellulosic substrate such as paper through which fluid flows by wicking. In some cases, the planar segments can be formed from a porous, cellulosic substrate such as paper through which fluid flows by wicking. The dimensions of the permeable regions 116 within the planar segments 104 are defined by a fluid impermeable boundary 118 that substantially permeates the thickness of the planar segment 104, so as to form a boundary that directs fluid flow along the fluid permeable column 106.
The fluid impermeable boundary 118 that defines the fluid permeable region 116 can be formed within a layer of a porous, cellulosic substrate (e.g., within the planar segment 104) using any suitable method known in the art. For example, the fluid impermeable boundary 118 can be formed by wax printing. In these methods, an inkjet printer is used to pattern a wax material on the porous, cellulosic substrate. Many types of wax-based solid ink are commercially available and are useful in such methods as the ink provides a visual indication of the location of the fluid impermeable boundary 118. However, it should be understood, that the wax material used to form the fluid impermeable boundary 118 does not require an ink to be functional. Examples of wax materials that maybe used include polyethylene waxes, hydrocarbon amide waxes or ester waxes. Once the wax is patterned, the porous, cellulosic substrate is heated (e.g., by placing the substrate on a hot plate with the wax side up at a temperature of 120° C.) and cooled to room temperature. This allows the wax material to substantially permeate the thickness of the porous, cellulosic substrate, so as to form a fluid impermeable boundary 118 that defines the dimensions of the fluid permeable region 116.
In some embodiments, the device can be a paper-based device formed from a porous, cellulosic substrate that is flexible. For certain applications, it is preferable that the cellulosic substrate can be folded, creased, or otherwise mechanically shaped to impart structure and function to the paper-based device formed from the cellulosic substrate. Examples of suitable porous, cellulosic substrates for the fabrication of paper-based devices include cellulose; derivatives of cellulose such as nitrocellulose or cellulose acetate; paper (e.g., filter paper, chromatography paper); woven cellulosic materials; and non-woven cellulosic materials.
In some embodiment, the porous, cellulosic substrate is paper. Paper is inexpensive, widely available, readily patterned, thin, lightweight, and can be disposed of with minimal environmental impact. Furthermore, a variety of grades of paper are available, permitting the selection of a paper substrate with the weight (i.e., grammage), thickness and/or rigidity and surface characteristics (i.e., porosity, hydrophobicity, and/or roughness), desired for the fabrication of a particular paper-based device. Suitable papers include, but are not limited to, chromatography paper, card stock, filter paper, vellum paper, printing paper, wrapping paper, ledger paper, bank paper, bond paper, blotting paper, drawing paper, fish paper, tissue paper, paper towel, wax paper, and photography paper.
In some embodiments, the devices described herein can be affixed to or secured within a polymer, metal, glass, wood, or paper housing to facilitate handling and use of the device. In some embodiments, the devices described herein are affixed to or secured within an inert, non-absorbent polymer such as a polyether block amide (e.g., PEBAX®, commercially available from Arkema, Colombes, France), a polyacrylate, a polymethacrylate (e.g., poly(methyl methacrylate)), a polyimide, polyurethane, polyamide (e.g., Nylon 6,6), polyvinylchloride, polyester, (HYTREL®, commercially available from DuPont, Wilmington, Del.), polyethylene (PE), polyether ether ketone (PEEK), fluoropolymers such as polytetrafluoroethylene (PTFE), perfluoroalkoxy, fluorinated ethylene propylene, or a blend or copolymer thereof. Silastic materials and silicon based polymers can also be used.
The devices described herein can be coupled to a power supply and optionally to one or more additional suitable features including, but not limited to, a voltmeter, an ammeter, a multimeter, an ohmmeter, a signal generator, a pulse generator, an oscilloscope, a frequency counter, a potentiostat, or a capacitance meter. The devices described herein can also be coupled to a computing device that performs arithmetic and logic operations necessary to analyze the samples from the device (e.g., to determine analyte concentration, etc.).
Also disclosed herein is a device 100 comprising: a plurality of planar segments 104, each planar segment 104 comprising a fluid permeable region 116 defined by a fluid impermeable boundary 118, wherein when the plurality of planar segments 104 are stacked such that the fluid permeable region 116 of each planar segment 104 is aligned, the fluid permeable region 116 of each planar segment collectively forms a fluid permeable column 106 traversing the stacked plurality of planar segments 104 from a first end 108 to a second end 110.
Also disclosed herein is a device 100 comprising: a stack 102 formed from a plurality of parallel segments 104; a fluid permeable column 106 traversing the stack 102 from a first end 108 to a second end 110; a first electrode 112 in electrical contact with the first end 108; and a second electrode 114 in electrical contact with the second end 110; wherein each parallel segment 104 comprises a fluid permeable region 116 defined by a fluid impermeable boundary 118; and wherein stacking of the plurality of segments 104 aligns the fluid permeable region 116 within each of the plurality of parallel segments 104 to form the fluid permeable column 106.
In some embodiments, the devices disclosed herein can comprise two or more fluid permeable columns. Referring now to
Referring now to
Referring now to
The device 100 can optionally further comprise a loading slip layer 170. The loading slip layer 170 can comprise a first fluid permeable region 172 defined by a first fluid impermeable boundary 174, wherein the loading slip layer can be translocated from a retracted position to a deployed position. In the retracted position, the first fluid permeable region 172 of the loading slip layer 170 is fluidly independent from the first fluid permeable column 106 and the second fluid permeable column 156. In the deployed position, the first fluid permeable region 172 of the loading slip layer 170 is in fluid contact with the first fluid permeable column 106, the second fluid permeable column 156, or a combination thereof. In some embodiments, the loading slip layer 170 can further comprise a second fluid permeable region 176 defined by a second fluid impermeable boundary 178, wherein in the retracted position the second fluid permeable region 176 of the loading slip layer 170 is fluidly independent from the first fluid permeable column 106 and the second fluid permeable column 156 and in the deployed position, the second fluid permeable region 176 of the loading slip layer 170 is in fluid contact with the second fluid permeable 156 column.
Referring now to
Also disclosed herein is a device 100 comprising: a plurality of planar segments 104, each planar segment 104 comprising a first fluid permeable region 116 defined by a first fluid impermeable boundary 118 and a second fluid permeable region 150 defined by a second fluid impermeable boundary 152; wherein when the plurality of planar segments 104 are stacked such that the first fluid permeable region 116 of each planar segment 104 is aligned and the second fluid permeable region 150 of each planar segment 104 is aligned, the first fluid permeable region 116 of each planar segment 104 collectively forms a first fluid permeable column 116 traversing the stacked plurality of planar segments (e.g., the stack 102) from a first end 108 to a second end 110 and the second fluid permeable region 150 of each planar segment 104 collectively forms a second fluid permeable column 156 traversing the stacked plurality of planar segments (e.g., the stack 102) from a third end 158 to a fourth end 160.
Also disclosed herein is a device 100 comprising: a stack 102 formed from a plurality of parallel segments 104; a first fluid permeable column 106 traversing the stack 102 from a first end 108 to a second end 110; a second fluid permeable column 156 traversing the stack 102 from a third end 158 to a fourth end 160; a first electrode 112 in electrical contact with the first end 108, the third end 158, or a combination thereof; and a second electrode 114 in electrical contact with the second end 110, the fourth end 160, or a combination thereof; wherein each parallel segment 104 comprises a first fluid permeable region 116 defined by a first fluid impermeable boundary 118 and a second fluid permeable region 150 defined by a second fluid impermeable boundary 152; wherein stacking of the plurality of segments 104 aligns the first fluid permeable region 116 within each of the plurality of parallel segments 104 to form the first fluid permeable column 106; and wherein stacking of the plurality of segments 104 aligns the second fluid permeable region 150 within each of the plurality of parallel segments 104 to form the second fluid permeable column 156.
Methods
Also disclosed herein are methods of use of the devices disclosed herein. In some embodiments, the method can comprise introducing a sample to the fluid permeable column of the device and applying a potential to the fluid permeable column. In some embodiments, the method can comprise electrophoresis (e.g., the device can be configured to electrophoretically localize and/or separate the sample). In some embodiments, the method can comprise isotachophoresis (e.g., the device can be configured to separate, localize and/or concentrate the sample).
The sample can comprise any fluid sample of interest. By way of example the fluid sample can be a bodily fluid. “Bodily fluid”, as used herein, refers to a fluid composition obtained from or located within a human or animal subject. Bodily fluids include, but are not limited to, urine, whole blood, blood plasma, serum, tears, semen, saliva, sputum, exhaled breath, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, interstitial fluid, lymph fluid, meningal fluid, amniotic fluid, glandular fluid, feces, perspiration, mucous, vaginal or urethral secretion, cerebrospinal fluid, and transdermal exudate. Bodily fluid also includes experimentally separated fractions of all of the preceding solutions, as well as mixtures containing homogenized solid material, such as feces, tissues, and biopsy samples.
In some embodiments, the sample can comprise an analyte. The analyte can be, for example, a biomarker (i.e., a molecular indicator associated with a particular pathological or physiological state) present in the bodily fluid (e.g., the sample) that can be assayed to identify risk for, diagnosis of, or progression of a pathological or physiological process in a subject. Examples of biomarkers include proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA, and combinations thereof. The analyte can be, for example, an antibody, peptide (natural, modified, or chemically synthesized), protein (e.g., a glycoprotein, a lipoprotein, or a recombinant protein), polynucleotide (e.g., DNA or RNA, an oligonucleotide, an aptamer, or a DNAzyme), lipid, polysaccharide, small molecule organic compound (e.g., a hormone, a prohormone, a narcotic, or a small molecule pharmaceutical), pathogen (e.g., bacteria, virus, or fungi, or protozoa), or combination thereof
The potential can be any potential consistent with the devices and methods described herein. In some embodiments, the potential applied to the fluid permeable column can be substantially less than the voltage applied in traditional electrophoresis. In some embodiments, the potential can be 40 volts (V) or less (e.g., 38 V or less, 36 V or less, 34 V or less, 32 V or less, 30 V or less, 28 V or less, 26 V or less, 24 V or less, 22 V or less, 20 V or less, 18 V or less, 16 V or less, 14 V or less, 12 V or less, or 10 V or less).
In some embodiments, introducing the sample to the fluid permeable column can comprise translocating the slip layer to the deployed position, wherein the sample is initially located in the fluid permeable region of the slip layer.
In some embodiments, the method can further comprise separating the analyte from the sample (e.g., the method can comprise electrophoretically separating the analyte from the sample).
In some embodiments, the method can further comprise accumulating the sample, the analyte, or a combination thereof in a section of the fluid permeable column. The section can comprise one or more of the parallel segments, the slip layer, or a combination thereof. In some embodiments, the method can further comprise removing the section of the fluid permeable column to isolate the sample, the analyte, or a combination thereof
In some embodiments, the method can further comprise analyzing the sample, analyte, or a combination thereof to determine a property of the sample, the analyte, or a combination thereof. Analyzing the sample, the analyte, or a combination thereof can comprise performing any type of analysis known in the art. Examples of analysis techniques include, but are not limited to, spectroscopic analysis (e.g., atomic absorption spectroscopy, atomic emission spectroscopy, atomic fluorescence spectroscopy, energy dispersive spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, Raman spectroscopy, X-Ray fluorescence spectroscopy, IR spectroscopy, laser induced breakdown spectroscopy, nuclear magnetic resonance spectroscopy, etc.), chromatographic analysis (e.g., thin layer chromatography, gas chromatography, etc.), colorimetry, voltammetry, potentiometry, calorimetry (e.g., differential scanning calorimetry), flow injection analysis, electron paramagnetic resonance, gas chromatography-mass spectrometry (GC-MS), gas chromatography-IR spectroscopy (GC-IR), mass-spectrometry, transmission electron microscopy, scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, X-ray microscopy, and combinations thereof. In some embodiments, analyzing the sample, analyte, or a combination thereof can comprise spectroscopic analysis (e.g., fluorescence spectroscopy) of the sample, analyte, or a combination thereof. In some embodiments, analyzing the sample, analyte, or a combination thereof can comprise analyzing the sample, analyte, or a combination thereof comprises electrochemical analysis of the sample, analyte, or a combination thereof. In some cases, this can comprise, for example, electrochemical detection of an electrochemical tag or label, such as a metal nanoparticle, conjugated to the analyte.
The property can be any property of interest of the sample, the analyte or a combination thereof. For example, the property can comprise the concentration of the sample and/or the analyte, determining the presence or absence of a particular analyte within the sample, determining the identity of the analyte, determining the number of analytes within the sample, or a combination thereof.
Also disclosed herein are methods of using devices that comprise a first fluid permeable column and a second permeable column. For example, the method can comprise a multichannel analysis of one or more sample, such as that illustrated in
In some embodiments, the method can comprise introducing a sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof; and applying a potential to the first fluid permeable column, the second fluid permeable column, or a combination thereof. Introducing the sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof can comprise, for example, translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
In some embodiments, the sample can comprise a first analyte. In some embodiments, the method can further comprise separating the first analyte from the sample. In some embodiments, the method can further comprise accumulating the sample, the first analyte, or a combination thereof in a section of the first fluid permeable column, the second fluid permeable column, or a combination thereof. In some embodiments, the section can comprise one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof. In some embodiments, the method can further comprise removing the section of the first fluid permeable column, the second fluid permeable column, or a combination thereof to isolate the sample, the first analyte, or a combination thereof. In some embodiments, the method can further comprise analyzing the sample, the first analyte, or a combination thereof to determine a property of the sample, the first analyte, or a combination thereof.
In some embodiments, the section of the first fluid permeable column can comprise the collection slip layer in the first position. In some embodiments, the method can further comprise translocating the collection slip layer to the second position. In some embodiments, the method can further comprise applying a potential to the second fluid permeable column. In some embodiments, the sample the first analyte, or a combination thereof further can comprise a second analyte. In some embodiments, the method can further comprise separating the second analyte from the sample, the first analyte, or a combination thereof. In some embodiments, the method can further comprise accumulating the sample, the first analyte, the second analyte or a combination thereof in a section of the second fluid permeable column. In some embodiments, the method can further comprise removing the section of the second fluid permeable column to isolate the sample, the first analyte, the second analyte, or a combination thereof. In some embodiments, the section of the second fluid permeable column can comprise one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof. In some embodiments, the method can further comprise analyzing the sample, the first analyte, the second analyte, or a combination thereof to determine a property of the sample, the first analyte, the second analyte, or a combination thereof.
In some embodiments the method can comprise introducing a first sample to the first fluid permeable column, introducing a second sample to the second fluid permeable column, and applying a potential to the first fluid permeable column and the second fluid permeable column (e.g.,
In some embodiments, the first sample can comprise a first analyte. In some embodiments, the second sample can comprise a second analyte. In some embodiments, the method can further comprise separating the first analyte from the first sample, separating the second analyte from the second sample, or a combination thereof. In some embodiments, the method can further comprise accumulating the first sample, the first analyte, or a combination thereof in a section of the first fluid permeable column; accumulating the second sample, the second analyte or a combination thereof in a section of the second fluid permeable column; or a combination thereof. In some embodiments, the section can comprise one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof. In some embodiments, the method can further comprise removing the section of the first fluid permeable column to isolate the first sample, the first analyte, or a combination thereof; removing the section of the second fluid permeable column to isolate the second sample, the second analyte, or a combination thereof; or a combination thereof. In some embodiments, the method can further comprise analyzing the first sample, the second sample, the first analyte, the second analyte, or a combination thereof to determine a property of the first sample, the second sample, the first analyte, the second analyte, or a combination thereof.
The devices and methods described herein are inexpensive, user friendly, sensitive, portable, robust, efficient, thin (e.g., column is ˜2 mm in length), rapid (completion of analysis in ˜5 min), and use low voltage (e.g., 10-20 V). As such, the device and methods are well suited for use in numerous sensing applications.
For example, the devices and methods described herein can be used in clinical and healthcare settings to detect and/or quantify biomarkers to identify risk for, diagnosis of, or progression of a pathological or physiological process in a subject. Examples of biomarkers include proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA, and combinations thereof.
The devices and methods described herein can be used in POC applications to diagnose infections in a patient (e.g., by measuring serum antibody concentrations or detect antigens). For example, the devices and methods described herein can be used to diagnose viral infections (e.g., HIV, hepatitis B, hepatitis C, rotavirus, influenza, polio, measles, yellow fever, rabies, dengue, or West Nile Virus), bacterial infections (e.g., E. coli, C. tetani, cholera, typhoid, diphtheria, tuberculosis, plague, Lyme disease, or H. pylori), and parasitic infections (e.g., toxoplasmosis, Chagas disease, or malaria). The devices and methods described herein can be used to rapidly assesses the immune status of people or animals against selected vaccine-preventable diseases (e.g. anthrax, human papillomavirus (HPV), diphtheria, hepatitis A, hepatitis B, haemophilus influenzae type b (Hib), influenza (flu), Japanese encephalitis (JE), measles, meningococcal, mumps, pertussis, pneumococcal, polio, rabies, rotavirus, rubella, shingles (herpes zoster), smallpox, tetanus, typhoid, tuberculosis (TB), varicella (chickenpox), yellow fever). The devices and methods described herein can be used to rapidly screen donated blood for evidence of viral contamination by HIV, hepatitis C, hepatitis B, and HTLV-1 and -2. The devices and methods described herein can also be used to measure hormone levels. For example, the devices and methods described herein can be used to measure levels of human chorionic gonadotropin (hCG) (as a test for pregnancy), Luteinizing Hormone (LH) (to determine the time of ovulation), or Thyroid Stimulating Hormone (TSH) (to assess thyroid function). The devices and methods described herein can be used to diagnose or monitor diabetes in a patient, for example, by measuring levels of glycosylated hemoglobin, insulin, or combinations thereof. The devices and methods described herein can be used to detect protein modifications (e.g., based on a differential charge between the native and modified protein and/or by utilizing recognition elements specific for either the native or modified protein). The devices and methods described herein can be used to administer personalized medical therapies to a subject (e.g., in a pharmacogenomic assay performed to select a therapy to be administered to a subject).
The devices and methods described herein can also be used in other commercial applications. For example, the devices and methods described herein can be used in the food and beverage industry, for example, in quality control applications or to detect potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. The devices and methods described herein can be used to detect and/or measure the levels of proteins of interest in foods, cosmetics, nutraceuticals, pharmaceuticals, and other consumer products. The devices and methods described herein can also be used to rapidly and accurately detect narcotics and biothreat agents (e.g., ricin).
Certain example embodiments are provided below.
A device comprising:
a stack formed from a plurality of parallel segments;
a fluid permeable column traversing the stack from a first end to a second end;
a first electrode in electrical contact with the first end; and
a second electrode in electrical contact with the second end;
wherein each segment comprises a fluid permeable region defined by a fluid impermeable boundary; and
wherein stacking of the plurality of segments aligns the fluid permeable region within each of the plurality of parallel segments to form the fluid permeable column.
The device of embodiment 1, further comprising a first reservoir in fluid contact with the first end, a second reservoir in contact with the second end, or a combination thereof.
The device of embodiment 2, further comprising a first separator in fluid contact with the first reservoir and the first end, a second separator in fluid contact with the second reservoir and the second end, or a combination thereof.
The device of embodiment 3, wherein the first separator is located between the first reservoir and the first end, second separator is located between the second reservoir and the second end, or a combination thereof.
The device of any one of embodiments 1-4, further comprising a slip layer, wherein the slip layer comprises:
a fluid permeable region defined by a fluid impermeable boundary;
wherein the slip layer can be translocated from a retracted position to a deployed position;
wherein in the retracted position the fluid permeable region of the slip layer is fluidly independent from the fluid permeable column; and
wherein in the deployed position, the fluid permeable region of the slip layer is in fluid contact with the fluid permeable column.
The device of any one of embodiments 1-5, wherein the plurality of segments are joined together in a sheet, and the stack is formed by folding the sheet.
The device of embodiment 6, wherein folding the sheet comprises accordion folding the sheet.
The device of any one of embodiments 1-7, wherein the plurality of parallel segments comprises at least 3 parallel segments.
The device of any one of embodiments 1-8, wherein the fluid permeable column is 10 mm or less in length.
The device of any one of embodiments 1-9, wherein the device is paper based.
The device of any one of embodiments 1-10, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the fluid permeable column of the device of any one of embodiments 1-11; and
applying a potential to the fluid permeable column.
The method of embodiment 12, wherein the potential is 40 V or less.
The method of any one of embodiments 12-13, wherein the sample comprises an analyte.
The method of embodiment 14, further comprising separating the analyte from the sample.
The method of any one of embodiments 12-15, further comprising accumulating the sample, the analyte, or a combination thereof in a section of the fluid permeable column.
The method of embodiment 18, further comprising removing the section of the fluid permeable column to isolate the sample, the analyte, or a combination thereof.
The method of any one of embodiments 18-19, wherein the section can comprise one or more of the parallel segments, a slip layer, or a combination thereof.
The method of any one of embodiments 12-18, further comprising analyzing the sample, analyte, or a combination thereof to determine a property of the sample, the analyte, or a combination thereof.
The method of any one of embodiments 12-19, wherein introducing the sample to the fluid permeable column comprises translocating the slip layer to the deployed position, wherein the sample is initially located in the fluid permeable region of the slip layer.
A device comprising a plurality of planar segments, each planar segment comprising a fluid permeable region defined by a fluid impermeable boundary,
wherein when the plurality of planar segments are stacked such that the fluid permeable region of each planar segment is aligned, the fluid permeable regions of the plurality of planar segments collectively forms a fluid permeable column traversing the stacked plurality of planar segments from a first end to a second end.
The device of embodiment 21, further comprising a first electrode in electrical contact with the first end, a second electrode in electrical contact with the second end, or a combination thereof.
The device of any one of embodiments 21-22, further comprising a first reservoir in fluid contact with the first end, a second reservoir in fluid contact with the second end, or a combination thereof.
The device of embodiment 23, further comprising a first separator in fluid contact with the first reservoir and the first end, a second separator in fluid contact with the second reservoir and the second end, or a combination thereof.
The device of embodiment 24, wherein the first separator is located between the first reservoir and the first end, the second separator is located between the second reservoir and the second end, or a combination thereof.
The device of any one of embodiments 21-25, further comprising a slip layer, wherein the slip layer comprises a fluid permeable region defined by a fluid impermeable boundary, wherein the slip layer can be translocated from a retracted position to a deployed position, wherein in the retracted position the fluid permeable region of the slip layer is fluidly independent from the fluid permeable column, and wherein in the deployed position, the fluid permeable region of the slip layer is in fluid contact with the fluid permeable column.
The device of any one of embodiments 21-26, wherein the plurality of planar segments are joined together in a sheet, and the plurality of planar segments are stacked by folding the sheet.
The device of embodiment 27, wherein folding the sheet comprises accordion folding the sheet.
The device of any one of embodiments 21-28, wherein the plurality of planar segments comprises at least 3 planar segments.
The device of any one of embodiments 21-29, wherein the fluid permeable column is 10 mm or less in length.
The device of any one of embodiments 21-30, wherein the device is paper based.
The device of any one of embodiments 21-31, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the fluid permeable column of the device of any one of embodiments 21-32; and
applying a potential to the fluid permeable column.
The method of embodiment 33, wherein the potential is 40 V or less.
The method of any one of embodiments 33-34, wherein the sample comprises an analyte.
The method of embodiment 35, further comprising separating the analyte from the sample.
The method of any one of embodiments 33-36, further comprising accumulating the sample, analyte, or a combination thereof in a section of the fluid permeable column.
The method of embodiment 37, further comprising removing the section of the fluid permeable column to isolate the sample, the analyte, or a combination thereof.
The method of any one of embodiments 37-38, wherein the section can comprise one or more of the planar segments, a slip layer, or a combination thereof.
The method of any one of embodiments 33-39, further comprising analyzing the sample, analyte, or a combination thereof to determine a property of the sample, the analyte, or a combination thereof.
The method of any one of embodiments 33-40, wherein introducing the sample to the fluid permeable column comprises translocating the slip layer to the deployed position, wherein the sample is initially located in the fluid permeable region of the slip layer.
A device comprising:
a plurality of planar segments, each planar segment comprising:
a top surface;
a bottom surface; and
a fluid permeable region defined by a fluid impermeable boundary extending through the planar segment from the top surface to the bottom surface so as to form a fluid permeable pathway extending through the planar segment from the top surface to the bottom surface;
wherein when the plurality of planar segments are stacked such that the bottom surface of a first planar segment is in intimate contact with the top surface of a second planar segment, the fluid permeable regions together form a fluid permeable column within the stacked plurality of planar segments extending from a first end to a second end;
wherein the first end comprises the fluid permeable region at the top surface of the first planar segment;
wherein the second end with the fluid permeable region at the bottom surface of the last planar segment;
a first electrode in electrical contact with the first end;
a second electrode in electrical contact with the second end.
The device of embodiment 42, further comprising a first reservoir in fluid contact with the first end, a second reservoir in fluid contact with the second end, or a combination thereof.
The device of embodiment 43, further comprising a first separator in fluid contact with the first reservoir and the first end, a second separator in fluid contact with the second reservoir and the second end, or a combination thereof.
The device of embodiment 44, wherein the first separator is located between the first reservoir and the first end, the second separator is located between the second reservoir and the second end, or a combination thereof.
The device of any one of embodiments 42-45, further comprising a slip layer, wherein the slip layer comprises a fluid permeable region defined by a fluid impermeable boundary, wherein the slip layer can be translocated from a retracted position to a deployed position, wherein in the retracted position the fluid permeable region of the slip layer is fluidly independent from the fluid permeable column, and wherein in the deployed position, the fluid permeable region of the slip layer is in fluid contact with the fluid permeable column.
The device of any one of embodiments 42-46, wherein the plurality of planar segments are joined together in a sheet, and the plurality of planar segments are stacked by folding the sheet.
The device of embodiment 47, wherein folding the sheet comprises accordion folding the sheet.
The device of any one of embodiments 42-48, wherein the plurality of planar segments comprises at least 3 planar segments.
The device of any one of embodiments 42-49, wherein the fluid permeable column is 10 mm or less in length.
The device of any one of embodiments 42-50, wherein the device is paper based.
The device of any one of embodiments 42-51, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the fluid permeable column of the device of any one of embodiments 43-52; and
applying a potential to the fluid permeable column.
The method of embodiment 53, wherein the potential is 40 V or less.
The method of any one of embodiments 53-54, wherein the sample comprises an analyte.
The method of embodiment 55, further comprising separating the analyte from the sample.
The method of any one of embodiments 53-56, further comprising accumulating the sample, the analyte, or a combination thereof in a section of the fluid permeable column.
The method of embodiment 57, further comprising removing the section of the fluid permeable column to isolate the sample, the analyte, or a combination thereof.
The method of any one of embodiments 57-58, wherein the section can comprise one or more of the planar segments, a slip layer, or a combination thereof.
The method of any one of embodiments 53-59, further comprising analyzing the sample, the analyte, or a combination thereof to determine a property of the sample, the analyte, or a combination thereof.
The method of any one of embodiments 53-60, wherein introducing the sample to the fluid permeable column comprises translocating the slip layer to the deployed position, wherein the sample is initially located in the fluid permeable region of the slip layer.
A device comprising:
a stack formed from a plurality of parallel segments;
a first fluid permeable column traversing the stack from a first end to a second end;
a second fluid permeable column traversing the stack from a third end to a fourth end;
a first electrode in electrical contact with the first end, the third end, or a combination thereof; and
a second electrode in electrical contact with the second end, the fourth end, or a combination thereof;
wherein each segment comprises a first fluid permeable region defined by a first fluid impermeable boundary and a second fluid permeable region defined by a second fluid impermeable boundary; and
wherein stacking of the plurality of segments aligns the first fluid permeable region within each of the plurality of parallel segments to form the first fluid permeable column and the second fluid permeable region within each of the plurality of parallel segments to form the second fluid permeable column.
The device of embodiment 62, further comprising:
a first reservoir in fluid contact with the first end, the third end, or a combination thereof;
a second reservoir in fluid contact with the second end, the fourth end, or a combination thereof; or
a combination thereof
The device of embodiment 63, further comprising:
a first separator in fluid contact with the first reservoir and the first end, the third end, or a combination thereof;
a second separator in fluid contact with the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of embodiment 64, wherein
the first separator is located between the first reservoir and the first end, the third end, or a combination thereof;
the second separator is located between the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of any one of embodiments 62-65, further comprising a loading slip layer, wherein the loading slip layer comprises:
a first fluid permeable region defined by a first fluid impermeable boundary;
wherein the loading slip layer can be translocated from a refracted position to a deployed position;
wherein in the retracted position the first fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the first fluid permeable region of the loading slip layer is in fluid contact with the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The device of embodiment 66, wherein the loading slip layer further comprises a second fluid permeable region defined by a second fluid impermeable boundary;
wherein in the retracted position the second fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the second fluid permeable region of the loading slip layer is in fluid contact with the second fluid permeable column.
The device of any one of embodiments 66-67, further comprising a collection slip layer, wherein the collection slip layer comprises:
a fluid permeable region defined by a fluid impermeable boundary;
wherein the collection slip layer can be translocated from a first position to a second position;
wherein in the first position, the fluid permeable region of the collection slip layer is in fluid contact with the first fluid permeable column and fluidly independent from the second fluid permeable column; and
wherein in the second position, the fluid permeable region of the collection slip layer is in fluid contact with the second fluid permeable column and fluidly independent from the first fluid permeable column.
The device of any one of embodiments 62-68, wherein the plurality of segments are joined together in a sheet, and the stack is formed by folding the sheet.
The device of embodiment 69, wherein folding the sheet comprises accordion folding the sheet.
The device of any one of embodiments 62-70, wherein the plurality of parallel segments comprises at least 3 parallel segments.
The device of any one of embodiments 62-71, wherein the first fluid permeable column, the second fluid permeable column, or a combination thereof is 10 mm or less in length.
The device of any one of embodiments 62-72, wherein the device is paper based.
The device of any one of embodiments 62-73, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof of the device of any one of embodiments 62-74; and
applying a potential to the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 75, wherein the potential is 40 V or less.
The method of any one of embodiments 75-76, wherein the sample comprises a first analyte.
The method of embodiment 77, further comprising separating the first analyte from the sample.
The method of any one of embodiments 75-78, further comprising accumulating the sample, the first analyte, or a combination thereof in a section of the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 79, further comprising removing the section of the first fluid permeable column, the second fluid permeable column, or a combination thereof to isolate the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 79-80, wherein the section comprises one or more of the parallel segments, a loading slip layer, a collection slip layer, or a combination thereof
The method of any one of embodiments 75-81, further comprising analyzing the sample, the first analyte, or a combination thereof to determine a property of the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 75-82, wherein introducing the sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
The method of embodiment 79, wherein the section of the first fluid permeable column comprises the collection slip layer in the first position.
The method of embodiment 84, further comprising translocating the collection slip layer to the second position.
The method of embodiment 85, further comprising applying a potential to the second fluid permeable column.
The method of embodiment 86, wherein the potential is 40 V or less.
The method of any one of embodiments 84-87, wherein the sample, the first analyte, or a combination thereof further comprises a second analyte.
The method of embodiment 88, further comprising separating the second analyte from the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 85-89, further comprising accumulating the sample, the first analyte, the second analyte or a combination thereof in a section of the second fluid permeable column.
The method of embodiment 90, further comprising removing the section of the second fluid permeable column to isolate the sample, the first analyte, the second analyte, or a combination thereof.
The method of any of embodiments 90-91, wherein the section of the second fluid permeable column comprises one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof.
The method of any one of embodiments 85-92, further comprising analyzing the sample, the first analyte, the second analyte, or a combination thereof to determine a property of the sample, the first analyte, the second analyte, or a combination thereof.
The method of any one of embodiments 85-93, wherein introducing the sample to the first fluid permeable column comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
A device comprising:
a plurality of planar segments, each planar segment comprising a first fluid permeable region defined by a first fluid impermeable boundary and a second fluid permeable region defined by a second fluid impermeable boundary;
wherein when the plurality of planar segments are stacked such that the first fluid permeable region of each planar segment is aligned and the second fluid permeable region of each planar segment is aligned, the first fluid permeable regions of the plurality of planar segments collectively forms a first fluid permeable column traversing the stacked plurality of planar segments from a first end to a second end, and the second fluid permeable regions of the plurality of planar segments collectively forms a second fluid permeable column traversing the stacked plurality of planar segments from a third end to a fourth end.
The device of embodiment 95, further comprising:
a first electrode in electrical contact with the first end, the third end, or a combination thereof;
a second electrode in electrical contact with the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of any one of embodiments 95-96, further comprising:
a first reservoir in fluid contact with the first end, the third end, or a combination thereof;
a second reservoir in fluid contact with the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of embodiment 97, further comprising:
a first separator in fluid contact with the first reservoir and the first end, the third end, or a combination thereof;
a second separator in fluid contact with the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of embodiment 98, wherein
the first separator is located between the first reservoir and the first end, the third end, or a combination thereof;
the second separator is located between the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of any one of embodiments 95-99, further comprising a loading slip layer, wherein the loading slip layer comprises:
a first fluid permeable region defined by a first fluid impermeable boundary;
wherein the loading slip layer can be translocated from a refracted position to a deployed position;
wherein in the retracted position the first fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the first fluid permeable region of the loading slip layer is in fluid contact with the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The device of embodiment 100, wherein the loading slip layer further comprises:
a second fluid permeable region defined by a second fluid impermeable boundary;
wherein in the retracted position the second fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the second fluid permeable region of the loading slip layer is in fluid contact with the second fluid permeable column.
The device of any one of embodiments 100-101, further comprising a collection slip layer, wherein the collection slip layer comprises:
a fluid permeable region defined by a fluid impermeable boundary;
wherein the collection slip layer can be translocated from a first position to a second position;
wherein in the first position, the fluid permeable region of the collection slip layer is in fluid contact with the first fluid permeable column and fluidly independent from the second fluid permeable column; and
wherein in the second position, the fluid permeable region of the collection slip layer is in fluid contact with the second fluid permeable column and fluidly independent from the first fluid permeable column.
The device of any one of embodiments 95-102, wherein the plurality of planar segments are joined together in a sheet, and the plurality of planar segments is stacked by folding the sheet.
The device of embodiment 103, wherein folding the sheet comprises accordion folding the sheet.
The device of any one of embodiments 95-104, wherein the plurality of planar segments comprises at least 3 planar segments.
The device of any one of embodiments 95-105, wherein the first fluid permeable column, the second fluid permeable column, or a combination thereof is 10 mm or less in length.
The device of any one of embodiments 95-106, wherein the device is paper based.
The device of any one of embodiments 95-107, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof of the device of any one of embodiments 95-108; and
applying a potential to the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 109, wherein the potential is 40 V or less.
The method of any one of embodiments 109-110, wherein the sample comprises a first analyte.
The method of embodiment 111, further comprising separating the first analyte from the sample.
The method of any one of embodiments 109-112, further comprising accumulating the sample, the first analyte, or a combination thereof in a section of the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 113, further comprising removing the section of the first fluid permeable column, the second fluid permeable column, or a combination thereof to isolate the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 113-114, wherein the section comprises one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof.
The method of any one of embodiments 109-115, further comprising analyzing the sample, the first analyte, or a combination thereof to determine a property of the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 109-116, wherein introducing the sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
The method of embodiment 113, wherein the section of the first fluid permeable column comprises the collection slip layer in the first position.
The method of embodiment 118, further comprising translocating the collection slip layer to the second position.
The method of embodiment 119, further comprising applying a potential to the second fluid permeable column.
The method of embodiment 120, wherein the potential is 40 V or less.
The method of any one of embodiments 118-121, wherein the sample, the first analyte, or a combination thereof further comprises a second analyte.
The method of embodiment 122, further comprising separating the second analyte from the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 119-123, further comprising accumulating the sample, the first analyte, the second analyte or a combination thereof in a section of the second fluid permeable column.
The method of embodiment 124, further comprising removing the section of the second fluid permeable column to isolate the sample, the first analyte, the second analyte, or a combination thereof.
The method of any of embodiments 124-125, wherein the section of the second fluid permeable column comprises one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof.
The method of any one of embodiments 118-126, further comprising analyzing the sample, the first analyte, the second analyte, or a combination thereof to determine a property of the sample, the first analyte, the second analyte, or a combination thereof.
The method of any one of embodiments 118-127, wherein introducing the sample to the first fluid permeable column comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
A device comprising:
a plurality of planar segments, each planar segment comprising:
a top surface;
a bottom surface; and
a first fluid permeable region defined by a first fluid impermeable boundary extending through the planar segment from the top surface to the bottom surface so as to form a first fluid permeable pathway extending through the planar segment from the top surface to the bottom surface;
a second fluid permeable region defined by a second fluid impermeable boundary extending through the planar segment from the top surface to the bottom surface so as to form a second fluid permeable pathway extending through the planar segment from the top surface to the bottom surface;
wherein when the plurality of segments are stacked such that the bottom surface of a first planar segment is in intimate contact with the top surface of a second planar segment:
the first fluid permeable regions together form a first fluid permeable column within the stacked plurality of planar segments extending from a first end to a second end; and
the second fluid permeable regions together form a second fluid permeable column within the stacked plurality of planar segments extending from a third end to a fourth end;
wherein the first end comprises the first fluid permeable region at the top surface of the first planar segment;
wherein the second end comprises the first fluid permeable region at the bottom surface of the last planar segment;
wherein the third end comprises the second fluid permeable region at the top surface of the first planar segment;
wherein the fourth end comprises the second fluid permeable region at the bottom surface of the last planar segment;
a first electrode in electrical contact with the first end, the third end, or a combination thereof;
a second electrode in electrical contact with the second end, the fourth end, or a combination thereof.
The device of embodiment 129, further comprising:
a first reservoir in fluid contact with the first end, the third end, or a combination thereof;
a second reservoir in fluid contact with the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of embodiment 130, further comprising:
a first separator in fluid contact with the first reservoir and the first end, the third end, or a combination thereof;
a second separator in fluid contact with the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of embodiment 131, wherein
the first separator is located between the first reservoir and the first end, the third end, or a combination thereof;
the second separator is located between the second reservoir and the second end, the fourth end, or a combination thereof; or
a combination thereof.
The device of any one of embodiments 129-132, further comprising a loading slip layer, wherein the loading slip layer comprises:
a first fluid permeable region defined by a first fluid impermeable boundary;
wherein the loading slip layer can be translocated from a refracted position to a deployed position;
wherein in the retracted position the first fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the first fluid permeable region of the loading slip layer is in fluid contact with the first fluid permeable column, the second fluid permeable column, or a combination thereof
The device of embodiment 133, wherein the loading slip layer further comprises:
a second fluid permeable region defined by a second fluid impermeable boundary;
wherein in the retracted position the second fluid permeable region of the loading slip layer is fluidly independent from the first fluid permeable column and the second fluid permeable column; and
wherein in the deployed position, the second fluid permeable region of the loading slip layer is in fluid contact with the second fluid permeable column.
The device of any one of embodiments 133-134, further comprising a collection slip layer, wherein the collection slip layer comprises:
a fluid permeable region defined by a fluid impermeable boundary;
wherein the collection slip layer can be translocated from a first position to a second position;
wherein in the first position, the fluid permeable region of the collection slip layer is in fluid contact with the first fluid permeable column and fluidly independent from the second fluid permeable column; and
wherein in the second position, the fluid permeable region of the collection slip layer is in fluid contact with the second fluid permeable column and fluidly independent from the first fluid permeable column.
The device of any one of embodiments 129-135, wherein the plurality of planar segments comprises a planar sheet and the plurality of planar segments is stacked by folding the planar sheet.
The device of embodiment 136, wherein folding the planar sheet comprises accordion folding the planar sheet.
The device of any one of embodiments 129-137, wherein the plurality of planar segments comprises 3 planar segments or more.
The device of any one of embodiments 129-138, wherein the first fluid permeable column, the second fluid permeable column, or a combination thereof is 10 mm or less in length.
The device of any one of embodiments 129-139, wherein the device is paper based.
The device of any one of embodiments 129-140, wherein the device further comprises a housing.
A method comprising:
introducing a sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof of the device of any one of embodiments 129-141; and
applying a potential to the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 142, wherein the potential is 40 V or less.
The method of any one of embodiments 142-143, wherein the sample comprises a first analyte.
The method of embodiment 144, further comprising separating the first analyte from the sample.
The method of any one of embodiments 142-145, further comprising accumulating the sample, the first analyte, or a combination thereof in a section of the first fluid permeable column, the second fluid permeable column, or a combination thereof.
The method of embodiment 146, further comprising removing the section of the first fluid permeable column, the second fluid permeable column, or a combination thereof to isolate the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 146-147, wherein the section comprises one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof.
The method of any one of embodiments 142-148, further comprising analyzing the sample, the first analyte, or a combination thereof to determine a property of the sample, the first analyte, or a combination thereof.
The method of any one of embodiments 142-149, wherein introducing the sample to the first fluid permeable column, the second fluid permeable column, or a combination thereof comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof.
The method of embodiment 146, wherein the section of the first fluid permeable column comprises the collection slip layer in the first position.
The method of embodiment 151, further comprising translocating the collection slip layer to the second position.
The method of embodiment 152, further comprising applying a potential to the second fluid permeable column.
The method of embodiment 153, wherein the potential is 40 V or less.
The method of any one of embodiments 151-154, wherein the sample, the first analyte, or a combination thereof further comprises a second analyte.
The method of embodiment 155, further comprising separating the second analyte from the sample, the first analyte, or a combination thereof
The method of any one of embodiments 152-156, further comprising accumulating the sample, the first analyte, the second analyte or a combination thereof in a section of the second fluid permeable column.
The method of embodiment 157, further comprising removing the section of the second fluid permeable column to isolate the sample, the first analyte, the second analyte, or a combination thereof.
The method of any of embodiments 157-158, wherein the section of the second fluid permeable column comprises one or more of the parallel segments, the loading slip layer, the collection slip layer, or a combination thereof.
The method of any one of embodiments 151-159, further comprising analyzing the sample, the first analyte, the second analyte, or a combination thereof to determine a property of the sample, the first analyte, the second analyte, or a combination thereof.
The method of any one of embodiments 151-160, wherein introducing the sample to the first fluid permeable column comprises translocating the loading slip layer to the deployed position, wherein the sample is initially located in the first fluid permeable region of the loading slip layer, the second fluid permeable region of the loading slip layer, or a combination thereof
The examples below are intended to further illustrate certain aspects of the systems and methods described herein, and are not intended to limit the scope of the claims.
The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process.
Presented herein is an origami paper-based electrophoretic device (oPAD-Ep), which can achieve rapid (˜5 min) separation of fluorescent molecules and proteins. The driving voltage was ˜10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep used multiple, thin (180 μm/layer) folded paper layers as the supporting medium for electrophoresis. This approach can shorten the distance between the anode and cathode, and this in turn can account for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep can enable sample introduction by use of a slip layer, as well as product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. The use of oPAD-Ep for simple separation of proteins in bovine serum is demonstrated (
Introduction
Herein, an electrophoretic (Ep) device, which can be integrated into paper analytical devices (PADs) (Maxwell E J et al. MRS Bull. 2013, 38, 309-314; Martinez A W et al. Anal. Chem. 2010, 82, 3-10) is discussed, and separation of fluorescent molecules and proteins at low voltages is demonstrated. The device, which is referred to herein as an oPAD-Ep (the “o” stands for origami) (Liu H and Crooks R M. J. Am. Chem. Soc. 2011, 133, 17564-17566), can be easy to construct (
In the 1930s, Tiselius developed the first Ep system, the “Tiselius apparatus”, for analysis of colloidal mixtures (Tiselius A. Trans. Faraday Soc. 1937, 33, 0524-0530). This technique has evolved over time to take advantage of physical and chemical differences between targets (such as proteins or DNA). For example, the supporting medium may be filter paper, natural gels, or synthetic gels (Martin N H and Franglen G T. J. Clin. Pathol. 1954, 7, 87-105; Scopes R K. Biochem. J. 1968, 107, 139-150; Thorne H V. Virology 1966, 29, 234-239; Meyers J A et al. J. Bacteriol. 1976, 127, 1529-1537; Giri K V. Nature 1957, 179, 632-632; Bachvaroff R and McMaster P R. Science 1964, 143, 1177-1179; Chrambach A and Rodbard D. Science 1971, 172, 440-451). The apparatuses used to carry out these separations can also vary widely, for example SDS-PAGE, capillary Ep, and isoelectric focusing (Weber K and Osborn M. J. Biol. Chem. 1969, 244, 4406-4412; Schägger H and Von Jagow G. Anal. Biochem. 1987, 166, 368-379; Pedersen-Bjergaard S and Rasmussen K E. Anal. Chem. 1999, 71, 2650-2656; Neuhoff V et al. Electrophoresis 1988, 9, 255-262, Bjellqvist B et al. J. Biochem. Bioph. Methods 1982, 6, 317-339).
In recent years, simple forms of paper Ep have been developed that can be incorporated into POC devices. For example, Ge et al. introduced a paper-based electrophoretic device for amino acid separation by imitating the design of conventional electrophoretic systems (Ge L et al. Chem. Commun. 2014, 50, 5699-5702). Using wax printing (Carrilho E et al. Anal. Chem. 2009, 81, 7091-7095), they patterned two reservoirs connected by a ˜20 mm-long channel on paper. A voltage of 330 V was applied across the channel, which achieved an electro-migration speed of a few mm/min for amino acids. Using an alternative design, Chen et al. achieved a similar electric field, but avoided the necessity of using a high applied voltage by placing the anode and cathode in close proximity (˜2 mm) (Chen S S et al. Lab Chip 2014, 14, 2124-2130). However, the device designs mentioned above involve either a high voltage, which is not suitable for POC applications, or challenging operational characteristics. Moreover, a constant pH was not maintained in either of these two devices, raising concerns about nonuniform Ep of amphoteric molecules, whose mobilities can be strongly dependent on the solution pH. The multilayer oPAD-Ep design described herein addresses these types of issues.
Three-dimensional (3D) PADs were first reported by Whitesides and coworkers in 2008 (Martinez A W et al. Proc. Natl. Acad. Sci. 2008, 105, 19606-19611). In these devices, multiple paper layers were stacked and held together with double-sided tape. More recently, a simpler method for achieving similar functionality was introduced by using the fabrication principles of origami; that is, folding a single piece of paper into a 3D geometry (Liu H and Crooks R M. J. Am. Chem. Soc. 2011, 133, 17564-17566). This family of sensors is referred to herein as oPADs. Since their inception, a number of oPADs have been reported for various applications, including: detection of biomolecules, paper-based batteries, and a microscope (Liu H et al. Angew. Chem., Int. Ed. 2012, 51, 6925-6928; Scida K et al. Anal. Chem. 2013, 85, 9713-9720; Ge L et al. Lab Chip 2012, 12, 3150-3158; Chen S S et al. Lab Chip 2014, 14, 2124-2130; Cybulski J S et al. PLoS ONE 2014, 9, e98781). In contrast to earlier systems, the oPAD-Ep takes advantage of the thinness of the paper used for device fabrication. This can result in a short distance between the anode and cathode (˜a few millimeters), which can lead to electric fields of ˜2 kV/m with an input voltage of 10 V. When subjected to this field, fluorescent molecules or proteins can penetrate each paper layer at a speed of 1-3 layers/min. Herein, the fundamental characteristics of the oPAD-Ep design are discussed, the separation of fluorescent molecules based on their different electrophoretic mobilities is demonstrated, and it is shown that bovine serum albumin (BSA) can be separated from calf serum within 5 min.
Tris-HCl buffer (1.0 M, pH=8.0), phosphate buffered saline (PBS, pH=7.4), and Whatman Grade 1 chromatography paper, were purchased from Fisher Scientific. Silver wire (2.0 mm in diameter), calf serum from formula-fed bovine calves, albumin (lyophilized powder, ≧95%, agarose gel Ep) and IgG (reagent grade, ≧95%, SDS-PAGE, essentially salt-free, lyophilized powder) from bovine serum, and FluoroProfile protein quantification kits were purchased from Sigma-Aldrich. The following fluorescent molecules were used as received: Ru(bpy)3Cl2 (Fluka), 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY2−, Invitrogen), 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt (MPTS3−, Anaspec), 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTS4−, Fisher Scientific), Rhodamine 6G (Acros), methylene blue (Sigma-Aldrich), and rhodamine B (Fluka). All solutions were prepared using deionized water having a resistivity of 18.2 MSΩ·cm from a Milli-Q Gradient System (Bedford, Mass.). Serum protein solutions were prepared with PBS.
Device Fabrication.
oPAD-Eps were fabricated in three steps: (1) the slip layer and origami paper were patterned using wax printing (Carrilho E et al. Anal. Chem. 2009, 81, 7091-7095), (2) the plastic buffer reservoirs were fabricated using a laser cutter, and (3) the oPAD-Eps were assembled as shown in
As shown in
Operation of the oPAD-Ep.
Before use, the two reservoirs of the oPAD-Ep were filled with 300.0 μL of buffer and then a Ag/AgCl electrode was inserted into each of them. For fluorescent molecule Ep, 0.20 M Tris-HCl (pH=8.0) was used, while PBS (pH=7.4) was used for protein Ep. Ag/AgCl electrodes were prepared by immersing Ag wires in commercial bleach overnight (Lathrop D K et al. J. Am. Chem. Soc. 2010, 132, 1878-1885), and rinsed thoroughly with DI water before use. The surface of the Ag wires turned dark brown after being oxidized to AgCl. A 0.50 μL aliquot of sample solution was loaded at the designated zone on the slip layer, and then introduced by pulling the slip layer into alignment with the origami paper. A BK Precision DC regulated power supply (model 1621A) was used to apply a voltage between the two Ag/AgCl electrodes. After Ep, the buffer and electrodes were removed from the reservoirs, and the screws were loosened to unfold the origami paper for analysis.
Fluorescence Analysis.
A Nikon AZ100 multi-purpose zoom fluorescence microscope was used to acquire fluorescent images of each oPAD-Ep layer, including the slip layer, and ImageJ software (NIH, Bethesda, Md.) was used to analyze the fluorescence intensity. For protein analyses, the FluoroProfile protein quantification kit from Sigma-Aldrich was used to label proteins with a fluorescent tag (Liu H and Crooks R M. J. Am. Chem. Soc. 2011, 133, 17564-17566). Following Ep, 0.50 μL FluoroProfile fluorescent reagent solution was spotted onto the origami paper and slip layer, both of which were placed in a humidity chamber for 30.0 min and then taken out and dried for an additional 30.0 min in a dark room. During this time period, epicocconone in the stain solution fully reacts with primary amine groups on proteins, producing a fluorescent conjugate having two excitation maxima at ˜400 nm and ˜500 nm, with emission at 610 nm (Bell P J and Karuso P. J. Am. Chem. Soc. 2003, 125, 9304-9305). An Omega XF204 filter (excitation: 540 nm and emission: 570-600 nm) was used to acquire the fluorescence images of stained proteins in the oPAD-Ep.
Results and Discussion
The Ep of single fluorescent molecules was examined, followed by the investigation of the use of the oPAD-Eps for more complex tasks including separation of fluorescent molecules and proteins. The fluorescent molecules used for demonstration purposes are listed in Table 1, along with their excitation and emission wavelengths (fluorescence spectra are provided in
For operation of the oPAD-Ep, the paper part of the device was folded, as shown in
In a series of experiments, a 23-layer paper device was used to study the migration of BODIPY2−. This experiment was carried out by placing a 0.50 μL aliquot of 1.0 mM BODIPY2− onto the slip layer, which was in turn placed between the second and third layers of the oPAD-Ep (e.g., Position 3 in
To provide a more quantitative analysis of the BODIPY2− migration experiment (
Δσ2=2Dpapert (1)
Here, Δσ2 is the mean square displacement at time t. A plot of σ2 vs. t is provided in
It has been shown previously that the electroosmotic velocity of albumin in barbital buffer in a variety of common papers ranges from ˜30% to 170% of its Ep velocity at pH 8.8 (Kunkel H G and Tiselius A. J. Gen. Physiol. 1951, 35, 89-118). In addition, Posner and coworkers observed significant electroosmotic flow (EOF) in nitrocellulose paper during their paper-based isotachophoretic preconcentration experiments. Specifically, they found that the fluorescent molecule AF488, which was focused between the leading and trailing electrolytes, moved faster (velocity increased from ˜30 μm/s to 150 μm/s) after adding 3% polyvinylpyrrolidone (PVP) to the leading electrolyte to suppress the EOF (Moghadam B Y et al. Anal. Chem. 2014, 86, 5829-5837). Clearly, the EOF in paper can vary over a wide range and can be dependent on experimental conditions such as paper structure and electrolyte. Therefore, the electroosmotic velocity of Rhodamine B, which is neutral in the pH range between 6.0 and 10.8 (Oh Y J et al. Lab Chip 2008, 8, 251-258), was measured to evaluate the EOF in oPAD-Eps. Rhodamine B (0.50 μL, 0.10 mM) was initially loaded onto the slip layer at Position 21, and the direction of applied electric field was from Position 23 toward Position 1 (
The following procedure was used to determine the value of E from equation 2. A multimeter was connected in series with the power supply to measure the current flowing through the oPAD-Ep with and without origami paper present in the device. At an applied voltage of 10.0 V, the values of the two currents were ˜1.7 mA and 6.0 mA, respectively. Using the difference between these currents and Ohm's law, the resistance of the origami paper was calculated to be ˜4.2 ka By multiplying this resistance by the current at 10.0 V, the voltage drop (ΔV) across the paper was determined to be ˜7 V. The value of E in the oPAD-Ep (˜1.7 kV/m at an applied voltage of 10.0 V) was then calculated by dividing ΔV by the total thickness (d=4.1 mm) of the 23-layer origami paper. Finally, using equation 2, μEp for BODIPY2− in the oPAD-Ep was calculated to be ˜2.2×10−9 m2/(s·V).
Following the procedure described for BODIPY2−, the Ep properties of three other dyes in the oPAD-Ep were evaluated: PTS4−, MPTS3−, and Ru(bpy)32+. Plots of the position of these dyes as a function of time are shown in
The separation of a mixture of two oppositely charged fluorescent molecules, MPTS3− and Ru(bpy)32+, which migrate in opposite directions upon the application of an electric field, was examined using the oPAD-Ep. The separation of MPTS3− and Ru(bpy)32+ was carried out as follows. A mixture containing 1.5 mM MPTS3− and 1.5 mM Ru(bpy)32+ was prepared by mixing equal aliquots of 3.0 mM MPTS3− and 3.0 mM Ru(bpy)32+. A 0.50 μL aliquot of the mixture was spotted onto the slip layer, and the slip layer was inserted at Position 11 of the oPAD-Ep. All other conditions were the same as in the previously described single-analyte experiments. When 10.0 V was applied between the two Ag/AgCl driving electrodes, MPTS3− moved from its initial position towards the anode and Ru(bpy)32+ migrated toward the cathode. After carrying out the separation, each layer of the oPAD-Ep was characterized spectroscopically using a different fluorescence filter (Table 1). Because the emission intensity is different for the two dyes, the results of this experiment, shown in
The separation of two dyes with the same charge was also examined. This demonstration of the oPAD-Ep involved the separation of two negatively charged dyes, BODIPY2− and PTS4−, which have the same charge but μEp values that differ by about 25%. In this case, a 0.50 μL aliquot of a mixture containing 1.5 mM PTS4− and 0.50 mM BODIPY2− was initially situated at Position 3 (
Ep is widely used to separate biomolecules such as DNA and proteins. One of the most common electrophoretic techniques is gel Ep, which uses a gel to suppress the thermal convection caused by Joule heating and to sieve biomolecules on the basis of their size. This method is routinely used in clinical laboratories to test for abnormalities in a variety of biological matrices, including: serum, urine, blood, and cerebrospinal fluid (Jeppson J et al. Clin. Chem. 1979, 25, 629-638). For example, in serum protein gel Ep, normal serum is separated into five different bands: (1) Albumin, which is approximately two-thirds of the total protein content (3-5 g/dL); (2) Alpha-1 (0.1-0.3 g/dL) and (3) Alpha-2 (0.60-1.0 g/dL), which are two groups of globulins mainly including heptoglobin, ceruloplasmin, and macroglobin; (4) Beta (0.7-1.2 g/dL), composed of transferrin and lipoprotein; and (5) Gama (0.6-1.6 g/dL), which contains primarily immunogolublins such as IgG (Kyle R et al. Clinical Indications and Applications of Electrophoresis and Immunofixation. In Manual of Clinical Immunology; Rose N et al., Eds.; ASM Press: Washington D.C. 2002; pp 66-70). An excess or insufficiency in any of these bands can indicate a need for medical attention. Commercially available devices for separating serum proteins can require a high voltage (200-300 V) and a long separation time (˜1 h), both of which are impractical for POC applications. In this section, it is shown that the oPAD-Ep can rapidly (5 min) separate serum proteins using a voltage of 10V.
The Ep properties of bovine serum albumin (BSA) and IgG (also from bovine serum) were initially evaluated separately in the oPAD-Ep. In these experiments, an 11-layer oPAD-Ep was first wetted with 1×PBS buffer (ionic strength 163 mM, pH=7.4). Next, 0.50 μL of a 0.1×PBS buffer (ionic strength 16.3 mM) containing either 5.0 g/dL BSA or 1.0 g/dL IgG was loaded at Position 3 of the oPAD-Ep. These conditions are different from those used for separating the fluorescent molecules: the oPAD-Ep consists of fewer layers and the buffer concentration is lower, both of which serve to increase the electric field within the device. This experimental flexibility (e.g., the number of layers in the device) can be an advantageous feature of the oPAD-Ep.
The mobility of BSA measured in the oPAD-Ep is an order of magnitude lower than the value reported in the literature using conventional paper Ep (Kunkel H G and Tiselius A. J. Gen. Physiol. 1951, 35, 89-118). In the previously reported experiments, however, Ep was carried out for 14 h (150 times longer than in these experiments) to achieve a reasonable separation of serum proteins. This long immersion time can cause deterioration of the paper structure, which can lead to faster migration of BSA. This contention is supported by the small difference (<8%) between the measured mobility of BSA in paper and in free solution noted in this prior report (Kunkel H G and Tiselius A. J. Gen. Physiol. 1951, 35, 89-118). In addition, the type of paper and the pH used is different, and the effects of electroosmosis were not considered in the calculations. After migration, remnants of BSA were observed on the paper from the oPAD-Ep (Positions 6-9,
In contrast to BSA, the distribution of IgG shifted only slightly toward the anode after 5.0 min (
Applying the same conditions used for the control experiments illustrated in
Paper zones with a diameter of 3.5 mm were used to obtain a BSA calibration curve. These paper zones were first wetted with 0.50 μL 0.1×PBS solution (ionic strength 16.3 mM), and then 0.50 μL of BSA solutions having different concentrations were spotted at the wet paper zone, followed by another 0.50 μL epicocconone to stain the protein. After that, the devices were kept in a humidity chamber for 30.0 min to allow epicocconone to fully react with BSA, and then moved to a dark room until the samples were dry (˜30 min). After taking fluorescence micrographs, the RFU of each paper zone was integrated in ImageJ and plotted as a function of BSA concentration (
A low-cost separation system based on folded paper has been described. This approach takes advantage of the thinness of origami paper (180 μm/layer) to achieve a high electric field strength (several kV/m) at a low applied voltage (˜10 V). The voltage required for the oPAD-Ep is more than an order of magnitude lower than that used in conventional electrophoretic devices. The simple construction, low voltage requirement, and ease of use can make the oPAD-Ep a candidate for POC applications (e.g., for separation of serum proteins as illustrated in
In this example, use of devices for isotachophoretic (ITP) preconcentration of charged molecules, such as DNA is described.
Isotachophoresis (ITP) is a special form of electrophoresis (Ep), and can be used for selective separation and preconcentration of analytes with specific ionic mobilities (e.g., DNA). ITP can use different electrolyte solutions in the cathode and anode buffer reservoirs: one with a higher ionic mobility, referred to as the leading electrolyte (LE), and the other one with a lower ionic mobility, referred to as the trailing electrolyte (TE) (
The ITP method using the devices described herein is illustrated generally in
Preconcentration of specific targets before analysis by the method discussed herein can lower the limit of detection (LOD) on a paper device, at least by 2 to 3 orders of magnitude (e.g.,
Herein, a paper isotachophoresis (ITP) platform fabricated using the principles of origami (Japanese paper folding) is described (Jung B et al. Anal. Chem., 2006, 78, 2319-2327). The device can be inexpensive, easy to assemble and operate, and can electrokinetically concentrate DNA. The design of this origami paper analytical device (oPAD) for isotachophoresis (oPAD-ITP) is illustrated in
The power requirements of the oPAD-ITP for the examples described herein were supplied by two 9 V batteries. This is a >20-fold lower voltage than previously reported, and therefore true point-of-care (POC) applications can be accessible and complications due to Joule heating can be minimized (Rosenfeld T and Bercovici M. Lab Chip, 2014, 14, 4465-4474; Moghadam B Y et al. Anal. Chem., 2014, 86, 5829-5837; Moghadam B Y et al. Anal. Chem., 2015, 87, 1009-1017). Further, the origami paper channel is fully enclosed by wax, and therefore solvent (e.g., water) evaporation can be minimized. The plastic slip layer can be used to establish a well-defined initial trailing electrolyte/leading electrolyte boundary. The oPAD-ITP is “digital” in the sense that the enriched product will be on individual paper layers and can be reclaimed by simply cutting off the desired layer(s). This opens up the possibility of coupling the oPAD-ITP with other detection systems to achieve lower limits of detection (LOD).
Due to their biocompatibility (Martinez A W et al. Anal. Chem., 2010, 82, 3-10), ease of fabrication (Carrilho E et al. Anal. Chem., 2009, 81, 7091-7095), and low-cost (Martinez A W et al. Anal. Chem., 2010, 82, 3-10; Carrilho E et al. Anal. Chem., 2009, 81, 5990-5998), PADs can be used as POC diagnostic devices and systems (Gubala V et al. Anal. Chem., 2011, 84, 487-515). A number of detection methods have been developed for PADs, including: electrochemistry (Scida K et al. Anal. Chem., 2013, 85, 9713-9720; Scida K et al. Anal. Chem., 2014, 86, 6501-6507; Dungchai W et al. Anal. Chem., 2009, 81, 5821-5826), photography (Ellerbee A K et al. Anal. Chem., 2009, 81, 8447-8452; Zhao W et al. Anal. Chem., 2008, 80, 8431-8437), luminescence (Ge L et al. Biomaterials, 2012, 33, 1024-1031; Ge L et al. Chem. Commun., 2014, 50, 5699-5702; Zhang X et al. Chem. Commun., 2013, 49, 3866-3868; Delaney J L et al. Anal. Chem., 2011, 83, 1300-1306), and others (Parolo C and Merkoci A. Chem. Soc. Rev., 2013, 42, 450-457; Yetisen A K et al. Lab Chip, 2013, 13, 2210-2251). Nevertheless, it still can be difficult to achieve sufficiently low limits of detection for some important applications, particularly those involving nucleic acid detection. One way around this problem is sample preconcentration, and, although there are many ways to approach this for bulk solutions, the number that have been reported for paper platforms is limited (Yetisen A K et al. Lab Chip, 2013, 13, 2210-2251).
Isotachophoresis (ITP) is an electrophoretic technique that can effectively concentrate ionic samples with minimal sample pretreatment (Jung B et al. Anal. Chem., 2006, 78, 2319-2327; Walker P A et al. Anal. Chem., 1998, 70, 3766-3769; Persat A et al. Anal. Chem., 2009, 81, 9507-9511). In a typical isotachophoresis experiment, the electric field profile across an electrophoretic channel is controlled by using electrolytes having different mobilities: a fast moving leading electrolyte and a slow moving trailing electrolyte (Everaerts F M et al. Isotachophoresis: theory, instrumentation and applications, Elsevier, 2011). When a voltage is applied across the channel, sample ions, initially present in the trailing electrolyte solution, out-pace the trailing electrolyte and accumulate at the trailing electrolyte/leading electrolyte boundary, which can result in a high local concentration.
Herein, an oPAD design for electrophoretic separations was adapted for isotachophoresis, such as for isotachophoresis concentration of DNA. Four experiments are described velow in more detail. First, isotachophoresis of 23-mer single-stranded DNA labeled with Cyanine5 (ssDNA) using the oPAD-ITP is discussed and this result is compared to a mathematical model (Rosenfeld T and Bercovici M. Lab Chip, 2014, 14, 4465-4474). Second, the effect of the initial concentration of sample DNA on isotachophoresis enrichment is examined. Third, some fundamental principles of the oPAD-ITP are examined, such as the electric field profile during sample focusing. Finally, an application of the oPAD-ITP for the isotachophoresis of a 100 bp dsDNA ladder, which is comprised of 100-1517 bp of double-stranded DNA (dsDNA), is discussed. The results of these studies suggest that the oPAD-ITP can provide additional functionality for a variety of paper-based detection platforms.
Whatman Grade 1 cellulose paper, HCl, acetic acid, and agarose were purchased from Fisher Scientific (Walthman, Mass.). Single-stranded DNA (ssDNA, 5′-AGT CAG TGT GGA AAA TCT CTA GC-Cy5-3′) was ordered from Integrated DNA Technologies (Coralville, Iowa) and purified by HPLC. The 100 bp dsDNA ladder was from New England BioLabs (Ipswich, Mass.). The following chemicals were from Sigma-Aldrich (St. Louis, Mo.) and used as received: 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris base), 2-aminoethanesulfonic acid (taurine), Ru(bpy)3Cl2, ethidium bromide (EtBr) solution (10 mg/mL), and EDTA.
The fabrication of the oPAD-ITP is similar to that of an electrophoretic device reported previously (Luo L et al. Anal. Chem., 2014, 86, 12390-12397), but there are some important differences. Whatman grade 1 cellulose paper (˜180 μm thick) was patterned with wax (CorelDRAW designs shown in
The trailing electrolyte and leading electrolyte solutions used in these experiments were 2.0 mM tris-taurine (pH 8.7) and 1.0 M tris-HCl (pH 7.3), respectively. After assembling the oPAD-ITP (origami paper, slip layer and reservoirs), the reservoirs were filled with 1.0 mL of the trailing electrolyte or leading electrolyte buffer as shown in
For the ssDNA concentration experiments, 40.0 nM of the ssDNA was initially present in the trailing electrolyte solution. For the dsDNA ladder experiments, 0.50 μg/mL of the dsDNA ladder was initially mixed in the trailing electrolyte solution. In the electric field profiling experiment, 30.0 μM Ru(bpy)32+ was initially mixed in the leading electrolyte solution. For these latter experiments, Ag/AgCl electrodes (Lathrop D K et al. J. Am. Chem. Soc., 2010, 132, 1878-1885), rather than Pt, were used to avoid generation of Cl2 due to the low resistance and high current level (˜17 mA). After isotachophoresis experiments, the solutions in both reservoirs were removed and the device was disassembled to analyze the content of each paper layer.
A Nikon AZ100 multi-purpose zoom fluorescence microscope with Nikon filters (ssDNA: 590-650 nm excitation and 663-738 nm emission; Ru(bpy)32+: 420-490 nm excitation and 510-700 nm emission) was used to acquire fluorescence images of each fold of the oPAD-ITP device. All images were then processed with ImageJ software to obtain integrated relative fluorescence unit (RFU) intensity for quantification of fluorescent molecules on each layer.
Gel electrophoresis was used to quantify the dsDNA content on each paper layer after isotachophoresis of the dsDNA ladder. Gel electrophoresis was chosen for two reasons. First, most common dsDNA stains, such as SYBR gold or EtBr, exhibit a high background on cellulose paper, and this can make it difficult to visualize and quantify the amount of dsDNA. Second, the dsDNA ladder is comprised of twelve dsDNA components having lengths ranging from 100 to 1517 bp. Gel electrophoresis can separate them and provides quantitative information for each component of the ladder.
The gel electrophoresis analyses were conducted as follows. First, each fold of the paper was cut off, dried, and then inserted into a 1.3% agarose gel containing 10 μg/mL EtBr (
The ssDNA focusing experiments were carried out as follows. An 11-layer oPAD-ITP was assembled as shown in
In microfluidic isotachophoresis experiments, sample ions are focused at the trailing electrolyte/leading electrolyte boundary, where there is a sharp change in the magnitude of the electric field. As the experiment progresses, this boundary migrates toward the leading electrolyte reservoir.
Here, a is the slope of the linear fit in
In isotachophoresis, the collection efficiency (C %) is defined as the percentage of the original sample that is accumulated by an isotachophoresis device during a defined period of time. The collection efficiency (C %) for the oPAD-ITP was calculated using equation 4 (Persat A et al. Anal. Chem., 2009, 81, 9507-9511).
Here, Cj is the concentration of ssDNA on the jth layer and Vj is the liquid capacity of one paper layer, ˜0.5 μL. Co and VTE are the original sample concentration and the volume of the trailing electrolyte solution, respectively. The calculated value of the collection efficiency (C %) is plotted as a function of time in
In isotachophoresis, the extraction efficiency is the ability of the device to concentrate a defined sample volume per unit electrical charge consumed. A descriptor, η, can be used represent the extraction efficiency and can be calculated using equation 5 (Rosenfeld T and Bercovici M. Lab Chip, 2014, 14, 4465-4474). A high value of η means less energy is required to concentrate a sample.
Here, NDNA(t) is the total moles of ssDNA focused by the oPAD-ITP after t min. In the experiments discussed herein, the current, i(t), remained almost constant at ˜0.53 mA during the focusing process (
According to classical peak-mode isotachophoresis theory, when the sample concentration is negligible compared with the concentration of either electrolyte (trailing electrolyte and leading electrolyte), the maximum peak sample concentration (Csample,peak) depends solely on the trailing electrolyte and leading electrolyte composition, and is independent of the initial sample concentration (CO) (Jung B et al. Anal. Chem., 2006, 78, 2319-2327). Therefore, the enrichment factor (EF), defined as the value of Csample,peak divided by Co, will be inversely proportional to the value of CO. Accordingly, the oPAD-ITP performance was examined using different initial ssDNA concentrations (CDNA,0), but otherwise the same experimental procedure described above.
In microfluidic devices, focusing of analyte at the trailing electrolyte/leading electrolyte boundary can result from a sharp transition of the electric field between the trailing electrolyte and leading electrolyte (Chambers R D and Santiago J G. Anal. Chem., 2009, 81, 3022-3028). To investigate if the same is true for the oPAD-ITP, the electric field profile during isotachophoresis focusing was measured, according to a previously reported approach (Chambers R D and Santiago J G. Anal. Chem., 2009, 81, 3022-3028). Specifically, Ru(bpy)32+, a nonfocusing fluorescent tracer (NFT) (Chambers R D and Santiago J G. Anal. Chem., 2009, 81, 3022-3028), was added to the leading electrolyte solution, and then its distribution across the paper folds in the oPAD-ITP was determined after focusing. In principle, the nonfocusing fluorescent tracer will migrate through the channel during isotachophoresis and leave behind a concentration distribution that is inversely proportional to the local electric field strength.
There is a possibility that the step-shaped distribution of nonfocusing fluorescent tracer shown in
Based on the results in this section, ssDNA focusing can be caused by the electric field transition at the trailing electrolyte/leading electrolyte boundary, and, to a lesser extent, by boundary effect.
Even though short DNA strands (usually several tens of bases) are widely used as model targets for developing DNA sensing technologies, real-world DNA, for example, in viruses or bacteria, is usually composed of thousands of base pairs (Lodish H F et al. Molecular cell biology, Citeseer, 2000; Davis L. Basic methods in molecular biology, Elsevier, 2012). Accordingly, it can be desirable for an isotachophoresis device to be capable of focusing DNA strands longer 100 bp. In this section, the oPAD-ITPs were used for focusing a 100 bp dsDNA ladder containing 100-1517 bp dsDNA. The same experimental setup and buffer conditions used in the previous section were used for these experiments. That is, the dsDNA ladder was loaded into the trailing electrolyte buffer, and the voltage was switched on for 10 min.
After isotachophoresis, each fold of paper was removed from the channel, and gel electrophoresis was used to elute its dsDNA content (
Herein, an origami paper-based device suitable for carrying out low-voltage isotachophoresis, the oPAD-ITP, was used for focusing of DNA samples having lengths ranging from 23 to at least 1517 bp. DNA was concentrated by more than two orders of magnitude within 4 min. The device uses a 2 mm-long, 2 mm-wide circular paper channel formed by concertina folding a paper strip and aligning the circular paper zones on each layer. Due to the short channel length, a high electric field of ˜16 kV/m can be generated in the paper channel using two 9 V batteries. The multiplayer architecture can also enable reclamation and analysis of the sample after isotachophoresis focusing by opening the origami paper and cutting out the desired layers. The electric field in the origami paper channel during isotachophoresis experiments was profiled using a nonfocusing fluorescent tracer. The result showed that focusing can rely on formation and subsequent movement of an electric field boundary between the leading and trailing electrolyte.
This approach can resolves several issues of previously reported paper-based isotachophoresis designs, including high operating voltage, solvent evaporation, and difficult sample reclamation. Using the oPAD-ITP, a >100-fold enrichment of ssDNA and dsDNA having lengths of up to 1517 bps was demonstrated. The time required for enrichment was ˜10 min, the paper device can accommodate solution volumes of up to 1.0 mL, and is battery operated (18 V). The collection efficiency ranged from ˜15-20%. The electric field profiling experiments, using Ru(bpy)32+ as a tracer, indicated that the focusing mechanism in the oPAD-ITP can be the same as in bulk liquid solutions: accumulation of sample at the boundary between the trailing electrolyte and leading electrolyte.
The oPAD-ITP can be coupled with other paper-based detection system to achieve lower limits of detection (Scida K et al. Anal. Chem., 2014, 86, 6501-6507). The structure of the paper channels can be tailored to achieve better sample enrichment.
In the examples above, fluorescence has been used to analyze analytes on each paper layer of an origami device. Here, electrochemical methods were used for quantitative analysis of the analytes on a paper layer. As shown in
Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
This application claims benefit of U.S. Provisional Application No. 62/084,076, filed Nov. 25, 2014, which is hereby incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. CBET1402242 awarded by the National Science Foundation. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62084076 | Nov 2014 | US |
