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WO2023019105A1 - Dispositif d'électrophorèse sur gel à cartouche pour séparation de biomolécules - Google Patents

Dispositif d'électrophorèse sur gel à cartouche pour séparation de biomolécules Download PDF

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Publication number
WO2023019105A1
WO2023019105A1 PCT/US2022/074661 US2022074661W WO2023019105A1 WO 2023019105 A1 WO2023019105 A1 WO 2023019105A1 US 2022074661 W US2022074661 W US 2022074661W WO 2023019105 A1 WO2023019105 A1 WO 2023019105A1
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WIPO (PCT)
Prior art keywords
separation
cartridge device
electrophoresis
chamber
gel
Prior art date
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Ceased
Application number
PCT/US2022/074661
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English (en)
Inventor
Mark Donald Mcclure
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Keydev Inc
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Keydev Inc
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Filing date
Publication date
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Publication of WO2023019105A1 publication Critical patent/WO2023019105A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials

Definitions

  • the present disclosure relates to a gel cartridge, system, and method for conducting electrophoresis tests.
  • Slab gel electrophoresis is used to separate molecules based on size, shape, and charge.
  • Traditional slab gel electrophoresis is a time and labor intensive process requiring careful skill, taking approximately 1 to 8 (or more) hours to prepare and run.
  • Pre-manufactured gel cartridges solve some of the labor issues by shifting the gel preparation steps to the factory. Examples are discussed in United States Patent Nos. 3,715,295 to Tocci, 5,582,702 to Cabilly et. al., and 6,905,585 to Goncalves. Customers purchase a pre-cast gel, which eliminates the lengthy preparation steps.
  • a multi-lane gel cartridge with a console system for improved handling, reduced cost, and faster results in the electrophoresis and separation of biomolecules using gel electrophoresis is shown and described herein.
  • One aspect, object, feature, and/or advantage of the present disclosure is a gel cartridge with at least one separation column comprising a cathode chamber, a sample input chamber, a separation lane, and an anode chamber.
  • the cartridge is designed so that when a molten or liquid separation gel is placed into the anode chamber and cathode chamber, surface wetting causes the molten or liquid gel to fill the separation lane up to the sample input chamber boundary.
  • the cartridge contains either geometric modifications or surface hydrophobic modifications that inhibit gel from migrating into and filling the sample input chamber. No gravity, pump pressure, pipette pressure, or vacuum pressure is required to fill the separation lane.
  • Another aspect, object, feature, and/or advantage of the present disclosure is a small total void volume of cathode chamber, anode chamber and separation lane.
  • the total volume can be between 0.25 mL to 2 mL. This allows the user to apply only a small amount of gel per separation, resulting in vastly improved cost savings per run.
  • the small volume also allows molten separation media, such as agarose gel, to rapidly cool and solidify, resulting in short (less than 5 minutes) overall cartridge preparation time, allowing the users to have a complete preparation and separation time of less than 10 minutes.
  • Another aspect, object, feature, and/or advantage of the present disclosure is the inclusion of 3-dimensional features into the cartridge that prevent the migration of gel via surface wetting forces into the sample input chamber. This allows the user to fill the separation lane by placing a few drops of molten or liquid gel into the anode chamber and cathode chamber. The sample input chamber remains empty and gel-free, allowing for subsequent input of the sample for analysis.
  • Another aspect, object, feature, and/or advantage of the present disclosure is a hydrophobic modification of the surfaces of the plastic walls of the sample input chamber, which inhibits the migration of gel via surface wetting forces into the sample input chamber. This allows the user to fill the separation lane by placing a few drops of molten or liquid gel into the anode chamber and cathode chamber. The sample input chamber remains empty and gel-free, allowing for subsequent input of the sample for analysis.
  • Another aspect, object, feature, and/or advantage of the present disclosure is the use of low-cost materials such as thermoplastic films, designed for high-speed, high-volume construction, allowing a low cost of the final cartridge.
  • Another aspect, object, feature, and/or advantage of the present disclosure has an anode chamber and cathode chamber that when placed in a vertical position are higher than the separation lane. This allows for a passive, gravity-assist filling of the separation lane when liquid gel is placed into the anode and cathode chambers.
  • Another aspect, obj ect, feature, and/or advantage of the present disclosure is a gel cartridge that allows the user to apply their own gel for the separation and analysis of biomolecules.
  • Either commercial or custom gels i.e. with the addition of surfactants, denaturants, dyes, etc.
  • surfactants i.e. with the addition of surfactants, denaturants, dyes, etc.
  • gel cartridge that allows the user to apply their own gel for the separation and analysis of biomolecules.
  • surfactants i.e. with the addition of surfactants, denaturants, dyes, etc.
  • Another aspect, object, feature, and/or advantage of the present disclosure is an improved bufferless gel electrophoresis system.
  • the reagents used to fill the cartridge are electrophoresis separation gel only, as well as the input sample for separation. There is no liquid water overlay, liquid buffer overlay, or liquid buffer reservoir. Bufferless operation is achieved by (a) sizing electrode chamber volumes to provide sufficient buffering (i.e., for ions and pH) relative to the separation channel volume (c) sizing electrode chamber surface area for sufficient heat dissipation d) sizing the separation channels to have a small cross sectional area (e.g. , 2.25 square millimeters) to allow for less electrical power usage and less heat buildup (e) enclosing the separation channel to limit evaporation of the buffer in the gel.
  • a small cross sectional area e.g. , 2.25 square millimeters
  • the separation channel is split into non-continuous segments of gel at the sample loading chamber. This allows the sample to be the only material to form the electrical path between the gel segments in the separation channel, resulting in a more consistent electrical field across the sample with sharper separation bands with minimal smearing or smiling. Contrasted with prior art, previous methods cast a sample well using the gel to contain the sample.
  • Another aspect, object, feature, and/or advantage of the present disclosure is a cartridge design with a thin plastic film construction of approximately 20 microns to 400 microns that allows users to easily excise a specific portion of gel after the electrophoresis is complete. This allows users to obtain purified samples for downstream use.
  • Another aspect, object, feature, and/or advantage of the present disclosure is a console that allows users to run the electrophoresis of the prepared gel cartridge, as well as to analyze and visualize the electrophoresis as it occurs, in real time using a fluorescent imaging system.
  • Another aspect, object, feature, and/or advantage of the present disclosure is a process for analyzing biomolecules using the gel cartridge and analysis console.
  • the steps comprise (a) melting a small amount of gel (preferably between 0.5 and 2.0 mL), (b) placing the gel cartridge into the analysis console, (c) placing a few drops of molten gel into the anode chamber and cathode chamber, and allowing the gel to migrate into the separation lane up to the boundaries of the sample input chamber, (d) allowing the gel to solidify (typically 3-5 minutes), (e) placing between 0.5-100 pL of sample into the sample input chamber, (f) applying a voltage for desired time (typically ⁇ 5 minutes), (g) viewing and recording the results, and h) excising a sample from the cartridge, if desired.
  • Figure 1A is a perspective illustration of an electrophoresis cartridge.
  • Figure IB is a perspective exploded illustration of a single gel separation column of the cartridge.
  • Figure 2A is an exploded perspective illustration of the components used in the construction of the electrophoresis cartridge.
  • Figure 2B is a close-up view of an electrode, showing indentations that allow contact with separation gel.
  • Figure 3A is an illustration of a single gel separation column emphasizing the sample loading chamber.
  • Figure 3B is an illustration of a portion of a single gel separation lane showing a side view of a sample loading chamber.
  • Figure 3C is an illustration of a portion of a single gel separation lane showing atop view of a sample loading chamber.
  • Figure 3D is an illustration of a portion of a single gel separation lane showing a side view of an alternative sample loading chamber.
  • Figure 3E is an illustration of a portion of a single gel separation lane showing a top view of an alternative sample loading chamber.
  • Figure 3F is an illustration of a portion of a single gel separation lane showing a side view of a hydrophobically modified sample loading chamber.
  • Figure 3G is an illustration of a portion of single gel separation lane showing a top view of a hydrophobically modified sample loading chamber.
  • Figure 4 is a black and white photo of a gel image of a DNA separation generated using the electrophoresis cartridge of Figure 1A.
  • Figure 5A is a cut-away view of a gel separation column showing a partial gel-fill operation on the anode side of the cartridge.
  • Figure 5B is a cut-away view of a gel separation column showing a fuller partial gel-fill operation on the anode side of the cartridge.
  • Figure 5C is a cut-away view of a gel separation column showing a complete gel-fill operation on the anode side of the cartridge.
  • Figure 5D is a cut-away view of a gel separation column showing a partial gel-fill operation on the cathode side of the cartridge.
  • Figure 5E is a cut-away view of a gel separation column showing a complete gel-fill operation on the cathode side of the cartridge.
  • Figure 6A is a perspective view of the electrophoresis console.
  • Figure 6B is an alternate exploded perspective view of the electrophoresis console.
  • Figure 7A is a perspective illustration of the electrophoresis console with a camera hood.
  • Figure 7B is a perspective illustration of the electrophoresis console with a camera hood showing the camera, electronics, and camera lens.
  • Types of biological materials and biomolecules used can include double-stranded (ds) DNA, single-stranded (ss) DNA, dsRNA, ssRNA, MRNA, genomic DNA, proteins, and carbohydrates.
  • the separation gel may be of any type used in conventional slab gel electrophoresis.
  • Preferred gels include agarose and polyacrylamide.
  • the gel electrophoresis cartridge 101 has a label, 103 for identifying information such as part number, lot number, run number, sample, etc.
  • a conductive strip 107 is the anode.
  • Conductive strip 108 is the cathode.
  • the cartridge contains at least one separation column 109 ( Figure IB) comprising anode chamber 106, cathode chamber 105, separation lane 102, and sample input chamber 104.
  • the anode chamber, 106, cathode camber 105, and separation lane 102 can be filled with a separation electrophoresis gel.
  • the gel serves a dual purpose as a conducting interface with the electrodes and as a separation medium.
  • Sample input chamber 104 is not filled with separation gel, but is instead left empty for subsequent input of sample.
  • Sample input chamber 104 is located within separation lane 102, and can be located closer to the cathode chamber 105 than the anode chamber 106.
  • the sample input chamber 104 is located 5 percent of the length of the separation lane from the cathode chamber 105 side.
  • the sample input chamber 104 is located between 1 percent and 33 percent of the length of separation from the cathode chamber 105 side.
  • Sample input chamber 104 is distanced from the cathode to reduce heat transfer from the electrophoresis electricity into the sample.
  • Bufferless slab gel electrophoresis cartridges are known.
  • the present disclosure improves over known bufferless systems in that no liquid or buffer overlay is required. Electrode chambers
  • This electrode chamber to channel ratio provides enough buffering to the electrophoresis separation for ions and pH while also keeping the gel cool and limiting it from drying out as the electrophoresis current is applied.
  • Figure IB shows a perspective exploded view of an individual separation column 109 comprising anode chamber 106, cathode chamber 105, separation lane 102, and sample input chamber 104.
  • anode chamber 106, cathode chamber 105 and separation lane 102 are filled with an electrophoresis gel.
  • Sample input chamber 104 is not filled with the separation gel and is left empty for subsequent input of sample solution.
  • the preferred anode chamber 106 geometry is approximately 5 millimeters (mm) wide x 8 mm length x 5 mm deep.
  • the preferred cathode chamber 105 is approximately 5 mm wide x 10 mm length x 5 mm deep.
  • the preferred separation lane 102 is approximately 40 mm long x 1.5 mm wide and 1.5 mm deep.
  • the sample input chamber 104 has dimensions of approximately 2.3 mm wide x 1 mm long by 1.8 mm deep.
  • the preferred anode chamber 106 volume is approximately 180 microliters (pL).
  • the preferred cathode chamber 105 is approximately 250 microliters.
  • the preferred separation lane 102 is approximately 100 microliters.
  • the anode and electrode chambers are oversized in the event the operator wants to extend the duration of the separation beyond the recommended run time of 3 to 5 minutes.
  • the preferred sample input chamber 104 a volume is approximately 6 microliters.
  • the geometry of the anode and cathode chambers may be any dimensions as long as the ratio of the chamber volumes to channel are sufficient as previously discussed.
  • the volume of the sample input chamber 104 may be sized according to the volume of sample analyte and experiment or application type.
  • the sample input chamber 104 could be as large as 100 pL or more to recover relatively large amounts of concentrated and separated sample material. This capability is not possible with microfluidic chips as found in prior art.
  • the sample solution (matrix) in which the biological sample is dissolved, suspended, or dispersed may be of any type used in conventional slab gel electrophoresis.
  • Figure 2A shows the components of a thermoformed gel electrophoresis cartridge 201. Label 103 is added to the cartridge 201 for general information. Conductive strip 108 is the cathode. Conductive strip 107 is the anode. Indentation 206 of the conductive strips 107 and 108 allows the conductive material to contact the conductive electrophoresis gel matrix.
  • Figure 2B shows electrodes 107 or 108 with a section folded down to allow contact with the conducting separation gel matrix.
  • Clear plastic substrate 205 forms the cartridge body, containing the separation lanes 102, cathode chambers 105, anode chambers 106, and sample input chambers 104.
  • Substrate 205 is a translucent or clear moldable plastic.
  • Clear plastic film 203 covers the separation lanes, but partially leaves open the anode chambers 106 and cathode chambers 105 for input and filling of gel matrix.
  • Clear plastic film cover 203 also has a slit opening 202 which allows for sample placement into sample input chambers 104.
  • An alternate clear plastic film cover 204 covers the separation lanes, contains slits 209 as openings to the sample input chambers 104, contains holes 208 for input of gel electrophoresis matrix by pipette tip into the anode chambers 106 and cathode chambers 105, and contains slits 207 and 210 to vent air when filling the anode and cathode chambers with gel.
  • Clear plastic film 203 should cover a majority of cathode chamber 105 and anode chamber 106 while still providing access to add gel.
  • the channel may not necessarily have 3 distinct walls.
  • the channel may be any shape, including U-shaped, round, v-shaped, rectangular shaped, fileted, chamfered, patterned, or irregularly shaped as long as it has 3 dimensions of geometry to sufficiently hold the separation material. All components of the channel and reservoir may have tapered or drafted walls with rounded comers or filets to ease manufacture and mold release.
  • Materials used for conductive strips 107 or 108 include but is not limited to copper, platinum, gold, silver, aluminum, steel, tin, graphite, carbon, silver plated conductors, gold plated conductors, nickel plated conductors, tinned conductors, conductive polymers such as polyaniline conductive composites, conductive inks, or conductive adhesives. Conductive materials may be applied using any process including but not limited to vapor deposition, gluing, resist/etching, inkjetting, screen printing, or pad printing. A preferred material is copper or aluminum tape.
  • Substrate 205 and film 203 or 204 materials include any thermoset or thermoplastic or any combination of plastic materials that is transparent for the desired analysis.
  • Preferable materials are the thermoplastic resins.
  • An even more preferable material is polypropylene or polystyrene.
  • Layers may be attached to each other using any adhesive known to the art, such as acrylic adhesive or silicone adhesive.
  • the adhesive on film 203 may face the inner cavity of the separation lane 102 and hydrophillically enable surface weting and filling of the gel into the separation lane 102.
  • the adhesive can be substantially transparent to the wavelengths of interest.
  • substrate 205 and film 203 may be made from a UV- transparent plastic such as the TopasTM COCTM polyethylene resins.
  • Figure 3B shows the top view of a separation column 109 ( Figure IB), with cathode chamber 105, anode chamber 106, separation lane 102 and sample input chamber 104.
  • Figures 3A-G show some possible configurations for the walls of the separation chamber.
  • the introduction of geometric features into the substrate 205 ( Figure 2A) walls in and around the sample input chamber 102 and features in film 203 or 204 prevents the electrophoresis gel from migrating past the boundaries of the sample input chamber, allowing for the creation of an empty chamber for subsequent sample input.
  • An alternate form uses a hydrophobic chemical coating or surface treatment for hydrophobic modification of the walls of the sample input chamber section, which prevents the electrophoresis gel from migrating past the boundaries of the sample input chamber 104, allowing the creation of an empty chamber for subsequent sample input.
  • Figure 3B shows a cutaway side view of one possible configuration of the sample input chamber 104 within the separation lane showing an abrupt widening and indentation 307 of substrate 205, which forms a sharp ledge 302 at the side of the sample input chamber.
  • Doted lines 301 show the boundary of the sample input chamber 104 within the separation lane, which is formed when gel is placed into the separation lanes.
  • Figure 3C shows a top view of the substrate modification shown in FIG. 3B.
  • Doted lines 301 show the interface between the sample input chamber 104 and the gel placed in the separation lanes.
  • sharp ledge 302 prevents gel from migrating into the sample input chamber when gel is placed into the anode and cathode chambers of a separation column.
  • Figure 3D shows an alternate modification of substrate 205, showing a cutaway view with an upward indentation forming sharp ledge 303, which alters surface weting characteristics and prevents gel from migrating into the sample input chamber when gel is placed into the anode and cathode chambers of a separation column.
  • Doted lines 301 show the boundary of the sample input chamber, which is formed when gel is placed into the separation lanes.
  • Figure 3E shows a top view of the substrate modification shown in Figure 3D.
  • Dotted lines 301 show the wall interface between the sample input chamber and the gel placed in the separation lanes.
  • sharp ledge 303 prevents gel from migrating into the sample input chamber when gel is placed into the anode and cathode chambers of a separation column.
  • Figure 3D shows a cutaway side view of another alternate modification of substrate 205, showing a chemical or surface-treatment hydrophobic modification of the walls (shown as shaded area) of the sample input chamber 104, which alters surface wetting characteristics and prevents gel from migrating into the sample input chamber when gel is placed into the anode and cathode chambers of a separation column.
  • Figure 3G shows a top view of the substrate modification as shown in side view Figure 3F.
  • Dotted lines 301 show the wall interface between the sample input chamber and the gel placed in the separation lanes.
  • Chemical or surface treatment hydrophobic modification of the walls of the sample input chamber 104 (shown as shaded area) alters surface wetting characteristics and prevents gel from migrating into the sample input chamber when gel is placed into the anode and cathode chambers of a separation column.
  • any combination top or side view surface wetting modification examples may be used to prevent sample gel from migrating into the sample input chamber 104.
  • Methods of hydrophobically modifying the surface include, but are not limited to, plasma treatment or coating with paraffin, polytetrafluoroethylene (PTFE), polyamide, polycarbonate, polyacrylonitrile, silicone polymer, or fluorocarbon wax.
  • PTFE polytetrafluoroethylene
  • polyamide polyamide
  • polycarbonate polycarbonate
  • polyacrylonitrile polyacrylonitrile
  • silicone polymer or fluorocarbon wax
  • Figure 4 shows an image of the separation of a 100 bp-15,000 bp DNA ladder using the electrophoresis device. This is discussed in the example section.
  • Figures 5A-5E shows an individual separation column 109 being filled using a pipette 502.
  • Figure 5A shows pipette 502 with electrophoresis gel matrix 503 placed into anode chamber 106. Electrophoresis gel matrix 503 migrates down and fills the separation lane 102 via surface interaction shown in Figure 5B.
  • Figure 5C shows that when the electrophoresis gel matrix 503 reaches ledge 302 ( Figure 5C) formed by a shape of the substrate 502, migration of the electrophoresis gel matrix 503 stops, leaving the sample input chamber empty.
  • the wall of the stopped gel 503 defines a wall of the sample input chamber 104.
  • Figure 5D shows pipette 502 with electrophoresis gel matrix 503 placed into cathode chamber 105. Electrophoresis gel matrix 503 migrates down and fills the separation lane 102 via surface interaction.
  • Figure 5E shows that when the electrophoresis gel matrix 503 reaches the ledge 302 ( Figure 3C) formed by a of the substrate 502, migration of the electrophoresis gel matrix 503 stops, leaving the sample input chamber empty. The wall of the stopped gel 503 defines a wall of the sample input chamber 104.
  • Electrophoresis can be performed on a sample with a negative charge.
  • a positive sample charge can be utilized however requires the electrical polarity on chambers 105 and 106 to be reversed.
  • a particularly beneficial aspect of the present disclosure is the ability of the lab practitioner to load their own custom gel into gel electrophoresis cartridge 101. Users are not tethered to a single gel defined by the cartridge manufacturer, such as agarose, but instead can use custom variants that they currently use in their own standard slab gel electrophoresis, such as agarose or polyacrylamide with varying concentrations, added denaturing agents, or surfactants.
  • FIG. 6A shows an electrophoresis console 601 for running the electrophoresis gel cartridge 101.
  • Electrophoresis cartridge 101 is slid into electrophoresis console 601, where electrodes 108 and 107 make contact with the instrument via spring pressure connection 610 ( Figure 6B).
  • Magnets 607 allow for attachment of emission filter 604, which has magnets 605.
  • the semi-transparent emission filter 604 removes the excitation wavelength light, but allows other light to pass, enabling a user to view electrophoresis separation real time during the electrophoretic separation.
  • a preferred transparent filter is orange transparent plastic, which filters out 450-500 nanometer excitation light.
  • Emission filter 604 also serves as a cover to protect the user from accidental contact with the high voltages applied to the gel electrophoresis cartridge 101.
  • Holes 606 are optionally placed into the emission filter 604 to allow air flow from the fans 609 up and around the excitation filter 608, electrophoresis cartridge 101, and up through the emission filter 604. Air from the fans also flows through the electrophoresis console holes 617, 619 ( Figure 6B) located on the rear and back of the unit.
  • LED Lights 602 which may have a variety of different colors for different operational states indicate the status and progress of the electrophoresis run. For example, LED light 602 may turn red for a timed “electrophoresis on” state, whereas no light may indicate an “electrophoresis off’ state.
  • Buttons 603 select the set of conditions used for the electrophoretic separation. For example, one button may have a separation of 3 minutes to 5 minutes with an applied voltage of 165 Volts (V). The system is designed so that pre-programmed separation conditions are entered into the control board 613 ( Figure 6B).
  • Figure 6B shows a perspective exploded view of the instrument with different components, including a bottom chassis frame 618 that contains an excitation light LED array 616 (e.g. , 460-470 nanometers emission wavelength).
  • Fans 609 provide air flow to cool heat generated by the control board 613, LED light array 616, and gel electrophoresis cartridge 101 containing gel.
  • the control board 613 carries a high voltage electrophoresis power supply, LED driver, microprocessor, and voltage regulation circuitry.
  • Excitation filter 608 allows the excitation wavelengths of interest to pass, blends, and homogenizes the light from the LED array 616 so that electrophoresis cartridge 101 is uniformly illuminated during the electrophoresis.
  • Control board 613 with a high voltage power supply controls the duration of the electrophoresis, as well as the applied voltage.
  • Top cover 612 covers the control board 613 and chassis frame 618.
  • Enclosure 611 includes a support for the electrophoresis cartridge 101, as well as mounting magnets 607 for the attachment of an emission filter top filter plate 604, or camera attachment hood 702 ( Figures 7A-7B).
  • the electrophoresis console 601 applies a voltage to electrophoresis cartridge 101 (Figure 1A) via high voltage power supply on the control board 613 ( Figure 6B) and electrodes (107 & 108 Figure 1A).
  • the applied voltage ranges from IV to 250V.
  • the voltage may be applied with a preferred time range of 3-7 minutes.
  • Figure 7A shows a camera attachment hood 702 that attaches to the electrophoresis console 601. Electrophoresis cartridge 101 is shown installed in the chassis 601 for this exemplary illustration. Cord 703 supplies power and data to the attached digital camera 705 Figure 7B).
  • Figure 7B shows a Camera hood 702, with digital camera, electronics 705 and camera lens 704. Magnets 705 allow for magnetic mounting with magnetic mounts 607 on electrophoresis console 601.
  • the operator may turn on the excitation backlight 616 (Figure 6B) and visualize the sample migration through the instrument’s emission filter 604 ( Figure 6A).
  • the operator may remove the sample and use common tools (razor blade, scissors) to excise the sample peaks/bands/fragment(s) of interest from the separation lane.
  • the operator can cut the separation column and use another fresh separation column or allow a used lane to dry out, severing electrical connectivity on the used separation column 109.
  • the operator can use a fresh cartridge 101 ( Figure 1A).
  • Biological samples may be tracked using any visual dye or fluorescent dye currently used in slab gel electrophoresis or capillary electrophoresis.
  • the dye may be a direct staining dye spiked into the sample, or added to the gel. If added to the gel, it has a short shelf life of approximately 2 weeks.
  • the dye may conjugate/associate/intercalate with the DNA/RNA/protein.
  • Examples of gel dye include but are not limited to Ethidium Bromide, SYBR Gold, SYBR Green, SYBR Safe, Eva Green, and Gelstar. Multiple dyes with different wavelengths may be multiplexed within the same sample and run. Different optical filters may be required for different dyes.
  • Other dyes may include but are not limited to FAM, NED, ROX, VIC, HEX, TAMRA, JOE.
  • a preferred dye is the type that is added directly to the sample.
  • a solution of 1% agarose was prepared using 5mL of DI water, lOOuL of 50x TAE buffer, and 45mg of Fisher Scientific UltraPureTM Agarose. The solution was stirred and microwaved twice for 20 seconds until the solution was water clear. This stock solution of gel is good for several weeks, and can be melted/cooled over 10 times without adverse effect on performance.
  • Cartridge 101 consists of 4 separation columns 109 which are capable of holding 250 pL of gel per column on the anode chamber and cathode chamber sides, therefore making an entire cartridge 101 consume only 2 mL of gel.
  • the sample was prepared by mixing 2 pL of a proprietary direct DNA stain dye with 1.5 pL InvitrogenTM 1Kb Plus DNA Ladder, and 48 pL of DI water.
  • the gel cartridge 101 ( Figure 1A) was loaded into the electrophoresis console 601 ( Figure 6A).
  • the electrophoresis cartridge was then loaded with gel by pipetting approximately 250 pL of the heated (approximately 70-100 degrees Celsius) prepared molten agarose gel into the anode chamber and 250 pL gel into the cathode chamber.
  • the gel migrated into and filled the separation lane, up to the capillary valve points.
  • a sample chamber 104 ( Figure 5E) was fully formed, with the walls of the gel forming the walls of the sample chamber.
  • the gel was allowed to cool and solidify for 3 minutes. 6 pL of the prepared ladder sample solution was loaded into the sample input chamber.
  • console 601 ( Figure 6A) button 603 was pressed enabling a voltage of 165 volts for 5 minutes.
  • the gel was imaged using the LED illuminator of console 601 ( Figure 6A).
  • An alternate method for tracking the progress of the DNA sample is to add blue loading dye into the sample input chamber prior to run initiation.

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Abstract

L'invention concerne une cartouche et une console d'électrophorèse sur gel destinées à être utilisées pour effectuer des séparations électrophorétiques. La cartouche comprend au moins une colonne de séparation comportant une chambre cathodique, une chambre d'introduction d'échantillon, un couloir de séparation et une chambre anodique, qui est remplie de gel de séparation par électrophorèse par l'utilisateur à l'aide d'un procédé passif de mouillage de surface sans pompe. La chambre d'introduction d'échantillon dans le couloir de séparation contient soit des modifications géométriques, soit un traitement hydrophobe des parois pour empêcher le remplissage avec du gel, laissant une chambre vide adaptée à l'introduction d'un échantillon biologique. Le gel contenu dans la cartouche pleine entre en contact avec une anode et une cathode sans tampon ni superposition de liquide, ce qui donne une cartouche d'électrophorèse sans tampon améliorée. Une console d'électrophorèse peut être utilisée pour l'application d'une tension de séparation à la cartouche, ainsi que pour le suivi en temps réel de la progression de la séparation électrophorétique.
PCT/US2022/074661 2021-08-08 2022-08-08 Dispositif d'électrophorèse sur gel à cartouche pour séparation de biomolécules Ceased WO2023019105A1 (fr)

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