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WO2025233688A1 - Système d'électrophorèse à base de microplaque - Google Patents

Système d'électrophorèse à base de microplaque

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Publication number
WO2025233688A1
WO2025233688A1 PCT/IB2025/050665 IB2025050665W WO2025233688A1 WO 2025233688 A1 WO2025233688 A1 WO 2025233688A1 IB 2025050665 W IB2025050665 W IB 2025050665W WO 2025233688 A1 WO2025233688 A1 WO 2025233688A1
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WO
WIPO (PCT)
Prior art keywords
electrophoresis
separation
sample
cell
channels
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/050665
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English (en)
Inventor
Abizar Lakdawalla
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Axor Biosystems Inc
Original Assignee
Axor Biosystems Inc
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Filing date
Publication date
Application filed by Axor Biosystems Inc filed Critical Axor Biosystems Inc
Publication of WO2025233688A1 publication Critical patent/WO2025233688A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • 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
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • the disclosure relates to the field of electrophoresis for analyzing and purifying materials based on their chemical and physical properties.
  • Analytical and preparative electrophoresis techniques represent cornerstone methodologies, spanning biomedical research to industrial applications, including pharmaceuticals, environmental analysis, molecular biology, and clinical diagnostics. These methods encompass a diverse array of electrophoresis devices and approaches, such as capillary electrophoresis, gel electrophoresis, and microchip electrophoresis, facilitating the characterization of a wide spectrum of biological entities, including nucleic acids (Lee PY, 2012), proteins (Srinivas PR, 2019), carbohydrates (Suzuki S, 1988), cellular organelles, viruses, and cells (Subirats X, 2011).
  • Methods for both analytical and preparative gel electrophoresis entail numerous steps and the use of multiple pieces of equipment (Green MR, 2012). These methods include sample preparation, sample loading into designated wells within a separation matrix or gel, followed by the application of an electric field to induce the migration of sample constituents through the matrix. Subsequent visualization and analysis of the separated sample constituents require staining, imaging, and data analysis (Kurien BT, 2018).
  • Preparative electrophoresis involves additional steps for isolating and purifying specific sample constituents (Sutton RMC, 2000). This often requires manual excision of gel material containing the specific sample constituent after electrophoretic separation, followed by a multiple-step extraction and concentration procedure, that are labor- intensive and time-consuming (Bier M, 1962).
  • Preparative electrophoresis systems from Coastal Genomics and Sage Science Inc utilize physically separated lanes, each containing a sample well and a purification chamber (Uyaguari-Diaz Ml, 2015) with each lane subjected to independent electric potentials. These systems aim to ensure separation and simultaneous entry of a specific sample constituent from multiple samples of interest into the purification chambers (Quail MA, 2012).
  • Analytical and preparative electrophoresis methods are labour-intensive with limited sample throughput and low compatibility with high-throughput workflows and robotic systems.
  • Different electrophoretic technologies and systems are generally required for different sample types and applications.
  • the inability to process different sample types such as DNA, RNA, proteins, cell organelles, viruses and cells in parallel, on a single device, coupled with the lack of integration with laboratory robotic systems, hinders the adoption of electrophoresis.
  • there is a pressing need for innovative solutions that can overcome the limitations of electrophoresis methods and systems to enable efficient and scalable separation and purification of diverse sample types in parallel, while also facilitating integration with automated laboratory workflows.
  • the present disclosure describes an electrophoresis microplate system that adheres to laboratory standards for microplates, facilitating seamless integration into automation workflows.
  • the electrophoresis microplates comprise up to 96-independent electrophoresis cells, arranged in a rectangular grid format.
  • electrophoresis cells are provided as electrophoresis strips offering further flexibility to mix-and-match different sample types and applications. These strips, held in a holding frame, enable efficient handling and processing of samples while conforming to established microplate standards.
  • each electrophoresis cell is supported by an electrophoresis instrument, that independently controls each cell in the microplate, and provides realtime analysis of analytical, preparative and/or affinity electrophoresis results during the run.
  • the disclosed system integrates pre-concentration of samples, electrophoretic separation and purification of multiple components from a sample.
  • Diverse samples types ranging from eukaryotic and prokaryotic cells to molecules such as DNA, RNA, and proteins can be analyzed on a single electrophoresis microplate.
  • a system for performing electrophoresis comprising an electrophoresis microplate.
  • the electrophoresis microplate comprises a plurality of electrophoresis cells (preferably 6, 12, 24, 48, or 96 electrophoresis cells), arranged in a planar rectangular grid.
  • the planar rectangular grid of electrophoresis cells has a ratio of 2:3 (rows:columns). More preferably, the electrophoresis microplate preferably conforms to the ANSI SLAS 1-2004 (R2012) laboratory microplate standard dimensions.
  • Each electrophoresis cell in said microplate operates independently of the other cells.
  • voltage differentials may be configured to be applied to each electrophoresis cell independently.
  • Each electrophoresis cell may comprise one or more separation channels, within which electrophoretic separation is configured to take place.
  • the separation channel(s) may be linear or comprise at least one turn, for example wherein the one or more separation channels are spiral, serpentine, in the form of a coil, or a pre-determined two- or three- dimensional form configured to maximize the dimensions of the said separation channel in the electrophoresis cell.
  • the separation channel may be comprised of more than one layer of separation channels fluidically connected to achieve two or more-dimensional electrophoretic separation of sample constituents, for example with said channels arranged orthogonally.
  • the separation channel(s) may comprise a separation matrix with zones comprised of affinity reagents configured to selectively bind to constituents in the sample.
  • Each electrophoresis cell may further comprise one or more side channels, fluidically coupled to the one or more separation channels.
  • the side channels may enable transfer of reagents and/or sample constituents with the separation channel.
  • Each electrophoresis cell may be configured for a voltage differential to be applied across said electrophoresis cell, wherein the voltage differential applied to said electrophoresis cell is configured to operate independently of the other electrophoresis cells.
  • the voltage differential may be configured to be applied across said one or more separation channels.
  • the voltage differential may be configured to be applied across said one or more side channels.
  • an electrophoresis strip for performing electrophoresis.
  • the electrophoresis strip comprises a plurality of electrophoresis cells per strip (preferably 2, 3, 4, 5, 6, 8 or 12 of electrophoresis cells per strip), arranged in a planar rectangular grid.
  • the planar rectangular grid has one or more rows or columns per strip, preferably 1 , 2, 3 or 4 rows or columns per strip.
  • Each electrophoresis cell in said strip operates independently of the other cells.
  • the electrophoresis cells of the strip may comprise the same features as the electrophoresis cell discussed above in relation to the microplate.
  • the electrophoresis cells comprises one or more separation channels, and optionally one or more side channels.
  • Each electrophoresis cell may be configured for a voltage differential to be applied across said electrophoresis cell, wherein the voltage differential applied to said electrophoresis cell is configured to operate independently of the other electrophoresis cells.
  • the voltage differential may be configured to be applied across said one or more separation channels.
  • the voltage differential may be configured to be applied across said one or more side channels.
  • a system for performing electrophoresis may comprise one or more of the electrophoresis strips and a holding frame configured to retain a plurality of said strips.
  • the holding frame, retaining one or more electrophoresis strips, may form a microplate as described above.
  • an electrophoresis cell comprises fluidic channels layer, comprising one or more separation channels, within which electrophoretic separation is configured to take place such that the electrophoresis cell is configured to facilitate analytical, preparative, and/or affinity electrophoresis within the one or more separation channels.
  • the electrophoresis cell comprises may also comprise an upper cover.
  • the upper cover may be configured to attach to a top surface of the electrophoresis cell, for example via a bottom surface. This may be is configured to shield said cell’s contents, for example from contamination, oxidation, evaporative loss, and unwanted mixing of reagents during storage, transport, and handling, while allowing the introduction of samples and electrodes.
  • the bottom surface of the upper cover is sealed to a top surface of the electrophoresis cell.
  • the upper cover may also comprise one or more features to guide entry of pipettes, for example the one or more features being arranged on a top surface of the upper cover. This may be advantageous to facilitate fluidic loading and/or unloading of sample into the electrophoresis cell.
  • upper cover may also comprise one or more features configured to guide entry of electrical contacts into the electrophoresis cell, for example through the upper cover.
  • the upper cover may comprise one or more electrode guide features configured to enable electrical contact in the electrophoresis cell through the upper cover.
  • the electrophoresis cell further comprises a fluidic chambers layer and a fluidic ports layer.
  • the fluidic chambers layer may comprise one or more of fluidic chambers (preferably 0,
  • fluidic chambers 1 , 2, 3, 4, 5, 6, 7, or 8 fluidic chambers), wherein said fluidic chambers are configured to serve as containers for samples, buffers and reagents.
  • the top surface of said fluidic chambers may be attached to the bottom surface of the top cover.
  • the fluidic ports layer may comprise a one or more of fluidic ports, that provide fluidic connections within the electrophoresis cell; for example wherein the top surface of the fluidic ports layer is attached to the bottom surface of the fluidics chamber layer.
  • the fluidic channels layer may comprise a plurality of separation channels (preferably 1 ,
  • the fluidic channels layer may further comprise one or more fluidic channels (preferably 0, 1 , 2, 3, 4, 5, 6, 7 or 8 fluidic channels) connecting fluidic chambers, fluidic ports and/or separation channels.
  • fluidic channels preferably 0, 1 , 2, 3, 4, 5, 6, 7 or 8 fluidic channels
  • the top surface of the fluidic channels layer may be attached to the bottom surface of the fluidic ports layer.
  • a bottom cover may also be provided.
  • the bottom cover may be a planar layer constructed of materials that are transparent to light.
  • the bottom cover may comprise a top surface that attaches to the bottom of the fluidic channels layer, configured to seal in the contents of the fluidic channels, and a bottom surface, configured to protect the cell from external contamination.
  • the electrophoresis cell is configured to facilitate analytical, preparative, and/or affinity electrophoresis on various sample types.
  • the upper cover may comprise one or more gas permeable structures or vents. This may be advantageous to allow gases generated during electrophoresis to escape.
  • the electrophoresis cell may further comprise one or more sample wells (for example, 1 , 2, 3, or 4 sample wells) placed in the separation channels.
  • sample wells bring configured to hold a sample to be subjected to electrophoresis.
  • the separation channel may be linear, or comprise at least one turn, such as a spiral, serpentine, coil, or other pre-determined two- or three-dimensional forms to maximize the dimensions of the said separation channel in the electrophoresis cell.
  • the separation channel may be comprised of more than one layer of separation channels, preferably with said plurality of channels arranged orthogonally.
  • a plurality of separation channels may advantageously achieve two or more-dimensional electrophoretic separation of sample constituents.
  • the electrophoresis cell may also further comprise electrodes, such as but not limited to conductive carbon compositions, conductive ceramics, conductive polymers, or metals and metal alloys.
  • the electrodes may be placed within the fluidic chambers, fluidic ports, and/or fluidic channels.
  • the electrophoresis cell may also further comprise buffers for performing electrophoresis.
  • the buffers may be placed within the fluidic chambers, fluidic ports, and/or fluidic channels.
  • the electrophoresis cell may also further comprise a separation matrix, for example placed within the fluidic channels and fluidic ports; wherein an electrical conduction path is formed between the fluidic chambers, ports and channels.
  • the electrophoresis cell may further comprise one or more side channels, fluidically coupled to the separation channel, for example wherein the one or more side channels are configured to facilitate transfer of reagents and/or sample constituents with the separation channel.
  • a side channel may be provided as a purification channel.
  • the electrophoresis cell may further comprise one or more chambers, fluidically connected to the separation channel.
  • the one or more chambers may be configured to enable fluidic loading and/or unloading of reagents and/or sample constituents between the one or more chambers and the separation channel.
  • the one or more chambers may be input chamber(s), output chamber(s), purification chamber(s), side chamber(s), and/or sample chamber(s), etc.
  • a system for performing electrophoresis comprising the electrophoresis cell described above.
  • the system may comprise the system of the first aspect, wherein the microplate comprises a plurality of the electrophoresis cells described above.
  • the system could also comprise the system or electrophoresis strip(s) of the second aspect, wherein the microplate comprises a plurality of the electrophoresis cells described above.
  • an electrophoresis instrument comprises an electrophoresis microplate compartment.
  • the microplate compartment may be configured for loading and unloading of an electrophoresis microplate, the electrophoresis microplate comprising a plurality of electrophoresis cells.
  • the electrophoresis microplate compartment may comprise a means configured to facilitate loading and unloading of electrophoresis strips and/or an electrophoresis plate, such as the electrophoresis strips and/or the electrophoresis plate discussed above.
  • the electrophoresis microplate compartment may also comprise a means configured to securely hold said strips and/or plate during the electrophoresis process.
  • the electrophoresis microplate compartment may further comprise a means configured to maintain pre-determined temperatures in said compartment.
  • the electrophoresis microplate compartment may further comprise at least one sensor to sense at least one of: (i) presence of strips or plate, (ii) position of strips or plate, (iii) temperature, and (iv) incident illumination intensities.
  • the electrophoresis instrument further comprises an electrode interface module.
  • the electrode interface module comprises a plurality of electrical contacts configured to establish electrical contact with each electrophoresis cell.
  • this may comprise an array of electrodes that penetrate through the top cover of an electrophoresis cell (such as the electrophoresis cell discussed above) to establish electrical contact with fluidic chambers, fluidic ports and fluidic channels within electrophoresis cell.
  • the electrode interface module may comprise an array of electrical contacts configured to electrically engage with electrodes positioned within the electrophoresis cell.
  • the electrophoresis instrument may further comprise an illumination module comprising one or more light sources configured to illuminate the electrophoresis microplate when said electrophoresis strips and microplate is positioned within the electrophoresis microplate compartment.
  • Light wavelength selection filters may also be arranged between the one or more light sources and the microplate holder.
  • the electrophoresis instrument also comprises an imaging module.
  • the imaging module may comprise a camera or an array of cameras configured for imaging of the said electrophoresis microplate in the said electrophoresis microplate holder.
  • Light wavelength selection filters may also be arranged between the microplate and the one or more cameras.
  • the electrophoresis instrument may further comprise a control module.
  • the control module may be configured to regulate various aspects of electrophoresis methods and systems.
  • the control module may comprise a central processing unit (CPU) or other microprocessor.
  • the CPU may comprise one or more data storage devices storing instructions that are executable by one or more processors.
  • the CPU may also comprise one or more communication interfaces, and/or one or more computer servers.
  • the control module may further comprise power supplies, for example including an electrophoresis power supply, configured to interface with the electrode interface module and configured to provide a voltage differential between the electrodes.
  • the electrophoresis power supply is configured to independently control the voltage differentials for each electrophoresis cell.
  • the power supplies may also comprise a second power supply, such as a low voltage power supply, configured to supply the remaining components of the electrophoresis instrument, such as electronics, electromechanical components, the central processing unit, the imaging module, and the illumination module.
  • the control module may further comprise electro-mechanical control.
  • the electromechanical assembly may be configured to move the plurality of electrical contacts to interface with different formats of electrophoresis microplates.
  • the electrophoresis instrument may further comprise an analysis module.
  • the analysis module may comprise one or more microcontrollers and/or microprocessors for processing electrophoretic data and imaging data; one or more memory devices for storing data and instructions; and a computer program product that when executed is configured to analyze information from the control module, imaging module, and, optionally, other sensors.
  • a method for performing electrophoresis on a sample may first comprise the steps of mixing a sample with an electrically conductive fluid and a stain (such as, but not limited to, a fluorescent stain), and loading the mixed sample into an input chamber of an electrophoresis cell.
  • a stain such as, but not limited to, a fluorescent stain
  • the electrophoresis cell is the cell discussed above and herein.
  • the method then comprises loading an electrophoresis strip and plate into the electrophoresis microplate compartment of an electrophoresis instrument.
  • the electrophoresis instrument is the instrument discussed above and herein.
  • the method then further comprises positioning of the electrodes by the electrode interface module of the electrophoresis instrument to pre-determined locations within the electrophoresis cell to establish electrical contact.
  • the method may comprise applying an electrical potential between a first electrode and a third electrode mediated by the control module in the electrophoresis instrument to concentrate samples onto the third electrode.
  • the method then comprises monitoring the concentration of sample at said third electrode with the illumination and imaging modules of the electrophoresis instrument until concentration is completed.
  • the first electrode may be an input electrode
  • the third electrode may be a concentration electrode, or vice versa.
  • the method then comprises applying the electric potential between the said first electrode and a second electrode causing the electric current to pass from the first electrode, through the separation matrix in the separation channel, and to an output electrode thereby separating the constituents in the samples along said separation channel.
  • the first electrode may be an input electrode
  • the second electrode may be an output electrode, or vice versa.
  • the method further comprises imaging the separation channel during the separation process with the imaging module, and optionally the illumination module.
  • the method then further comprises analyzing the images with the analysis module to determine the molecular weights of the components.
  • the method further comprising the steps for purification of a sample, comprising separating the sample constituents in the separation channel, till a selected sample constituent enters a junction formed by the separation channel and a first chamber, such as a first purification chamber, or a first side channel, such as a first purification channel, in the electrophoresis cell.
  • a first chamber such as a first purification chamber
  • a first side channel such as a first purification channel
  • the method then comprises applying an electric potential between a second side channel electrode, such as a second purification electrode, positioned in a second chamber, such as a second purification chamber, or a second side channel, such as a first purification channel, that is fluidically connected to the separation channel, and a first side channel electrode, such as a first purification electrode, positioned in the first chamber or the first side channel, that is fluidically connected to the separation channel; resulting in the migration of selected sample constituent from the separation channel into the first chamber or first side channel.
  • a second side channel electrode such as a second purification electrode
  • the method may then further comprise removing said selected sample constituent from said first chamber or first side channel.
  • a second sample constituent may also be purified by migrating a second sample constituent into the second chamber or second side channel.
  • the method may then optionally comprising removing said second sample constituent from said second chamber or second side channel.
  • the electrophoresis cell is preferably the electrophoresis cell as discussed above.
  • said electrophoresis cell may optionally comprise: an upper cover, comprising; a top surface comprising features to guide entry of pipettes; a bottom surface; that attaches to the top surface of the electrophoresis cell to shield said cell’s contents from external contaminants, oxidation, and evaporation; a fluidic chambers layer comprising 0, 1 , 2, 3, 4, 5, 6, 7, or 8 fluidic chambers; wherein said fluidic chambers serve as containers for samples, buffers and reagents; wherein the top surface of said fluidic chambers is attached to the bottom surface of the top cover; a fluidic ports layer; said fluidic ports forming apertures to provide fluidic connections within the electrophoresis cell; wherein the top surface of the fluidic ports layer is attached to the bottom surface of the fluidics chamber layer; a fluidic channels layer; comprising 1 , 2, 3, or 4 separation channels, within which electrophoretic separation takes place; where
  • the separation channel may be comprised of a separation matrix, for example wherein the separation matrix comprises zones comprised of affinity reagents that selectively bind to constituents in the sample.
  • the separation channel may be comprised of a sample well.
  • the electrophoresis instrument is preferably the electrophoresis instrument as discussed above.
  • said electrophoresis instrument may comprise: an electrophoresis microplate compartment, comprising; mechanisms to facilitate loading and unloading of the electrophoresis strips and electrophoresis plate; mechanisms to securely hold said strips and plate during the electrophoresis process; devices to maintain pre-determined temperatures in said compartment; and sensors to sense presence and position of strips or plate, temperature, and incident illumination intensities; an electrode interface module, comprising; an array of electrodes that penetrate through the top cover of the electrophoresis cell to establish electrical contact with the fluidic chambers, fluidic ports and fluidic channels within the electrophoresis cell; an illumination module, comprising; light sources configured to illuminate the electrophoresis microplate when said electrophoresis strips and microplate is positioned within the electrophoresis microplate compartment; light wavelength selection filters arranged between the light element and the microplate holder; an imaging module, comprising; a camera or an array of cameras to
  • the electrophoresis kit comprises: a plurality of electrophoresis strips; an electrophoresis strip holder; a sample suspension buffer; molecular weight fiducials; control samples; and instructions.
  • the sample suspension buffer preferably comprises staining reagents to stain sample constituents, and electrophoresis buffers.
  • the buffer may also comprise thickening agents and/or dyes.
  • Fig. 1 shows perspective representations of electrophoresis microplates.
  • A shows a perspective representation of an electrophoresis microplate with 96 independent electrophoresis cells
  • B shows 24 independent electrophoresis cells
  • C with 6 independent electrophoresis cells.
  • Fig. 2 shows perspective views of electrophoresis strips with multiple electrophoresis cells.
  • Fig. 3 shows perspective views of the electrophoresis cell, such as an electrophoresis cell for use in Fig. 2, with different functional layers.
  • Fig. 4 shows perspective views of the components of the upper cover of the electrophoresis cell of Fig. 3.
  • Fig. 5 shows top, side views and a perspective schematic view of fluidic channels, wells and fluidic chambers in the electrophoresis cell of Fig. 3.
  • Fig. 6 illustrates the fluidic port layers and placements of the fluidic ports in the electrophoresis cell of Fig. 3 in perspective views.
  • Fig. 7 shows schematic illustrations showing top and side views of electrode placements in an electrophoretic cell, such as that shown in Fig. 3.
  • Fig. 8 illustrates the top and side views of an electrophoresis cell with a sample well and a perspective view of an electrophoresis cell with a concentration electrode.
  • Fig. 9 shows schematic representations of separation channel variants in electrophoresis microplates with 96-, 24- and 6-electrophoresis cells, such as the electrophoresis microplates shown in Fig. 1.
  • Fig. 10 illustrates separation channel designs with straight (A), serpentine (B, C, D), spiral (E), two-dimensional, three-dimensional (F, G), branched (H, I), and capillary structures (J) shown in perspective views.
  • Fig. 11 shows schematic representations of the process of capillary electrophoresis in the electrophoresis cell with capillary width separation channels, shown in a perspective and top views.
  • Fig. 12 illustrates the process of two-dimensional electrophoresis in the electrophoresis cell with two dimensions of separation channels, shown in perspective views.
  • Fig. 13 shows a schematic representation of the process of electrophoretic purification in electrophoresis cells with a sample well, shown in top views.
  • Fig. 14 shows schematic representations of the process of electrophoretic purification in electrophoresis cells with a concentration electrode, shown in top views.
  • Fig. 15 shows schematic representations of the process of affinity electrophoresis with zones of immobilized binding reagents, shown in top views.
  • Fig. 16 shows schematic representations of an electrophoresis instrument.
  • Fig. 17 shows a schematic representation of the control module of the electrophoresis instrument of Fig. 16.
  • Fig. 18 shows a schematic outline of a process to determine the molecular weights of sample constituents from images of the electrophoresis cells.
  • Fig. 19 presents the process flow describing the operation of an electrophoresis instrument, such as the electrophoresis instrument of Fig. 16.
  • Fig. 20 shows the results of electrophoretic concentration of dyes.
  • Fig. 21 shows the results from electrophoretic separation of dyes and of DNA.
  • Fig. 22 shows the results from the electrophoretic purification of a dye.
  • Fig. 23 illustrates the results of a simulation of DNA fragments migrating past a junction of fluidic channels.
  • Second fluidics port layer (in the two-dimensional electrophoresis cell)
  • Second separation channel layer (in the two-dimensional electrophoresis cell)
  • Agarose a purified colloidal linear polysaccharide prepared from seaweed, is made up of repeats of agarobiose (alternating units of galactose and 3,6-anhydrogalactose). Agarose contains no charged groups and is thus useful as a separation matrix for electrophoresis.
  • Algorithms Mathematical procedures or instructions embedded within the electrophoresis Instrument to optimize electrophoretic conditions and data analysis for different sample types and applications.
  • Analysis module The system within the electrophoresis instrument responsible for the analysis of images and other data gathered during an electrophoresis run.
  • Analytical electrophoresis Determination of the migration pattern of constituents in a sample under the influence of an electrical potential difference. The resulting separation pattern provides an indication of size and electrical charge characteristics of constituents in the sample.
  • Binding reagent Non-limiting list of entities such as aptamers, antibodies, dyes, lectins, metals nucleic acid probes, that selectively bind to a specific constituent of a sample.
  • Control module The system within the electrophoresis instrument responsible for regulating and controlling various parameters during electrophoresis.
  • Detector Device that is capable of detecting an optical, thermal, chemical, or electrical signal, but is not limited hereto.
  • Electrodes Conductive materials that transmit electrical currents to drive the movement of charged molecules during electrophoresis.
  • Electro-osmotic flow The bulk flow of liquid in the electrophoresis chamber from the effect of the applied electrical field.
  • Electrophoresis buffer Fluidic material that allows passage of an electrical current while resisting changes in pH.
  • Electrophoresis The process of moving charged molecules in an electric field.
  • Electrophoresis cell An element within the electrophoresis microplate containing the necessary components and structures for conducting electrophoresis on a sample.
  • Fluidic Materials that are not solid and that can flow such as liquids and gases.
  • Glycans Refers to carbohydrate-based polymers.
  • Electrophoresis instrument A device designed to facilitate and control electrophoretic processes in the electrophoresis cell, strip or microplate, typically comprising of a nonlimiting list of modules for plate handling, imaging, control, and analysis.
  • Electrophoresis microplate A microplate-like device comprising multiple independent electrophoresis cells arranged in a grid format compliant with laboratory microplate dimensional standards.
  • Fluidic chamber Containers within the electrophoresis cell used for holding samples, buffers, reagents, and other solutions required for electrophoretic processes.
  • Fluidic module A module within the electrophoresis instrument responsible for precise handling and dispensing of fluids into or from the electrophoresis cells.
  • Fluidic ports Apertures or conduits within the electrophoresis cell that enable fluidic connections between different chambers and channels, facilitating the movement and sensing of ions and sample constituents.
  • Imaging module A module equipped with sensors and imaging devices to monitor electrophoretic processes in real-time and capture high-resolution images of the electrophoresis cell, strip or microplate.
  • Illumination module A module equipped with lighting elements to illuminate the electrophoresis microplate for imaging and visualization purposes during electrophoresis.
  • Isotachophoresis An electrophoretic method wherein sample ions are introduced between ions with higher mobility and ions with lower mobility than that of any of the sample ions. The sample ions undergo a considerable concentration and sharpening between the higher and lower mobility ions.
  • Label A detectable moiety attached to a material.
  • the label can be light absorbing, luminescent, fluorescent, phosphorescent, magnetic, electrical, or modifying an electrical property such as impedance, but are not limited hereto.
  • Lane A path the constituents of a sample take through the gel matrix.
  • a matrix can also be formed from inorganic materials such as, glass, silica, carbon, and metals or combinations thereof, but are not limited hereto.
  • Microplate Planar device comprised of multiple containers referred to as "wells” or “cells” typically arranged in a 2:3 (rows: columns) rectangular grid to provide microplates with 6, 12, 24, 48, 96, 384 or 1536 wells or cells, respectively as described in the ANSI SLAS 1- 2004 (R2012) laboratory microplate standards.
  • Microplate compartment A module of the electrophoresis instrument designed for loading, holding and unloading the electrophoresis microplate.
  • Nucleic acids molecules comprised of nucleobases, sugar moieties, and phosphate moieties.
  • the nucleic acid can be comprised of nucleotides, oligonucleotides, polynucleotides, single stranded nucleic acid molecules, or double stranded nucleic acid molecules or of combinations thereof.
  • the nucleic acid can be naturally occurring or non- naturally occurring, e.g., a modified or engineered nucleic acid with modifications at the base moiety, sugar moiety, or phosphate moieties.
  • Preparative electrophoresis Purification of a component or group of constituents in the sample by electrophoretic processes.
  • Sample Animal and plant tissues, tissue homogenates, animal and plant cells, bacterial cells, cell lysates, viruses, cellular organelles, macromolecular complexes such as ribosomes, macromolecules such as carbohydrates, molecular complexes such as ribonucleoproteins, nucleic acids, molecules, ions, but not limited thereto.
  • Sample constituents Entities such as molecules, macromolecules such as peptides, carbohydrates, glycoproteins, DNA, RNA and proteins, molecular complexes, particles, organelles, cells and the like present in a sample.
  • Separation channels Channels within the electrophoresis cell for the separation of sample constituents based on their physico-chemical properties under the influence of an electric field.
  • Disclosed herein is a system for conducting analytical, preparative and affinity electrophoresis within a microplate format.
  • the various aspects of this disclosure are detailed in sections covering (1) an overview of the electrophoresis microplate, strip and cell; (2) essential components of the electrophoresis cells; (3) processes for analytical, preparative and affinity electrophoresis with the electrophoresis cell; (4) manufacturing of electrophoresis strips and plates; (5) overview of the electrophoresis instrument and instrument operation; (6) kits; and (7) applications and examples.
  • These sections serve organizational purposes and do not limit the scope of the disclosure.
  • Microplates are used extensively for the processing, storage, transfer and analysis of materials in laboratories. Microplates have been standardized by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS) to contain fluidic chambers or wells in a rectangular 2:3 (rows:columns) grid format. Microplates can contain 6, 12, 24, 48, 96, 384, 864, and 1536 wells per microplate, arranged in 2 rows: 3 columns rectangular format, though other formats are available.
  • ANSI American National Standards Institute
  • SLAS Society for Laboratory Automation and Screening
  • microplates can be increased by integrating electrophoresis processes within the standardized microplate architecture.
  • a microplate that integrates electrophoresis, as disclosed herein, would allow analytical, preparative and affinity electrophoresis to be performed in the familiar microplate format.
  • electrophoresis microplates 105 as represented in perspective views in Fig. 1A, 1 B, 1 C, comprised of a multitude of independent electrophoresis cells 110 in which analytical, preparative and affinity electrophoresis is performed.
  • the electrophoresis cells are arranged in a rectangular grid format, adhering to the 2:3 ratio of rows to columns specified in the ANSI SLAS 1-2004 (R2012) laboratory microplate standards, however the skilled person will understand that other formats and configurations of electrophoresis cells may be used.
  • the arrangement of electrophoresis cells 110 within the electrophoresis microplate 105 is tailored to fit standard microplate dimensions while accommodating various electrophoresis cell sizes.
  • FIG. 1A shows an electrophoresis microplate with ninety-six electrophoresis cells of dimensions of up to 9 mm by 9 mm.
  • Fig. 1 B shows an electrophoresis microplate with twenty-four electrophoresis cells of up to 18 mm by 18 mm dimensions.
  • Fig. 1C shows an electrophoresis microplate with six electrophoresis cells of dimensions up to 36 mm by 36 mm.
  • the number of electrophoresis cells per electrophoresis microplate can comprise 6, 12, 24, 48, 96 cells.
  • the examples for electrophoresis microplates disclosed herein are not limited to the illustrated examples; rather, these examples are intended to disclose the concepts to those skilled in this art.
  • Each electrophoresis cell 110 within the electrophoresis microplate 105 operates autonomously, enabling the simultaneous execution of analytical, preparative, or affinity electrophoresis methods on various sample types, including but not limited to eukaryotic and prokaryotic cells, viruses, cell constituents, macromolecular complexes, DNA, RNA, and proteins providing a versatile electrophoresis consumable compatible with laboratory automation.
  • the electrophoresis microplate is offered as electrophoresis strips, with each strip comprised of electrophoresis cells, as described below.
  • Fractional format of electrophoresis microplates can be achieved by providing the electrophoresis cells as electrophoresis strips 205, as shown in Fig. 2.
  • the strips are held in a holding frame that complies with the dimensional standards for SBS microplates.
  • the holding frame 210 is designed to retain the electrophoresis strip 205 in a fixed orientation and can feature fiducials such as a notch, among other possibilities.
  • the electrophoresis strip can comprise 2, 3, 4, 6, 8, or 12 electrophoresis cells per strip, though not limited thereto.
  • Fig. 2A, 2B, and 2C depict electrophoresis strips 205 arranged as vertical columns within the holding frame 210.
  • the fractional electrophoresis microplate can feature electrophoresis strips arranged in horizontal rows.
  • each strip can include one or more vertical columns or one or more horizontal rows of electrophoresis cells.
  • Fig. 2A presents a perspective view of the fractional electrophoresis microplate, comprised of the holding frame 210 and the electrophoresis strips 205.
  • Each strip is comprised of eight electrophoresis cells 110 arranged as a column, each cell with dimensions of up to 9 mm by 9 mm. In the illustrated example, six electrophoresis strips partially fill the holding frame.
  • An individual 8-cell electrophoresis strip is shown adjacent to the microplate in Fig. 2A. This configuration, accommodating up to ninety-six electrophoresis cells, effectively meets the demands of high-throughput laboratories.
  • Fig. 2B shows a partially filled holding frame 210, containing five electrophoresis strips 205, each comprised of four electrophoresis cells 110, arranged as a column, measuring up to 18 mm by 18 mm. An individual 4-cell electrophoresis strip 205 is shown next to the microplate. This configuration, accommodating up to twenty-four electrophoresis cells, caters to mid-throughput laboratories catering to preparative electrophoresis of samples with larger mass or volume.
  • Fig. 2C shows a partially filled holding frame 210, housing two electrophoresis strips 205, arranged as a column, each with two electrophoresis cells 110 measuring up to 36 mm by 36 mm.
  • This arrangement is particularly suited for applications necessitating electrophoretic processing of samples with greater mass and for specialized applications like preparative electrophoresis by isotachophoresis and for two-dimensional analytical electrophoresis.
  • the electrophoresis strip offers several benefits that contribute to its utility and versatility in laboratory settings.
  • the electrophoresis microplate can be configured with varying numbers of electrophoresis strips, allowing users to scale up or down depending on the sample size or throughput needs, thereby optimizing resource utilization. By allowing different application-specific electrophoresis strips to be run within each plate, this also may enable users to tailor experiments to specific applications such as performing analytical and preparative experiments in the same plate.
  • laboratories can scale their experiments according to sample mass, or throughput demands. Whether it's processing a large number of samples in parallel or handling larger sample volumes with specialized strip configurations, the system offers scalability to meet evolving research needs.
  • the electrophoresis strip format offers modularity, customizability, versatility, scalability, ease of use, and maximized efficiency, making it valuable for a wide range of electrophoresis applications in research and clinical laboratories.
  • Electrophoresis cell constitutes a fundamental enabling component of the disclosed electrophoresis microplate system. By adjusting the overall size and structure of the electrophoresis cell, it can be customized to accommodate various applications, sample volumes and throughput requirements. The adaptability of the electrophoresis cell to different sample types and electrophoretic applications is a key feature of the disclosed system.
  • Fig. 3A shows a representation of the electrophoresis cell 110, in perspective view.
  • Fig. 3C represents a second layer, comprised of fluidic chambers 310, serving to hold the sample and reagents required for electrophoretic processing. This second fluidic- chambers-layer is positioned below the upper cover 305.
  • Fig. 3D represents a third layer, comprised of fluidic ports 315 that provide fluidic connections between the second fluidic-chambers-layer 310 and a separation-channels- layer 320.
  • Fig. 3E shows the fourth layer, placed below the third fluidic-ports-layer, comprised of separation channels 320.
  • the separation channel is an essential component of the electrophoresis cell as it functions to separate the sample constituents during electrophoresis.
  • a lower cover 325 seals the bottom of the electrophoresis cell to ensure proper containment of components as represented in Fig. 3F.
  • the structures of the electrophoresis cell and additional components including but not limited to, electrodes, buffers, reagents, separation or affinity matrices, provide a self-contained consumable unit.
  • the integrated construction enhances convenience and efficiency in laboratory workflows, reducing the number of steps needed for electrophoretic analysis.
  • electrophoresis cell various structures and components, including but not limited to the upper cover, fluidic chambers, fluidic ports, electrodes, separation channels, and lower cover. These descriptions are designed to clarify each structure and component’s design and roles, thereby enhancing the overall grasp of how the electrophoresis cell functions as part of the electrophoresis microplate system. It should be noted that while the ensuing descriptions are specific to the electrophoresis cell, they are also relevant and applicable to both electrophoresis strips and plates that contain a multitude of electrophoresis cells.
  • the upper cover 305 of the electrophoresis cell plays a role in the electrophoresis cell, strip or plate, by acting as a protective barrier. It effectively shields the cell’s contents from external contaminants, oxidation, and evaporation. Additionally, it ensures that reagents in different fluidic chambers do not mix inadvertently during the cell’s storage, transport, or handling, while still permitting the insertion of samples and electrodes during the electrophoresis process.
  • the detailed explanations that follow regarding the upper cover of the electrophoresis cell are equally relevant to both the electrophoresis strip and plate. In essence, the upper cover can be placed on the electrophoresis strip or the electrophoresis microplate as a single piece.
  • the upper cover 305 as shown in Fig. 4A can be constructed of multiple layers, including a protective removable layer 405, shown in Fig. 4B.
  • This layer can be detached to access the underlying layer or layers, facilitating easy access while maintaining a secure seal that safeguards against damage and contamination.
  • the protective removable layer can be a planar structure, via detachable adhesive components 415, such as those offered by Remington Lamination Inc., featuring a permanent adhesive on one side for lasting attachment to the protective layer, and a peelable adhesive on the other, ensuring clean removal from the layer below.
  • the protective removable layer 405 of the upper cover integrates structural elements 410 that affixes to the elevated features or ridges of a guide layer 425 situated beneath it, as illustrated in Fig. 4B.
  • the upper cover comprises a second layer, the guide layer 425, as illustrated in Fig. 4D.
  • This layer is equipped with structures designed as pipette-tip guides 430 for efficient fluidic loading and unloading, as well as electrodes guides 435 to contact electrodes accurately within the cell.
  • the guide structures can be comprised of cylindrical, ellipsoidal, cubical or polygonal protrusions, but not limited thereto.
  • the guide layer 425 can incorporate both human and machine-readable information 420, such as the usage, contents, and specifications of the electrophoresis cell, strip, or plate. These details can be integrated through molding, etching, embossing, printing, or other methods directly onto the guide layer. Occasionally, as depicted in Fig.
  • this information might be added as a distinct layer.
  • Such data is important for the smooth operation of the system, ensuring precise identification of each component’s role and aiding in the monitoring of samples during the electrophoresis process.
  • a layer can feature, penetrable features 440, such as scoring or notching to facilitate easier penetration by pipette tips or electrodes and can incorporate gas-permeable structures 445, to allow the escape of gases generated during electrophoresis as shown in Fig. 4E.
  • Methods for attaching the upper cover assembly to the electrophoresis cell, strip or microplate can include pressure-sensitive adhesives or heat- fusible materials, such as polyacrylamides, polyesters, polypropylene, high-density polyethylene, low-density polyethylene, metalized plastics, aluminum foils, or combinations thereof.
  • Fluidic chambers Within the architecture of the electrophoresis cell 110, as illustrated in Fig. 3B, resides a layer equipped with fluidic chambers 310. These chambers serve as containers for various materials required for specific electrophoretic applications, including but not limited to; samples, cell suspension solutions, extraction reagents, sample labeling reagents, sample buffers, stacking buffers, concentration buffers, electrophoresis buffers, purification buffers, and other reagents.
  • the number of fluidic chambers within an electrophoresis cell can vary, ranging from none to a multitude of fluidic chambers depending on the specific requirements of the electrophoretic application.
  • Some designs of the electrophoresis cell may lack fluidic chambers, instead utilizing a sample well 505 positioned within one end of the separation channel 510, as shown schematically in top and side views in Fig. 5A.
  • a single input fluidic chamber 515 that is proximal to the sample well 505 or that serves as a container for the sample can connect to just one end of the separation channel 510, as shown in the top and side schematic views in Fig. 5B.
  • a sample well can be omitted as shown in Fig.
  • a sample well 505 can be included as shown in the top and side schematic views in Fig. 5E, F and G and in the perspective view of a fluidic chamber layer in Fig. 5H.
  • a pair of fluidic chambers can connect at both ends of the separation channel, as illustrated in Fig. 5C.
  • Some designs of the electrophoresis cell can incorporate additional fluidic chambers, termed as side chambers 525, forming fluidic connections with the separation channel along the channel’s length as shown in the top and side views in Fig. 5D and 5E.
  • These side chambers can serve as reservoirs for reagents needed for interfacing with the sample or sample constituents.
  • a subset of these side chambers can function as purification chambers 530 and 535, collecting the constituents of the sample that have been separated within the separation channel as shown in the top and side views in Fig. 5D, 5E, 5F and 5G and in the perspective view in Fig. 5H.
  • the side chambers 525 and purification chambers 530 and 535 can be positioned at staggered locations along the separation channel as illustrated in Fig. 5D and 5E, or can be positioned diametrically opposite to each other as shown for two purification chambers 530 and 535 in the top and side schematic views in Fig. 5E and 5F. In some designs, these side chambers can form fluidic connections directly with the separation channel as shown in Fig. 5D and 5E. In other designs, the purification chambers 530 or 535 can connect through ancillary channels 540 as shown in Fig. 5F or via branching channels 545 as shown in Fig. 5H.
  • the dimensions of the fluidic chambers within an electrophoresis cell can differ from each other as shown schematically in top and side views in Fig. 5D, 5E, 5F and 5G and in the perspective view in Fig. 5H based on specific requirements of an electrophoretic application.
  • fluidic chamber can be designed to accommodate larger volumes.
  • the input chamber 515 and output chamber 520 can hold a larger volume of fluids to alleviate changes in buffer pH, to accommodate electroosmotic flow of buffers from one chamber to another and to reduce temperature changes caused by electrophoresis.
  • the purification chambers 530 and 535 in the design represented in Fig. 5H are substantially smaller than the input 515 and output chambers 520 to avoid dilution of the purified sample constituent that has been drawn into a purification chamber 530 or 535.
  • the fluidic chambers are an integral component of the electrophoresis system, serving multiple functions. They are necessary for the storage of necessary reagents and enable the efficient introduction of samples into the system. These chambers act as reservoirs for reactants that facilitate sample processing and provide the ions necessary for the electrophoretic separation process. Additionally, they play a role in buffering against pH fluctuations, collecting and releasing gases, and accommodating the fluid movement caused by electroosmotic flow.
  • the input and output chambers are designed to contain electrophoresis buffers, which are crucial in maintaining the conditions required for the effective separation of sample constituents during electrophoresis. The forthcoming section will detail a partial list of such electrophoresis buffers.
  • An ideal buffer stabilizes the pH at all locations in the separation channel in the electrophoresis Cell, reduces or eliminates Hydrogen and Oxygen gas formation from the hydrolysis of water in the buffer, reduce electroosmotic flow, have a low conductivity, and do not degrade agarose or acrylamide gel matrix during extended storage.
  • Electrophoresis buffers based on ion exchange reagents provide a uniform pH throughout the cell since any proton buildup near the anode is compensated by absorption by a cation exchange matrix and hydroxyl buildup near the cathode is compensated by absorption by the anion exchange matrix.
  • a cation exchange material such as CM -25-120 Sephadex and a suitable anion exchange material, such as WA-30 (Millipore Sigma Inc.) (US Patent No. 5582702, 1996) and (US Patent No. 5865974, 1999).
  • MOPS Buffer Tris base 60.6 g MOPS 104.6g SDS 10.0g EDTA 3.0g Deionized water to 1000 ml, typically used for electrophoresis of protein samples.
  • Non-aqueous electrophoresis buffers based on solvents such as acetonitrile, methanol, formamide, and dimethylformamide, to which are added anhydrous acid or buffer salts can be used for the analysis of drugs, dyes, preservatives, surfactants, and inorganic ions • Sodium Borate Buffer; 10 mM sodium hydroxide, pH adjusted to 8.5 with boric acid (H3BO3) (Brody JR, 2004) typically used for analytical electrophoresis of DNA samples. Electrophoresis buffers that contain Borate are not typically suitable for preparative electrophoresis as Borate inhibits enzymatic activity (Paties Montagner G, 2023).
  • Tris Acetate EDTA (TAE) Buffer 40 mM Tris pH 7.6-8.0, 20 mM acetic acid, 1 mM EDTA.
  • TAE Tris Acetate EDTA
  • Buffers containing EDTA negatively impact the resolution of DNA (Sanderson BA, 2014) and can interfere with downstream enzymatic activity due to chelation of metal ions.
  • Tris Borate EDTA (TBE) Buffer 89 mM Tris pH 8.3, 89 mM boric acid, 2 mM EDTA, typically used for analytical electrophoresis of DNA and RNA samples.
  • Tris Glycine Buffer 25 mM Tris base, 192 mM glycine, 0.1 % SDS, pH 8.3, typically used for analytical and preparative electrophoresis of protein samples.
  • Tris Phosphate EDTA (TPE) Buffer 89 mM Tris pH 8.3, 89 mM phosphoric acid, 2 mM EDTA, typically used for analytical and preparative electrophoresis of DNA samples.
  • Tri Triethanolamine
  • TRICINE N-tris hydroxymethylmethylglycine
  • Buffers with pKa values of the weak acid and the weak base within 0.3 units of each provide electrical current stability, tolerance to higher voltage at lower working concentration resulting in lower current per unit Voltage, resulting in less heat generation (US Patent No. 6582574B1 , 2003) (Porath, 1955).
  • the buffers stored in the fluidic chambers connect with other structures within the electrophoresis cell through fluidic ports which are further described in the below section.
  • Sample suspension reagent compositions that are well known in the art can be used for electrophoresis in electrophoresis cells comprised of sample wells.
  • the sample suspension reagent can be comprised of buffers that can have lower ionic strength and/or lower pH than the electrophoresis Buffer resulting in the Sample constituents becoming concentrated at the beginning of the separation channel due to the phenomenon of sample stacking wherein constituents migrating from a low- conductivity buffer into a high-conductivity buffer slow down at the boundary of the two buffers forming a narrow zone (Slampova A, 2019).
  • the sample is concentrated on the Concentrating Electrode, salts, chaotropic agents, organic solvents, or detergents can be used to prevent aggregation and precipitation of samples prior to electrophoretic separation.
  • a non-limiting list of such agents which might be used solely or in combination includes urea, glycerol, dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), guanidine hydrochloride, and betaine.
  • compositions of a non-limiting list of sample suspension buffers and reagents are listed below.
  • Sample buffers for DNA A buffer comprised of 0.25% w/v Xylene cyanol FF, 0. 25% w/v of Bromophenol Blue, 5% v/v glycerol 10mM EDTA suspended in electrophoresis buffer can be added to a DNA sample in a 1 :10 ratio of sample reagent to DNA sample. The DNA sample mixed with the sample buffer can then be placed in the sample well of the electrophoresis cell.
  • Alternative formulations of DNA sample reagents can contain 4% Sucrose or 2.5% Ficol instead of glycerol. 1 % SDS can be included to eliminate protein-DNA interactions, preventing appearance of additional bands from protein-DNA complexes.
  • RNA sample is denatured at 65C for 5 minutes in the above-mentioned DNA sample suspension reagent composition to which formaldehyde at 5% is added.
  • Sample buffers for proteins A protein sample can be electrophoresed after denaturing the protein constituents with Sodium or Lithium dodecyl sulfate.
  • a common formulation of a denaturing Protein sample suspension reagent is provided here; 0.67g Tris HCI, 0.68g Tris Base, 0.80g Lithium dodecyl sulfate, 0.006g EDTA, 4g Glycerol, 0.75ml of a 1% solution of SERVA Blue G250, and 0.25ml of a 1 % solution of Phenol Red dissolved in a final volume of 10ml with water.
  • the protein sample is mixed with the Protein sample suspension reagent at a 1 :4 ratio of sample suspension reagent to Protein Sample, electrophoresis can be performed on non-denatured protein samples to detect native configurations of the protein, in such instances, the dodecyl sulfate salt is removed from the above Protein Sample Suspension Reagent.
  • additional reagents can be included in the sample suspension reagent such as staining reagents that stain constituents in the sample facilitating detection during electrophoresis.
  • Proteins can be stained by colorimetric stain such as Coomassie Blue or with fluorescent dyes.
  • the Fluorescent Chromeo Py-dyes from Active Motif that become fluorescent upon reacting with primary amines in proteins can be used to stain proteins in the sample.
  • Two examples from publications (Craig DB, 2005) and (Del Mar Barrios-Romero M, 2013).
  • DNA and RNA can be stained with fluorescent dyes well known in the art such as Ethidium Bromide, SYBR Green, Propidium Iodide, Gel Red (Hall AC, 2019).
  • the sample suspension reagent can be comprised of reagents that bind to specific constituents in the sample. Bound and unbound binding reagents will migrate at different velocities in the separation channel and the ratio of fluorescence signals can provide a quantitation of said constituents.
  • the constituents in the sample can be labeled with a detectable moiety or moieties and then mixed with the binding reagent.
  • binding reagent and the sample constituents can be labeled with detectable moiety or moieties.
  • binding reagents can be labeled with fluorescent moieties with different fluorescent colors.
  • the mixture of binding reagents is then mixed with the sample, concentrated in the concentration chamber and fractionated in the fractionation chamber.
  • Fluidic ports The electrophoresis cell is designed with strategically placed fluidic ports within its non- permeable constructs, enabling the controlled transit of fluids between distinct fluidic chambers, as well as to and from ancillary and separation channels, and between chambers and channels. These fluidic ports are pivotal in orchestrating the flow of reagents, ions, and sample constituents within the cell. They act as conduits linking various chambers and channels, thereby streamlining the processes of sample handling, component segregation, and subsequent purification.
  • the electrophoresis cell can be engineered with fluidic ports that can be positioned as a distinct layer 315, as exemplified in the perspective view in Fig. 3D, or incorporated within various other layers or constituents of the cell, such as the upper cover, fluidic chambers, separation channels, or the lower cover.
  • An example of the electrophoresis cell is comprised of two distinct layers of fluidic ports, a first layer 605 and a second layer 315.
  • the first layer of fluidic ports 605, as shown in Fig. 6B, is interposed between the upper cover 305, as seen in Fig. 6A and the fluidic chambers layer 310, as shown in Fig. 6C.
  • This first layer of fluidic ports 605 can either be an integral part of the upper cover 305, exist as a separate entity, or be affixed to the fluidic chambers layer 310, as shown in Fig. 6B.
  • the materials constituting the first layer of fluidic ports can be selected for ease of perforation, facilitating the injection of fluids into the electrophoresis cell and the establishment of electrical connectivity with the fluidic chambers or channels within the cell.
  • this layer can incorporate features that support penetration and subsequent resealing, or it can be comprised of self-healing polymers that allow for such functionality.
  • the first fluidic port layer can comprise of an input port 610 and an output port 615, along with two purification ports 620, 625.
  • Each port is aligned with the corresponding chamber in the fluidic chambers layer 310 shown in Fig. 6C; namely, the input port 610 is aligned with the input chamber 515, the output port 615 is aligned with the output chamber 520, and the purification ports 620, 625 are aligned with the purification chambers 530, 535; to ensure efficient transfer of fluids to and from the fluidic chambers.
  • the second layer of fluidic ports 315 is sandwiched between the fluidic chambers layer 310 (Fig. 6C) and the separation channel layer 320 (Fig. 6E).
  • the second layer of fluidic ports 315 is comprised of five fluidic ports, namely, an input port, in the second fluidics port layer 630, a sample port, in the second fluidics port layer 635, two purification ports, in the second fluidics port layer 640, 645, and an output port, in the second fluidics port layer 650, as shown in the perspective and top views in Fig. 6D.
  • the Input port, in second fluidics port layer 630 and second-port-layer output port 650 allow an electric current to pass through the separation channel 320.
  • the second-port-layer purification ports 640, 645 are placed directly above the separation channel 320 and will allow a sample constituent to be electrophoretically moved from the separation channel 510 into the corresponding purification chamber, 530, 535.
  • the fluidic ports in addition to being apertures, can comprise materials that are semi-permeable, permeable, or porous. These materials can be tailored to selectively permit the passage of sample constituents, based on size, charge, hydrophilicity, hydrophobicity, affinity, or other physicochemical characteristics.
  • Fluidic ports can be comprised of electrically conductive materials, enabling further control over the passage of sample constituents by subjecting them to repulsive or attractive electrical forces.
  • These electrically conductive electrodes can be porous, shaped as an annulus, or a mesh or similar structure that can allow fluids to flow through the port without hindrance unless an electrical charge is imposed on the electrodes contained in the fluidic port. This functionality allows for precise regulation of sample movement within the electrophoresis cell.
  • Ports with electrical connectivity can also be utilized to measure electrical properties, such as conductivity, between a set of ports. Ports with split electrodes, featuring two electrical connections, enable the precise measurement of changes in electrical properties during the passage of sample constituents through that port.
  • Electrophoretic separation is achieved by the application of electrical current through electrically conductive electrodes. These electrodes serve as essential components for facilitating the movement of sample constituents through the various structures in the cell, through the separation matrix, and into purification chambers. Within the electrophoresis cell, two or more electrodes can be placed in contact with a sample, a buffer or a reagent present in the fluidic chambers, the fluidic ports, or the separation channels as shown as simplified top and side view schematics in Fig. 7.
  • electrodes can be positioned directly at each end of the separation channel 510 in an electrophoresis cell, as diagrammed in the top and side views in Fig. 7A, though this configuration may damage the separation matrix present in the separation channel 510, due to gas production from the electrolysis of water, elevated temperatures at the electrodes, and fluid depletion from electroosmosis.
  • an ion exchange buffer system capable of minimizing the damage from the electrophoretic process can be employed, as exemplified in (US Patent No. 5582702A, 1996).
  • the ion exchange buffer system utilizes a cation exchange resin to serve as a sink for proton buildup near the anode buffering against pH change and H2 gas generation from the hydrolysis of water. Similarly, hydroxyl buildup near the cathode is compensated by absorption by an anion exchange matrix.
  • an electrode can be situated within the input chamber 515 as shown schematically in Fig. 7B, instead of within the separation channel 510 itself.
  • the electrode positioned in the input chamber 515, proximal to the sample well 505 is denoted as the input electrode 705.
  • the output electrode 710 is termed the output electrode 710 as shown in Fig. 7C.
  • the side chambers 525 in an electrophoresis cell can be equipped with a side chamber electrode 715 to electrophoretically drive reagents from the side chamber 525 into the separation channel 510 or from the separation channel into the side chamber.
  • the side chamber can be used as the first purification chamber 530 with an electrode placed in the first purification chamber referred to as the first purification electrode 720 as shown in Fig. 7D, 7E and 7F.
  • the purification electrode placed in the second purification chamber 535 is referred to as the second purification electrode 725.
  • the configuration with two purification chambers and associated purification electrodes enables the isolation of two sample constituents by shuttling a first component of interest from the separation channel into the first purification chamber 530 and a second component of interest into the second purification chamber 535.
  • concentration electrode 730 positioned within the input chamber at or close to the entrance of the separation channel as shown in the top and side views in Fig. 7E and 7F.
  • the concentration electrode 730 facilitates the electrophoretic pre-concentration of a dilute sample into a smaller space with an approximately 100x concentrated sample being placed at the entrance of the separation channel before the commencement of electrophoresis.
  • the concentration electrode can assume diverse geometries, such as a porous grid, as an annular, cylindrical or rectangular ring or other configurations designed to accumulate the sample without hindering ion flow through the separation channel.
  • An additional set of electrodes can be placed across a fluidic channel or fluidic port to serve as a sensor to detect passage of materials through the channel or port or to act as a gate to hinder or accelerate the flow of materials through the fluidic channel or fluidic port.
  • a configuration of such sensing and/or control electrodes 735 is shown in Fig. 7F flanking an ancillary fluidic channel 540.
  • An electrical property such as conductance, measured across any set of electrodes disclosed above can provide information about the composition of a material or materials present between those electrodes to further inform the analysis and purification of materials within the electrophoresis cell.
  • Fig. 8A shows the top view of an example of the electrophoresis cell with a sample well 505.
  • the cell is shown without the upper cover for clarity.
  • the sample well 505 is positioned within the input chamber 515.
  • the input electrode 705 is positioned in the input chamber 515 as shown in Fig. 8A.
  • the input port 630 fluidically connects the input chamber 515 and one end of the separation channel 510.
  • the output electrode 710 is positioned in the output chamber 520 with the output port 650 forming a fluidic connection between the output chamber 520 and the other end of the separation channel 510. Electrical current can pass between the input electrode 705 and the output electrode 710, via the input chamber 515, the input port 630, the separation channel 510, the output port 650, and the output chamber 520 enabling electrophoretic separation.
  • the configuration of the electrophoresis cell represented in Fig. 8A further shows the first purification electrode 720, positioned in the first purification chamber 530 with the first purification port 640 facilitating a fluidic connection between the first purification chamber 530 and the separation channel 510.
  • the second purification electrode 725, placed in the second purification chamber 535 with the second purification port 650 forms the fluidic connection between the second purification chamber 535 and the separation channel 510.
  • Fig. 8B shows the cross-sectional side view of the electrophoresis cell represented in Fig. 8A.
  • the input electrode 705 penetrates through the upper cover 305 of the cell into the input chamber 515.
  • the input chamber 515 is electrically and fluidically connected to the separation channel 510 in the separation channel layer 320 through the input port 630.
  • the input chamber 515 contains the sample well 505 through which the sample is introduced directly into the separation channel 510.
  • the output electrode 710 placed in the output chamber 520 forms the electrical connection through the output port 650 into the separation channel 510.
  • the input and output ports are localized within the fluidic ports layer 315 and the input and output chambers are localized within the fluidic chambers layer 310.
  • the lower cover 325 seals in the separation channel layer.
  • Fig. 8C shows a perspective view of an alternative configuration of the electrophoresis cell.
  • the electrophoresis cell includes an additional electrode, the concentration electrode 730 that is placed in the input chamber 515.
  • the sample well 505 shown in Fig. 8A and 8B is not included, instead sample constituents are concentrated by applying an electrical current between the input electrode 705 and the concentration electrode 730 resulting in the sample constituents being concentrated at the surface of the concentration electrode.
  • Fig. 8C shows the placement of the output electrode 710 in the output chamber 520, the first and second purification electrodes 720, 725 in the first and second purification chambers 530, 535, respectively.
  • the electrodes can take the form of strips, cylindrical, elliptical, triangular, rectangular rods in shape, but not limited thereof.
  • the electrodes can be fashioned as single-use, disposable units that are internally integrated into the electrophoresis cell, or alternatively, the cell can accommodate external electrodes that engage at designated regions within the cell. Some examples can incorporate a hybrid approach, wherein internal electrodes are embedded at certain locations within the electrophoresis cell, while at other locations, an interface for external electrodes is established.
  • Electrodes can include conductive non-metallic substances like carbon-based compounds, ceramics (Smyth DM, 1987), plastics (Bengtsson K, 2014) (Kumar A, 2023), or traditional conductive metals, depending on the particular needs of the application, but not limited thereof.
  • electrodes can be positioned in the fluidic chambers, preferably positioned above the separation channel rather than in the separation channel layer or below. This positioning helps mitigate risks from gases generated during electrophoresis, localized heating at the electrodes, and metal ions released from the electrodes.
  • electrodes perform a pivotal role in propelling electrical current through the chambers, ports and channels to achieve the desired electrophoretic analysis and purification steps.
  • Their precise placement and composition are paramount factors in ensuring the efficiency and efficacy of the electrophoresis process within the electrophoresis cell.
  • the separation channel within the electrophoresis cell serves as the conduit for electrophoretic separation of sample constituents based on their physico-chemical properties.
  • the separation channel s characteristics, including but not limited to its length, cross-sectional dimensions, and geometric configuration, are critical determinants of the electrophoresis cell’s analytical and purification efficacy.
  • the disclosure provides for a versatile range of channel lengths, extending from millimeters to several centimeters. This versatility is instrumental in enhancing the resolution of separation and in facilitating the analysis of a diverse set of applications and samples.
  • Fig. 9 Depicted in Fig. 9 are various examples of the separation channel layer, corresponding to electrophoresis cells with differing dimensions.
  • Fig. 9A illustrates electrophoresis cells with dimensions up to 9 mm by 9 mm, present in 96-cell electrophoresis microplates, which can provide separation channel lengths ranging from approximately 10 mm to 100 mm.
  • Electrophoresis cells within a 24-cell electrophoresis microplate, with dimensions up to 18 mm by 18 mm per cell, are capable of supporting channel lengths from about 20 mm to in excess of 200 mm, as shown in Fig. 9B.
  • Fig. 9C represents a 6-cell electrophoresis microplate configuration, where electrophoresis cells measure up to 36 mm by 36 mm and can provide channel lengths surpassing 40 centimeters.
  • the prescribed lengths of the channels are a function of the channel’s cross-sectional dimensions and the number of turns or folds of the serpentine channels within the allotted spatial constraints.
  • channels widths of 25, 50, 75, or 100 micrometers but not limited thereof, providing similar rapid analysis times and high resolution of capillary electrophoresis systems and can be suitable for DNA sizing and Sanger sequencing applications.
  • wider channels of millimeter dimensions providing increased sample loading capacity are preferred and are optimal for isolating DNA, RNA, proteins, cell organelles, viruses, bacteria, or cells from samples.
  • separation channels 510 within the electrophoresis cell.
  • These configurations include, but are not limited to, linear channels as exemplified in Fig. 10A.
  • serpentine channels 510 as depicted in Figs. 10B, 10C, and 10D, are designed to maximize the utilization of the spatial dimensions within the electrophoresis cell.
  • serpentine channels 510 By incrementally increasing the number of turns within the serpentine channels and correspondingly reducing the channel width, it is possible to significantly extend the total length of the channel, as demonstrated in Fig. 10D. Notwithstanding the reduction in channel width for extended channels, the sample loading capacity is preserved by augmenting the channel depth through the incorporation of thicker separation channel layers — a notable design feature of the disclosed electrophoresis cell.
  • a channel with a width of 2 mm and a depth of 10 mm is capable of accommodating a sample volume of approximately 40 pL in a 2 mm sample well.
  • the disclosure encompasses spiral separation channels 510, as illustrated in Fig. 10E, which capitalize on the available space within the cell to furnish the requisite channel length necessary for achieving optimal electrophoretic resolution.
  • the electrophoresis cell can be configured with a multi-layered separation channel structure, wherein two or more layers of separation channels are superimposed.
  • An example exemplified in Fig. 10F demonstrates an electrophoresis cell comprised of dual layers of separation channels. This design facilitates sequential electrophoretic separations along orthogonal axes, significantly enhancing the overall separation efficiency. Initially, the sample is introduced into the primary separation channel 510, where it undergoes electrophoretic separation along the ‘a’ to ‘b’ vector as depicted in Fig. 10F.
  • the separated sample constituents are electrophoretically conveyed along the ‘b’ to ‘c’ trajectory onto a slab gel located in an additional separation channel 510’, situated in a secondary layer beneath the initial one.
  • the sample constituents are then subjected to a further stage of separation, proceeding perpendicularly relative to the first separation channel, along the ‘c’ to ‘d’ vector, as illustrated in Fig. 10F.
  • the separation channels can be conceived as three-dimensional constructs as represented in Fig. 10G.
  • These separation channels can take the form of tubular structures bent into a U-shape, or alternatively, in the form of a singular coil-based separation channel 510 shown in Fig. 10G or stratified coils, but not limited thereof.
  • the coils can be fabricated from tubular segments with cross-sectional dimensions that span from the micrometer to the millimeter scale.
  • the selection of materials for these tubes is diverse, including but not limited to silica, glass, and a variety of plastics. This assortment of dimensions and materials empowers the electrophoresis cell with a wide spectrum of capabilities suitable for both analytical and preparative electrophoretic applications.
  • Fig. 10H illustrates an example featuring a linear form of the separation channel 510, with a first purification channel 540 and a second purification channel 542 fluidically connecting to the separation channel 510. This configuration enables the separated sample constituents to be transported from the separation channel 510 into the purification channels 540, 542 and into the purification chambers.
  • Fig. 101 depicts an alternative example employing a serpentine separation channel 510 design instead of a linear channel, with a first purification channel 540 and a second purification channel 542 connected into the serpentine separation channel for similar functionality.
  • Fig. 10J shows an example of the separation channel layer featuring side channels 1005 fluidically connected to a high-density serpentine separation channel 510.
  • An enlarged version of the 18 mm by 18 mm separation channel layer is included in Fig. 10J to show the configuration in greater detail.
  • the separation channel 510 in the displayed example measures 100 micrometers in width and is spaced at intervals of 250 micrometers apart. This configuration results in an approximate total channel length of 500 millimeters, within an 18 mm by 18 mm electrophoresis cell. This surpasses the lengths of separation channels found in microfluidic electrophoretic devices and matches or exceeds the lengths of capillaries utilized in capillary electrophoresis systems provided by Thermo Fisher Scientific.
  • the side channels 1005 in the example shown in Fig. 10J play a distinct role: introducing a small plug of the sample into the separation channel.
  • the sample is introduced through the side channel chamber (not shown), into the side channel and across the junction formed by the cross-over of the side channels and the main separation channel 510, by capillary action or by electrophoresis. Subsequently, the sample plug at the junction enters the separation channel and undergoes electrophoretic separation.
  • the surfaces of these channels can undergo dynamic or permanent modifications to regulate electrophoretic or electroosmotic properties (Dolnik V, 2004).
  • the channels can be coated with materials such as polyethylene glycol, polyvinyl alcohol, dextran, cyclodextrins, detergents, specific proteins, metal salts but not limited thereof. Such alterations serve to mitigate the binding of sample constituents, to selectively impede or deplete certain constituents within the sample, to selectively accelerate certain constituents in the sample, or modify electroosmotic flow in the channel.
  • the various coating methods well known in the art further enhance the application-specific performance of the electrophoresis cell.
  • the current disclosure introduces several innovative separation channel configurations presenting unique advantages for enhancing the efficiency and versatility of electrophoretic separations.
  • These separation channel configurations leverage varying geometries, structures, and dimensions to optimize separation performance in different scenarios. For instance, certain configurations enhance the resolution of separation, crucial for applications requiring precise identification of sample constituents. Additionally, specific channel geometries facilitate rapid and high-throughput preparative electrophoresis, ideal for purifying large quantities of sample constituents in a shorter timeframe, unlocking new possibilities in fields such as proteomics, genomics, drug discovery, and molecular diagnostics.
  • Separation channels are fundamental components in electrophoresis, often comprised of separation matrices that serve multiple essential functions. These matrices play a pivotal role in controlling the migration of sample constituents, while providing vital support and stability to the molecules undergoing separation. Importantly, they act as barriers, preventing the diffusion or denaturation of molecules during electrophoresis, thereby preserving the structural integrity of sample constituents for subsequent purification.
  • Separation matrix compositions can be tailored to different sample types to enhance the efficiency of analytical and preparative electrophoresis.
  • the separation matrices can comprise materials with electrostatic properties, varying porosity, hydrophilicity or hydrophobicity, and repulsive or affinity characteristics. If electrophoretic separation is carried out in solution, sample constituents can be separated according to their net surface charge density. If carried out in the presence of a retarding gel, sample constituents migrate according to both charge and size. Separation matrix can have retarding, affinity, or repulsive properties, facilitating separation based on physical, chemical, and biological properties. Materials such as agarose and acrylamide polymers offer adjustable retardation capabilities as well as serve as scaffolds for attaching molecules such as antibodies, oligonucleotides, metal ions, and dyes.
  • Agarose at concentrations of 0.3% to 3% agarose in appropriate electrophoresis buffers can be used for the analysis of high molecular weight DNA and RNA (length of >100 base) and proteins (> 200 kilodalton molecular weight), and particles, virus, and cells.
  • high molecular weight DNA and RNA length of >100 base
  • proteins > 200 kilodalton molecular weight
  • particles, virus, and cells For analysis of low molecular weight DNA and RNA (of less than 100 bases), and for proteins of less than 200 kilodaltons polyacrylamide-based separation matrix are well- documented in the field.
  • a solution containing monomeric acrylamide, a cross linker, a slow ion buffer, and a photocatalyst or photo-initiator, such as benzoin ethers, and benzophenone derivatives and an amine transfer agent, which initiates free-radical cross- linking when exposed to a source of UV light resulting in polymerization, can be used to polymerize polyacrylamide gels in the separation channel.
  • the separation channels can be comprised of a composite separation matrix comprised of a mixture of agarose and polyacrylamide.
  • the separation channel is filled with a mixture of the acrylamide monomeric solutions at 2x the required concentration, prewarmed to a temperature of 50°C, and agarose, at 80°C-90°C at 2x the required concentration, and allowed to cool. Polymerization of the acrylamide is then completed by allowing the cross-linking of acrylamide to complete (Melrose, 2023).
  • the composite separation matrix can be comprised of a mixture of polyacrylamide with a non-limiting list of materials such as celluloses, hydroxyethyl cellulose, starch, pectin, polyethylene glycols or derivatives such as cross-linked dextran.
  • the composite matrix can be comprised of agarose mixed with other polymers such as hydroxyethyl cellulose (Siles BA, 1997).
  • Additives such as dyes or stains that bind to DNA, RNA, or proteins are used to visualize separated sample constituents in separation matrices.
  • Hydrophilic or hydrophobic reagents including polar or non-polar solvents, ion exchange resins, and detergents like sodium dodecyl sulfate (SDS), along with substances such as formamide, formaldehyde, and labeled or unlabeled affinity or complexing reagents like dyes, oligonucleotides, antibodies, or aptamers, influence electrophoretic migration and enhance the versatility of separation matrices.
  • the composition of the separation matrix can remain consistent or vary across different separation channels.
  • Various compartments within the electrophoresis cell such as side channels, purification channels, fluidic ports, and fluidic chambers, can contain a separation matrix. In these compartments, the composition of the separation matrix can be consistent or diverse, depending on the specific needs of each area.
  • composition of the separation matrix within a compartment can be uniform throughout or implemented as a gradient.
  • one terminus of the separation channel can have a higher concentration of a matrix component, gradually decreasing towards the other terminus.
  • the separation channel can be divided into distinct zones, each containing separation matrix with different properties or chemical compositions. These zones can incorporate various labeled or unlabeled affinity or complexing reagents, such as oligonucleotides, antibodies, aptamers, lectins, serum proteins, enzymes, carbohydrates, metals, and other binding reagents, to capture different constituents within the sample.
  • affinity or complexing reagents such as oligonucleotides, antibodies, aptamers, lectins, serum proteins, enzymes, carbohydrates, metals, and other binding reagents
  • electrophoretic separation channel matrices provide customizable attributes such as electrostatic, porosity, and hydrophilic or hydrophobic properties, enhancing efficiency and accuracy for specific applications. Additionally, incorporating dyes or stains into the matrix facilitates visualization of separated molecules, improving the analysis and interpretation of results. Attaching molecules such as antibodies, oligonucleotides, metal ions, and dyes further expands the scope of applications, electrophoresis cells with multiple matrices, facilitate multiple analysis of a sample within a single cell. Gradient formation within the channels enhances separation resolution and enables analysis of complex mixtures with a broader range of molecular weights, for example.
  • Examples featuring distinct zones within channels, each with different matrix compositions, offer precise control over separation, facilitating selective capture, isolation and detection of specific sample constituents in complex mixtures. These collective benefits enhance the efficiency, versatility, and applicability of electrophoretic techniques for end-users, thereby enabling various applications in molecular biology, biochemistry, genetics, and other fields.
  • the electrophoresis cell can be comprised of a lower cover 325 that seals the base of the electrophoresis cell and can be comprised of a flexible or rigid transparent material comprised of a first surface and an opposite second surface that allows light to be transmitted to and from the separation channel while sealing the base of the electrophoresis cell.
  • the lower cover can be comprised of one or a multitude of planar layers sandwiched within the first and second surface; with at least one of the multitude of layers comprised of a bonding agent to attach the lower cover to the base of the electrophoresis cell; with at least one of the layer comprised of a transparent material that can transmit UV and visible wavelengths of light; with at least one of the multitude of layers comprised of an opaque material to mask certain regions of the base of the electrophoresis cell; with at least one of the multitude of layers comprised of machine and human-readable information; with at least one of the layers comprised of a removable layer to protect the bottom surface of the lower cover.
  • the core components of the electrophoresis cell have been described in the sections above.
  • additional layers, structures or components not described above can be incorporated into the electrophoresis cell.
  • two or more of the structural layers can be combined as a single layer.
  • a significant advantage offered by the various examples is the ability to perform a multitude of electrophoresis applications from high-resolution analytical electrophoresis, two-dimensional electrophoresis analysis, preparative electrophoresis, and affinity electrophoresis in parallel on a single system that is compatible with laboratory automation.
  • analytical capillary electrophoresis, two-dimensional electrophoresis, preparative electrophoresis, and affinity electrophoresis in the electrophoresis cells are described.
  • the separation channels in the analytical electrophoresis cell can be made with widths of 25, 50, 75 or 100 micrometers, but not limited thereof.
  • the separation channel can be made in electrophoresis cell of dimensions of 9 mm by 9 mm, 18 mm by 18 mm, and 36 mm by 36 mm, but not limited thereof.
  • the separation channel for analytical electrophoresis would be integrated into the electrophoresis strip 205 and would be held in the strip holder 210 to produce the electrophoresis microplate as shown in Fig. 2.
  • the separation channel design would be integrated directly into the electrophoresis microplate.
  • Electrophoresis cells, strips or plates featuring the channel dimensions for capillary electrophoresis can be manufactured from a variety of materials such as silica, glass, acrylic, and other plastics, utilizing diverse fabrication techniques including chemical or laser etching, micromachining, hot stamping or embossing, laser etching, molding, or employing capillary tubes affixed to the electrophoresis cell as depicted in Fig. 10G, without being limited solely to these methods.
  • FIG. 11 the process for conducting electrophoresis within electrophoresis cells featuring micrometer-scale separation channels is depicted.
  • Fig. 11A provides a perspective view of the high-density serpentine channel 510 showcased in Fig. 10J, for enhanced comprehension of Figs. 11 B, 11C, 11 D, and 11 E.
  • the serpentine-shaped separation channel 510 represented in Fig. 11A has a channel width of 100 micrometers.
  • the gap between the parallel runs within the serpentine channel design measures 250 micrometers, yielding a total channel length of 50 cm.
  • Two side channels 1005, 1005’ link to the separation channel 510 and an input port forming a fluidic junction 1105 as illustrated in Fig. 11A.
  • a sample buffer comprising agents such as fluorescent dyes to stain sample constituents like DNA, RNA, proteins, or other constituents.
  • the sample buffer can include fiducial markers labeled with a fluorescent dye distinct in its fluorescent properties from the dye employed to label the sample constituents, facilitating differentiation between the signal from sample constituents and the fiducial markers. These fiducial markers can be tailored to avoid overlapping with the molecular weights of sample constituents, further distinguishing them from the sample constituents.
  • Figs. 11 B, 11 C, 11 D, and 11 E present simplified schematics of the process of performing analytical electrophoresis in the electrophoresis cell. As shown in Fig.
  • the process starts by placing the suspended sample into a sample chamber 1110 that is fluidically connected to the side channel 1005.
  • the sample is drawn into the side channel by capillary forces filling the fluidic junction 1105 between the two side channels 1005 and 1005’ and the separation channel 510.
  • the sample can also be drawn into the fluidic junction 1105 by applying a negative voltage to the first side channel electrode 1115 and a positive voltage to the second side channel electrode 1120. The movement of the sample results in the formation of a small sample plug within the fluidic junction 1105 as shown in Fig. 11 C.
  • the analytical electrophoresis process is then initiated by applying a negative voltage to the input electrode 705 positioned within the input chamber 515 and a positive voltage to the output electrode 710 situated within the output chamber 520 as shown in Fig. 11C.
  • the current flow is continued until the sample constituents undergo separation within the separation matrix present within the separation channel 510, as illustrated in Fig. 11 D.
  • a weak positive voltage can be applied to the two side electrodes 1115 and 1120 aiding in the withdrawal of the excess sample into the side chambers.
  • An imaging system captures images of the separation layer containing the separation channel and side channels. These images are then analyzed to determine the molecular weights of the separated sample constituents, utilizing reference points provided by the molecular weight fiducials 1130 depicted in Fig. 11 D. These fiducials, integrated into the sample and subjected to identical electrophoretic conditions as the sample, serve as markers for calibration during the electrophoretic separation process.
  • Fig. 11 A, 11 B, 11C and 11 D represents one among numerous alternative designs for generating a sample plug.
  • the side channels can be positioned as a staggered configuration, leading to the production of a longer sample plug at the fluidic junction 1105 as shown in Fig. 11 E. While electrophoretic separation ensues as previously described, the elongated plug dimensions in this configuration may potentially result in lower resolution. Nonetheless, this approach facilitates the sampling of a larger aliquot of the sample.
  • Two-dimensional gel electrophoresis is widely employed for the quantification and analysis of proteins, enabling the study of protein composition, modifications, and interactions within complex protein extracts.
  • This process entails two successive electrophoresis separations (Lee PY S.-A. N., 2020). Initially, sample constituents are separated based on their isoelectric points — the pH at which a protein carries no net charge and ceases migration within the gel. Subsequently, these resolved constituents are transferred to a second rectangular gel, where a second electrophoresis is carried out. In this subsequent stage, constituents previously separated by their isoelectric points are further segregated based on their molecular weight.
  • the implementation of the two-dimensional gel electrophoresis process within the electrophoresis cell involves the fabrication of a specialized cell featuring two layers of separation channels, as illustrated in Fig. 12.
  • the fluidic chamber layer 310 of the two-dimensional electrophoresis cell is comprised of the sample well 505, the input chamber 515, and two output chambers 520, 520’.
  • Three electrodes are positioned within the fluidic chamber layer 310, namely, the input electrode 705, and two output electrodes 710 and 710’.
  • the upper cover and lower cover are not shown in Fig. 12.
  • the fluidic chamber layer 310 is separated from the first separation layer 320 by the first fluidic port layer 315 depicted in Fig. 12B.
  • the first fluidic port layer represented in Fig. 12B encompasses four fluidic ports facilitating fluidic connections between the fluidic chamber layer 310 and the first separation channel layer 320.
  • the input chamber 515 is linked to the input end of the first separation channel 510 via the input port 610, while the sample well port 635 allows for the introduction of the sample into the sample well 505 located in the first separation channel 510 as shown in Fig. 12C.
  • the first output port 615 connects the first output chamber 520 to the output end of the first separation channel 510.
  • a second output port 615’ establishes a fluidic connection between the second output chamber 520’ and the output end of the second separation channels 510’.
  • the two-dimensional analytical electrophoretic process commences by introducing the prepared sample into the sample well 505 depicted in Fig. 12A.
  • the sample then descends by gravity through the fluidic ports layer 315 comprised of the sample well port 635 as shown in Fig. 12B into the corresponding sample well 505 located in the first separation matrix within the first separation channel 510, as illustrated in Fig. 12C.
  • An electrical current is established by applying a negative voltage to the input electrode 705 positioned in the input chamber 515 and a positive voltage to the output electrode 710 positioned in the output chamber 520, driving the sample constituents through the separation channel 510 from point ‘a’ to ‘b’ direction, as indicated by the arrow in Fig. 12C. Though not shown in Fig.
  • the fluidic chambers contain the required electrophoresis buffers and the separation channels contain the required separation matrix.
  • the separated sample constituents from the first electrophoretic separation step are localized in the first separation matrix in the first separation channel 510 as shown in Fig. 12C.
  • the separated sample constituents are transferred from the first separation channel layer 320 to the second separation channel layer 320’ comprised of a rectangular separation channel 510’ as shown in Fig. 12 E.
  • a negative voltage is applied to both, the input electrode 705 and the first output electrode 710 and a positive voltage is applied to a transfer electrode 1210 positioned at the beginning of the second separation channel 510’.
  • the resulting electric current transports the separated sample constituents from the first separation channel 510 shown in Fig. 12C through a narrow rectangular transfer port 1205 situated in the second fluidics port layer 315’ shown in Fig.
  • a negative voltage is applied to the transfer electrode 1210 located in the second separation channel layer 320’ while a positive voltage is administered to the second output electrode 710’ positioned in the second output chamber 520, located in the fluidic chamber layer 310, for a brief period. This facilitates the release of the sample constituents from the transfer electrode 1210.
  • the input electrode 705 and the first output electrode 710 are both connected to the same negative voltage with both electrodes 705, 710 assuming the role of a combined input electrode.
  • the positive voltage is applied to the second output electrode 710’ situated in the second output chamber 520’.
  • the sample constituents, previously separated in the first electrophoresis separation are now further segregated in the ‘c’ to ‘d’ direction, resulting in an increase in electrophoretic resolution from the sequential first and second electrophoretic separations.
  • the electrophoresis cell designed for two-dimensional electrophoresis can be adapted for various applications by employing separation matrices with differing compositions for the first and second electrophoretic separations.
  • isoelectric focusing of proteins in the sample is utilized for the first dimension, followed by electrophoretic separation by molecular weights for the second dimension.
  • Alternative methods, such as stacking sample constituents by isotachophoresis in the first dimension followed by molecular weight-based separation in the second dimension can be explored.
  • novel applications can be investigated within the two-dimensional format, such as separating intact cells or cell components in the first dimension followed by the separation of constituents within these cells or cell components in the second dimension.
  • Fig. 13 Illustrated in Fig. 13 is a schematic overview of a process for the electrophoretic separation and purification of sample constituents.
  • the electrophoresis cell incorporates a sample well.
  • a separate process for electrophoretic separation and purification within an electrophoresis cell devoid of a sample well is elaborated upon in Fig. 14.
  • Fig. 13A presents a top view of an example of an electrophoresis cell, aiming to enhance comprehension of the preparative electrophoretic process delineated in the simplified schematic depicted in Figs. 13B, 13C, 13D, 13E and 13F.
  • the sample Prior to initiating the preparative electrophoresis process, the sample is suspended in a sample buffer containing a thickening agent, such as glycerol or sucrose, along with a visible tracking dye and a fluorescent dye intended for staining sample constituents such as DNA, RNA, proteins, or other components.
  • a thickening agent such as glycerol or sucrose
  • the sample buffer can incorporate fiducial markers labeled with a distinct fluorescent dye from the one employed to label the sample constituents. These fiducial markers are strategically designed to prevent overlap with the molecular weights of the sample constituents.
  • Fig. 13A presents a simplified top view of a preparative electrophoresis cell comprising the sample well 505, input chamber 515, output chamber 520, first purification chamber 530, second purification chamber 535, input port 630, output port 650, first and second purification ports 640, 645, separation channel 510 and four electrodes, the input electrode 705, the output electrode 710, the first purification electrode 720, and the second purification electrode 725.
  • the prepared sample is introduced into the sample well 505, descending by gravity into the well in the separation matrix located in the separation channel 510, as illustrated in the simplified schematic top view in Fig. 13B.
  • An electrical current is applied between the input electrode 705 and the output electrode 710, with the output electrode 710 set at a more positive voltage relative to the input electrode 705, as depicted in the top view in Fig. 13B.
  • the resulting ion flow is conveyed between the input electrode 705 placed in the input chamber 515 through the input port 630 into the separation channel 510 through the output port 650 and to the output electrode 710 placed in the output chamber 520 as represented in Fig. 13A and 13B.
  • negatively charged constituents in the sample migrate through the separation matrix within the separation channel 510, at velocities contingent on their charge and size. Typically, smaller constituents exhibit greater velocities compared to larger ones, thereby effecting separation by size within the separation matrix 510.
  • the flow of electrical current is sustained until the sample constituents have traversed the length of the separation channel.
  • Images of the separating sample constituents are captured and analyzed to determine the molecular weights of the sample constituents with reference to the migration of fiducial markers of known molecular weights, electrophoresis is continued till a sample constituent designated for purification reaches the region in the separation channel adjacent to the purification chambers 530, 535 (or purification channels in other example configurations) as shown in Fig. 13D. Subsequently, the electrical current flow between the input electrode 705 and the output electrode 710 is stopped. A positive potential is applied to the first purification electrode 720 while a negative potential is applied to the second purification electrode 725 facilitating the migration of the designated component into the first purification chamber 530. Once the designated component has migrated into the first purification chamber 530 the voltage between the first and second purification electrodes is deactivated.
  • the voltage between the input electrode 705 and the output electrode 710 is reactivated, allowing the continuation of electrophoretic separation until the subsequent designated sample constituent enters the vicinity of the second purification chamber 535. At this point, the voltage between the input electrode 705 and output electrode 710 is deactivated. Subsequently, a positive voltage is applied to the second purification electrode 725 and a negative voltage is applied to the input electrode 705, driving the designated second component into the second Purification chamber. Any constituents not designated for purification remain within the separation channel 510 or collect in the output chamber 520 as shown in Fig. 13F.
  • the electrophoresis cell serves the dual function of either purifying constituents from the sample or removing/subtracting constituents from the sample.
  • purification scenarios users extract the purified component from the corresponding purification chamber.
  • subtraction scenarios users retrieve the sample with subtracted constituents from the output chamber 520.
  • a concentration electrode 730 is positioned just upstream of the separation matrix within the separation channel 510.
  • sample buffer containing a fluorescent dye intended for staining sample constituents such as DNA, RNA, proteins, or other components.
  • sample buffer can incorporate fiducial markers labeled with a distinct fluorescent dye from the one employed to label the sample constituents. These fiducial markers are strategically designed to prevent overlap with the molecular weights of the sample constituents.
  • Fig. 14A presents a top view of an example of an electrophoresis cell, aiming to enhance comprehension of the preparative electrophoretic process delineated in the simplified schematic depicted in Figs. 14B, 14C, 14D, 14E and 14F.
  • Fig. 14A presents a simplified top view of a preparative electrophoresis cell comprising the input chamber 515, output chamber 520, first purification chamber 530, second purification chamber 535, input port 630, output port 650, first and second purification ports 640, 645, separation channel 510 and five electrodes, the input electrode 705, the concentration electrode 730, the output electrode 710, the first purification electrode 720, and the second purification electrode 725.
  • Fig. 14B represents the commencement of the pre-concentration step in the preparative electrophoretic process.
  • a dilute sample is introduced into the input chamber 515, and the electrical current flow is activated between the input electrode 705, set at a negative voltage, and the concentration electrode 730, set at a positive voltage, inducing the migration of sample constituents onto the surface or surfaces of the concentration electrode 730.
  • the concentration electrode's shape can be in the form of flat plates oriented in the direction of ionic flow, a mesh, U-shaped, or hollow circular, rectangular, or triangularshaped rings, among other configurations, ensuring unimpeded passage of electrical current, ions, liquids, and sample constituents.
  • Fig. 14C represents concentration of the sample constituents onto the concentration electrode 730.
  • the electrical current between the input electrode 705 and the concentration electrode 730 is stopped when no additional decrease in fluorescence intensity is seen in the input chamber 515. Subsequently, the electrical current between the input electrode 705 and the output electrode 720 is activated, propelling the concentrated sample constituents from the concentration electrode 730 into and through the separation matrix, thereby effecting separation by size and charge.
  • Migration progress is monitored by an imaging system capable of detecting the migration of stained constituents through the matrix.
  • an imaging system capable of detecting the migration of stained constituents through the matrix.
  • the electrical current between the two purification electrodes 725, set at a negative voltage, and 720, set at a positive voltage is subsequently activated, driving the desired component into the first purification chamber 530 as represented in Fig. 14D.
  • the voltage between the two purification electrodes 720, 725 is then deactivated.
  • Fig. 14E and 14F schematically show the process of purifying a second sample constituent. To recommence the migration of sample constituents through the separation channel, the electrical current between the input electrode 705 and the output electrode 710 is reactivated, and electrophoretic separation continues until the subsequent desired sample constituent enters the area adjacent to the second purification chamber 535.
  • the current between the input electrode 705 and the output electrode 710 is deactivated, and the migration of the second desired sample constituent into the second purification chamber is achieved by activating the current between the input electrode 705, set at a negative voltage, and the second purification electrode 725 driving the desired second component into the second purification chamber 535. Any remaining sample constituents are retained in the separation channel 510 or can migrate into the output chamber 520 if electrophoresis is sustained.
  • the process outlined herein can be utilized to purify desired sample constituents, as depicted in Fig. 13 and Fig. 14, and can also serve to eliminate sample constituents from the sample by effectively directing undesired sample constituents into one of the "purification" wells, subsequently collecting the remaining sample constituents from the output well upon electrophoresis completion.
  • the electrophoresis cell provides a powerful way to purify one or more sample constituents from a sample or to ‘subtract’ one of more sample constituents from a sample with a straightforward pipette-in and a pipette-out process, resulting in a substantial reduction in process costs.
  • the purification or subtraction process is applicable to a broad array of samples, ranging from cells to molecules and be used at different scales up to milligrams of sample contingent upon channel and cell dimensions.
  • Affinity electrophoresis a process conducted in the presence of affinity reagents that selectively bind to specific sample constituents, is a powerful process that combines the specificity provided by affinity reagents with the separation capability of electrophoresis.
  • Affinity reagents encompass a diverse range of molecules, including but not limited to; chemical compounds targeting known structures; dyes that bind to specific molecules, macromolecules, cell organelles, or cells; DNA or RNA probes that hybridize with known nucleic acid sequences (Gebhard J, 2022); natural or modified proteins that bind to molecules, macromolecules, viral particles, or cells; and antibodies or aptamers that recognize specific antigens (Groff K, 2015); (Tao X, 2020).
  • the affinity reagents are bound to the separation matrix.
  • Oligonucleotides synthesized with an Acrydite group and commercially available from Integrated DNA Technologies Inc., can be copolymerized directly to acrylamide monomers (Kenney, Ray, & Boles, 1998) creating a separation matrix to which oligonucleotides are covalently attached and that can now bind to complementary nucleic acid sequences present in the sample.
  • These Acrydite-attached affinity reagents can include a non-limiting list of; oligonucleotides, aptamers, or molecules such as antibodies or glycans containing the Acrydite moiety, either directly or through an oligonucleotide intermediary.
  • the Acrydite-containing acrylamide can be mixed with other separation matrices such as agarose to form a composite matrix that would exhibit the affinity binding property.
  • Agarose can be conjugated with affinity reagents utilizing well-established techniques in the field.
  • agarose modified to contain N-hydroxysuccinimide (NHS) functional groups commercially available from Thermo Fisher Scientific as the Pierce NHS-Activated Agarose (Catalog number: 26196)
  • NHS-Activated Agarose catalog number: 26196
  • the Pierce CDI-activated Agarose Resin catalog number: 20259
  • agarose featuring activated epoxy groups enabling covalent binding with free amines, thiol groups, or hydroxyl groups, can be employed to attach a variety of affinity reagents.
  • Fig. 15A illustrates the derivatization of internal channel surfaces 1505 with a dendrimeric chemical linker 1510, enhancing the loading capacity of affinity reagents 1515 onto the channel surface 1505.
  • Fig. 15B demonstrates the immobilization of affinity reagents 1515 onto components of the separation matrix 1520.
  • Affinity reagents affixed to modified agarose or other matrices and/or to the Acrydite moiety can be leveraged to construct multiple zones equipped with affinity reagents tailored to identify specific constituents within samples, including but not limited to, viruses, bacteria, cells, cellular components, macromolecules, glycans, proteins, nucleic acids, and other molecular entities.
  • Fig. 15C shows a schematic representation of an electrophoresis cell for affinity electrophoresis.
  • the separation channel 510 comprises multiple detection zones denoted as a, b, c ... g, h, i, j. These detection zones comprise different affinity reagents capable of detecting different distinct components within the sample by associating or binding with said components.
  • the stratification of various zones within the separation channel is prepared by the initial deposition of an unmodified separation matrix within the separation channel and allowing it to polymerize or solidify. Subsequently, a separation matrix comprised of an affinity reagent is layered atop the unmodified zone and allowed to polymerize. This process is iterated to generate multiple detection zones, each featuring distinct affinity reagents, interspersed with unmodified separation matrix within the separation channel, as illustrated in Fig. 15C.
  • the sample constituents, labeled with fluorescent tags, are loaded into the input chamber 515, depicted in Fig. 15C.
  • An electrical current is applied between the input electrode 705, set at a negative voltage, and the concentration electrode 730, set at a positive voltage, until the sample is concentrated onto the concentration electrode 730, as illustrated in Fig. 15D.
  • the electrical current is applied between the input electrode 705, set at a negative voltage, and the output electrode 710, set at a positive voltage, driving the migration of sample constituents into the separation channel 510.
  • the input electrode 705 set at a negative voltage
  • the output electrode 710 set at a positive voltage
  • zones ‘a’ to ‘j’ may contain immobilized affinity reagents designed to recognize and bind to various viral species and variants, with zones ‘a’ and ‘j’, for example, serving as calibration controls, while zones ‘b’ to T target different coronaviruses. Imaging of the electrophoresis channel post-electrophoresis enables rapid assessment of the presence of different viral species and variants within a sample, offering a quantitative readout without the necessity for complex sample preparation methods.
  • the method ensures high specificity in detecting target molecules, such as viruses, bacterial cells, mammalian cells, cell components, macromolecules, DNA, RNA sequences, proteins and other molecules of interest. This specificity minimizes false positives and enhances the accuracy of the assay.
  • Quantitative Analysis The correlation between fluorescent intensity and the concentration of specific sample constituents allows for quantitative analysis. This quantitative readout provides valuable information about the relative abundance of target constituents within the sample.
  • Rapid Assessment The method enables rapid assessment, offering a timely evaluation of the presence and concentration of target constituents. This rapid turnaround time is particularly advantageous in scenarios requiring prompt decision-making or screening of large sample sets.
  • the affinity electrophoresis approach can be integrated with automated systems for high-throughput analysis, further increasing efficiency and scalability.
  • affinity electrophoresis with the disclosed electrophoresis cell, strip and plate architectures and associated processes offers a powerful and efficient method for precise, quantitative, and rapid analysis of target molecules in complex samples, with potential applications in diverse fields such as clinical diagnostics, pharmaceutical research, and environmental monitoring.
  • the manufacturing methods utilized for laboratory multi-well strips and microplates can be effectively applied to produce electrophoresis cells, strips and electrophoresis microplates described herein. These methods encompass various techniques compatible with industrial manufacturing processes, ensuring scalability and cost-effectiveness in production.
  • Some of the components can be manufactured from materials compatible with electrophoretic separations and can include a non-limiting list of; glass, quartz, thermoplastics or thermosetting plastics such as polypropylene, acrylic, polycarbonate, polylactic acid, cyclic olefin copolymer, cyclic olefin polymer, polyethylene, or other polymeric plastics or composite plastic materials.
  • materials compatible with electrophoretic separations can include a non-limiting list of; glass, quartz, thermoplastics or thermosetting plastics such as polypropylene, acrylic, polycarbonate, polylactic acid, cyclic olefin copolymer, cyclic olefin polymer, polyethylene, or other polymeric plastics or composite plastic materials.
  • the holding frame can be manufactured from a metal such as aluminum or metal alloys or from composites such as plastic composites or other materials such as ceramics, that are well known in the art. These processes offer precise control over dimensions and material composition, facilitating customization for diverse electrophoretic applications.
  • Electrophoresis cells, strips, and microplates can be manufactured either as planar layers and subsequently assembled by joining individual units to form larger units, or as integrated single units.
  • electrophoresis cells into microplates can benefit from automation and robotics, streamlining production and ensuring consistency.
  • Techniques such as ultrasonic welding, adhesive bonding, or heat sealing can be employed to securely integrate the individual components of electrophoresis cells, guaranteeing leakproof operation.
  • Addition of components to the electrophoresis cells such as separation matrix, electrodes, and buffers can be performed on a serial assembly line wherein each component is added, allowed to polymerize or solidify in the case of the separation matrix, electrodes placed in the specified location and finally filled with remaining fluid components before sealing of the electrophoresis strips and plates followed by the attachment of human and machine-readable information, packaging and storage.
  • the electrophoresis strips and plates would be tested for performance by an initial physical examination based on high-throughput machine vision processes followed by random sampling for functional tests.
  • the strips or plates are then combined with other components to produce an application specific kit.
  • the electrophoresis instrument described herein and as shown in the simplified structural layout of an example in Fig. 16, is designed to operate the electrophoretic processes on the electrophoresis microplate. Comprising various components and functionalities, this instrument offers precise control and monitoring capabilities, ensuring efficient electrophoresis across multiple samples simultaneously while supporting the independent operation of each electrophoresis cells.
  • electrophoresis instruments can comprise capabilities to process multiple electrophoresis microplates serially or in parallel and can be part of a larger automation system or a standalone system.
  • the electrophoresis instrument can comprise the following non-limiting list of functional modules: An electrophoresis-microplate-compartment 1605, a liquid handling module 1610, an electrode interface module 1615, an illumination module 1620, an imaging module 1625, a control module 1630, an analysis module 1635.
  • the electrophoresis instrument can include user interface elements integrated with the instrument or provided separately such as a remotely accessed computer. These modules are described further in the sections below.
  • the electrophoresis-microplate-compartment 1605 shown in the schematic of the electrophoresis instrument in Fig. 16, facilitates the loading, positioning and unloading of the electrophoresis microplate 105.
  • the microplate compartment provides unobstructed visibility to the bottom of the electrophoresis cells to perform imaging.
  • thermophoretic compartment Within the microplate compartment, temperature regulation elements are incorporated to sustain an optimal temperature conducive to electrophoretic separation. Moreover, the compartment is equipped with a diverse array of sensors, including but not limited to, detecting microplate positioning, weight, and temperature. These sensors contribute important data points utilized by the control module to effectively monitor the conditions of the electrophoretic run.
  • the electrophoresis microplate compartment can be accessed through a lift-up lid configuration, providing convenient access.
  • the compartment can be accessed through a sliding tray mechanism, providing access to loading and unloading of microplates for robotic devices, but not limited thereto.
  • the electrophoresis instrument can comprise a liquid handling module 1610, as shown in Fig. 16. This module facilitates the precise placement and removal of samples from within the electrophoresis cells of the electrophoresis microplate.
  • the liquid handling module can be integrated within the electrophoresis instrument to provide a standalone instrument or the liquid handling function can be provided by external liquid handling automation wherein the electrophoresis instrument would interface directly with the external liquid handling device.
  • the liquid handling module can aspirate samples from a source liquid container that can be in a microplate format and dispense said samples into the appropriate location in the electrophoresis cells before the start of the electrophoresis process.
  • the liquid handling module would remove purified sample constituents, if required by the specified application, from the electrophoresis cells and dispense them into receiving liquid containers that can be in the form of a microplate.
  • Integrating a liquid handling capability into the electrophoresis instrument offers several significant benefits by reducing manual intervention, streamlining the workflow and minimizing the risk of errors associated with manual pipetting. This efficiency gains time for researchers, allowing them to focus on other critical tasks.
  • the liquid handling module enables high-throughput processing of samples, allowing multiple samples to be handled simultaneously or in rapid succession. This capability is particularly advantageous in laboratories with large sample volumes or where time-sensitive experiments are conducted. Minimizing manual pipetting reduces the risk of sample contamination, ensuring the integrity of experimental results. Closed liquid handling systems further mitigate contamination risks by preventing exposure to external contaminants.
  • the liquid handling module can integrate with laboratory information management systems for efficient tracking of samples, reagents, and experimental parameters, enhancing data integrity and traceability.
  • the electrophoresis instrument comprises of an electrode interface module 1615, as shown in Fig. 16, that forms an electrical connection with a multitude of components on the electrophoresis cell such as; the fluidic chambers, the separation channels, and/or the fluidic ports. In some examples, five electrical connections are required for each electrophoresis cell with up to 480 electrical connections for an electrophoresis microplate with 96- electrophoresis cells.
  • the electrode interface module comprises electrical contacts 1617 that penetrate through the top layer of the electrophoresis cells, into the fluidic chambers, channels and ports or, that engage disposable electrodes positioned within the electrophoresis cells.
  • Electrodes can be enclosed within insulating sleeves providing partial electrical insulation.
  • An electro-mechanical assembly within the electrode interface is configured to move the multiple electrodes into contact with corresponding locations that can contain a rotatable, movable and/or retractable mechanism to interface the electrodes with different formats of electrophoresis microplates.
  • the mechanism can provide adjustable inter-electrode spacing for use with electrophoresis strips containing cells sized 9 mm by 9 mm, 18 mm by 18 mm, and/or 36 mm by 36 mm, but not limited hereto.
  • the illumination module 1620 can comprise or consist of one or multiple light sources, as depicted in Fig. 16.
  • the light sources can be placed tangential to and below the microplate, in the same plane, and/or positioned above it.
  • the illumination module 1620 is fitted with illumination components 1622 designed to illuminate the electrophoresis microplate 105 for detecting sample constituents based on fluorescence, reflectance, or absorbance in the electrophoresis cell, strip, or microplate.
  • illumination components including but not limited to, laser diodes, LEDs, and/or fluorescent lamps, can be used. These illumination components may incorporate light shaping, light diffusion and light wavelength selection elements 1623 to maximize the signal generated from the sample constituents present in the electrophoresis cell, strip or microplate.
  • the electrophoresis instrument shown in Fig.16 comprises an imaging module 1625 to detect and monitor the process of electrophoresis of sample constituents based on the optical properties associated with said components, such as fluorescence, luminescence and absorbance of light.
  • the imaging module can be placed below the plane of the electrophoresis microplate as shown in Fig. 16, placed tangential to the microplate, or can be placed above the microplate. More than one imaging modules can be used with one placed below and one above the plane of the electrophoresis microplate, but not limited hereto.
  • the imaging module 1625 can be comprised of one or a multitude of cameras 1627 with each camera comprised of an imaging sensor, lenses, and optical filters.
  • the imaging sensor can comprise one or a multitude of point, line or two-dimensional imaging sensors such as CMOS cameras, Electron-Multiplying Charge-Coupled Devices (EMCCDs), avalanche photodiodes (APDs), or photomultiplier tubes (PMTs), that can be stationary or part of a scanning system, but not limited hereto.
  • the imaging sensor can include optical elements such as zoom and focusing lenses to expand the flexibility of the imaging system.
  • a multitude of cameras can provide higher spatial resolution (Duparre JW, 2020) with each camera imaging a fractional area of the microplate.
  • an array of 6 cameras arranged in a rectangular grid of 2 rows and 3 columns can be used to image the microplate with each camera capturing an image of 1/6th of the electrophoresis microplate with a resultant 6x increase in resolution.
  • the cameras can be set up to detect light from one or a multitude of wavelengths.
  • the imaging module can comprise fixed or switchable light wavelength selection filters 1629 serving to select specific wavelengths to be detected by the sensor.
  • light can be detected with a color (Red-Green-Blue) imaging sensor or other modalities that provide multispectral images (He X, 2020).
  • the imaging module 1625 captures a sequence of images of the electrophoresis cells during the electrophoretic process including but not limited to: a. The light intensity incident on the microplate or strips; b. The machine-readable information imprinted on the microplate, strips, or cells; c. The light intensity within the input chamber and at the concentration electrode to monitor the concentration; d. The distribution of light intensities along the separation channels to monitor the migration of components in the channels; e. The light intensities within the input, side, purification and output chambers to monitor movement of components to or from the chambers; f. The light intensities at the fluidic ports.
  • the imaging module is controlled by and provides image data to the analysis and control modules based on requests from the analysis and control modules.
  • the control module 1630 embedded in the electrophoresis instrument as shown in Fig. 16, regulates various aspects of methods and systems of the present disclosure.
  • the control module can capture sensor data comprising temperature of the electrophoresis microplate compartment, electrode voltages, electrophoresis current, illumination intensities, positions of the microplate, microplate compartment, electrode interface and other sensors to minimize electro-mechanical and optical failures.
  • the control module comprises low-voltage power supplies to supply the analysis and control modules, electro-mechanical devices, illumination module, imaging module, and user interfaces.
  • the control module comprises power supplies for electrophoresis, that can include one or a multitude of constant current, constant voltage, or pulsed power supplies with built in measurement functions for current, voltage, time, and temperature.
  • the control module in cohort with the imaging and analysis module dynamically adjusts the electric field across the electrodes during electrophoretic migration.
  • the electrical current can switch polarity at a defined frequency forcing the sample constituents to frequently reverse the direction of migration resulting in them traversing greater distances than provided by the channel length.
  • the switching characteristics can be adjusted to provide a net forward movement from the input electrode to the output electrode.
  • An offset electrical potential can be applied to some of the electrodes to establish a current flow opposing the separation and purification migration direction to reduce undesired leakage of untargeted species into a channel.
  • control module By modulating the voltage and directionality of the electric current, the control module can enhance separation efficiency, isolate targeted bands for purification, or alter the separation conditions in response to real-time data. This is a powerful and novel feature of the disclosures herein, because it can compensate for small variations in buffer, matrix, cell, and electrical components from cell to cell and run to run over time and can achieve better controlled separations and purifications.
  • the control module 1630 includes a central processing unit 1705, shown in Fig. 17, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the control module 1630 also includes memory or memory location 1710, electronic storage unit 1715, communication interface 1720 for communicating with one or more other systems, and peripheral devices 1725, such as other memory, data storage and/or electronic display adapters.
  • the memory 1710, storage unit 1715, interface 1720 and peripheral devices 1725 are in communication with the central processing unit 1705 through a communication bus.
  • the storage unit 1715 can be a data storage unit for storing data.
  • the control module 1630 can be operatively coupled to a computer network 1730 with the aid of the communication interface 1720.
  • the network 1730 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 1730 in some cases with the aid of the control module 1630, can implement a peer-to-peer network, which may enable devices coupled to the control module 1630 to behave as a client or a server.
  • the central processing unit 1705 can execute a sequence of instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 1710.
  • the instructions can be directed to the central processing unit 1705, which can subsequently program or otherwise configure the central processing unit 1705 to implement methods of the present disclosure.
  • the central processing unit in the control module can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the control module can be included in the circuit.
  • the circuit is an application specific integrated circuit.
  • the storage unit 1715 can store files, such as drivers, libraries and saved programs.
  • the storage unit 1715 can store user data, e.g., user preferences and user programs.
  • the control module 1630 in some cases can include one or more additional data storage units that are external to the control module, such as located on a remote server that is in communication with the control module through an intranet or the Internet.
  • the control module can communicate with one or more remote control modules through the network 1730.
  • the control module can communicate with a remotecontrol module of a user.
  • the user can access the control module via the network 1730.
  • Methods as described herein can be implemented by way of machine executable code stored on an electronic storage location of the control module, such as, for example, on the memory 1710 or electronic storage unit 1715.
  • the machine executable code can be provided in the form of software.
  • the code can be executed by the processor 1705.
  • the code can be retrieved from the storage unit 1715 and stored on the memory 1710 for ready access by the processor 1705.
  • the electronic storage unit 1715 can be precluded, and machine-executable instructions are stored on memory 1710.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • control module 1630 can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical networks and over various air-links.
  • the physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software.
  • Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the control module can include or be in communication with an electronic display 1735 that comprises a user interface 1740 for providing, for example, electrophoresis analysis information to a user.
  • a user interface 1740 for providing, for example, electrophoresis analysis information to a user.
  • user interface include, without limitation, a graphical user interface and web-based user interface.
  • control module The dynamic interface between the control module and the different electrophoresis instrument modules enables precise control and regulation of analytical, preparative and affinity electrophoresis steps ensuring consistent and reproducible results across experiments.
  • An analysis module 1635 is comprised of microcontrollers, microprocessors, and the like; memory devices for storing data and/or instructions, and algorithms to analyze information from the imaging and other sensors.
  • the module incorporates algorithms tailored for analytical, preparative, and affinity electrophoresis, for diverse sample types. These algorithms optimize electrophoretic conditions, enhancing separation efficiency and resolution.
  • the analysis module processes the machine-readable information associated with each electrophoresis cell to identify cell-specific attributes such as; separation channel dimensions and design, chamber dimensions, separation gel matrix, fiducial markers, reagent contents, application compatibility and other information.
  • Images captured during the electrophoretic process are analyzed by analysis algorithms to determine sample concentration, separation of sample constituents, and purification of sample constituents, but not limited hereto.
  • the sample concentration algorithm controls the electrical potential between the input electrode and the purification electrode by monitoring the light emission or light absorbance signal in the input chamber and at the concentration electrode. The electrical potential is maintained till the signal has plateaued at the concentration electrode.
  • the sample concentration algorithm then passes control to a separation-and-molecular- weight identification algorithm that processes the set of images acquired during the electrophoretic separation while controlling the electrical potential between the input electrode and the output electrode.
  • FIG. 18 A non-limiting exemplary overview of an algorithm to analyze migrating sample constituents is represented in Fig. 18.
  • Fig. 18A shows a representation of one of a sequence of images of the electrophoresis microplate with eighteen electrophoresis cells.
  • the image of the whole electrophoresis microplate is converted into multiple images of individual cells.
  • An image representation of the individual cell is shown in Fig. 18B.
  • the images of the cell are masked to remove all non-required image areas such as the interstitial spaces between the separation channels.
  • the masked images are pre-processed to reduce noise and enhance the image for post-processing as represented in Fig. 18C.
  • the enhanced image is processed to convert the serpentine, helical or other channel shape into a linear format while retaining the spatial locations of the optical signals or bands within the channel as shown in Fig. 18D.
  • Fig.18E represents the end-result of the process to calculate the molecular weight of each of the bands in the sample.
  • the molecular weight is calculated based on the migration distance of said band in comparison to the migration distance of the fiducial markers of known molecular weights.
  • the fiducial markers can have non-overlapping locations to the sample constituents and/or can be labeled with a different optical signal to distinguish the molecular weight fiducials from the sample constituents.
  • predictive analysis models can be used to determine the time at which a sample constituent to be purified enters the purification zone adjacent to the purification channel or chamber.
  • the separation and molecular weight algorithm switches to a purification algorithm that switches the electrical potential from the input and output electrode to the pair of purification electrodes. Migration of the desired sample constituent into the purification chamber is monitored by the imaging module. On completion of the purification module, a maintenance algorithm is activated to prevent diffusion or further migration of the purified materials.
  • a set of data compilation, analysis, and reporting algorithms synthesize the outcomes of the electrophoretic process for every electrophoresis cell, compiling the identified band identities, the conditions under which separation and purification is achieved, the signal intensities for every band or affinity zone, and any deviations or anomalies observed during the run. This comprehensive report provides valuable insights into the results of the electrophoresis process.
  • electrophoresis Instrument to perform analytical and preparative electrophoresis on the electrophoresis microplate with high precision, throughput, and automation compatibility for up to 96 electrophoresis cells in a single run.
  • This instrument represents a versatile and efficient tool for a wide range of electrophoretic applications in research, diagnostics, and biotechnology.
  • Fig. 19 shows an overview of the process to operate the electrophoresis instrument for purification of DNA fragments.
  • the electrophoresis instrument embodied in this workflow does not contain the fluidics module.
  • the process can be divided into two sets of actions, those performed by the user and those performed by the electrophoresis instrument.
  • Fig. 19A summarizes the preliminary steps performed by the user.
  • the user may have performed a library prep procedure for sequencing on a next generation sequencing platform and needs to remove primers, adapters, adapter-dimers from the reaction products commonly referred to as a “clean-up” process in the art.
  • the user mixes the library preparation reaction mix with an aliquot of a 10x sample loading buffer provided in the kit.
  • the sample loading buffer contains a fluorescent staining dye that binds to the DNA strands and molecular weight fiducial markers, in addition to other components required for loading and electrophoretic separation.
  • the prepared sample is then loaded into the electrophoresis cell of an electrophoresis strip, provided in the kit, and the strip is placed in the holding frame.
  • the holding frame with electrophoresis strips forms the electrophoresis microplate.
  • the user then places the electrophoresis microplate into the electrophoresis-microplate-compartment in the electrophoresis instrument. The user initiates the plate loading operation from the user interface.
  • Fig. 19B The instrument action now commences; the microplate compartment positions the electrophoresis microplate to be imaged by the imaging module.
  • the control module reads the sensors associated with the microplate compartment to confirm the number of strips loaded.
  • the control module then turns on the illumination module and the imaging module capturing an image of the bottom of the electrophoresis strips.
  • the analysis module in the electrophoresis instrument translates the machine-readable information on each electrophoresis cell and enters the information into a database.
  • Fig. 19C The system then prompts the user to enter information about the sample and the required run conditions.
  • Fig. 19D The user responds to the instrument prompt by entering the required sample and run parameters for each electrophoresis cell into the user interface.
  • the parameters can include sample specific information for each electrophoresis cell, application specific information such as molecular weights to be isolated or removed from the sample, but not limited thereof.
  • the required information may be performed on a remote interface by direct entry or by importing from pre-existing data in a tabular format to populate the required fields in the database.
  • the electrophoresis instrument captures the user provided information into the database and the analysis module compares the provided information with the machine- readable information for the cell, to validate that the electrophoresis cell is compatible with the sample, application, analysis, purification and other information entered by the user.
  • Fig. 19F The user approves the validation provided by the instrument and initiates the run by clicking “Start”.
  • the control module within the electrophoresis system guides the placement of the appropriate electrode array in the electrode interface module to be placed in contact with the requisite compartments within the electrophoresis cells.
  • the control module activates the required voltages to specific electrodes based on the cell and applicationspecific algorithms.
  • Fig. 19H The control module co-ordinates the illumination module, the imaging module, and the analysis module to perform the first step, sample concentration.
  • the input chamber is imaged to determine the level of fluorescence.
  • the electrical potential between the input electrode and the concentration electrode is turned on and the decrease in fluorescence in the input chamber is monitored till no further decrease in fluorescence levels is seen indicating that sample concentration onto the concentration electrode is complete
  • the control module switches to the electrophoretic separation algorithm, with the electric current being switched to the input and output electrodes - driving the sample constituents into the separation matrix in the separation channel. Separation is monitored by the imaging module and molecular weights are calculated by the analysis module.
  • Fig. 19J The results of the molecular weight analysis are displayed on the user interfaces for the user to review.
  • the user may choose to change the purification parameters based on the results of the electrophoretic separation or may approve the next stage, removal of DNA fragments of a defined size range.
  • the control module activates the purification algorithms wherein, separation is monitored till the required molecular weight bands reach the purification zone in the electrophoresis cell defined by the location of the purification channels and/or purification chambers within the electrophoresis cell.
  • the relevant electrodes are activated and the migration of the selected bands into the purification chambers is monitored by the imaging and analysis modules. Once the purification process is complete, the user is informed and instructed to remove the electrophoresis strip.
  • the instrument can keep the electrodes activated at reduced electric potentials to eliminate diffusion of the bands and prevent migration of purified materials.
  • Fig. 19L The user requests the plate compartment to release the electrophoresis microplate and removes the microplate.
  • the user would remove the ‘purified’ sample from the output well as the sample will have been subjected to the removal of primers and dimers from the sample.
  • kits that are comprised of materials and reagents necessary for performing analytical or preparative electrophoresis.
  • the kit can be labeled for a specific use: As examples; for high- resolution analysis of DNA, for purification of DNA fragments, or for detection of Coronavirus sub-types, but not limited hereto.
  • the kit can be comprised of sample preparation reagents comprised of sample suspension reagents, sample labeling reagents, reference fiducial reagents, instructions, but not limited hereto.
  • the kit can be comprised of electrophoresis strips specific to the purpose of the kit; analytical, preparative or affinity electrophoresis analysis of a specific sample type as described herein.
  • the electrophoresis strip is comprised of electrophoresis cells comprised of separation matrix, electrophoresis buffers, embedded electrodes and other components as needed for the specific purpose.
  • the kit can include a second, third or other additional container into which the additional components may be separately placed.
  • the kit may include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
  • the independent electrophoresis cell architecture presents a distinct advantage in its adaptability to specific applications for sample separation, analysis, or purification, eliminating the need for dedicated infrastructure tailored to each application.
  • the disclosed architecture allows for versatile utilization across a spectrum of tasks, from sample analysis to component purification. Samples encompassing small molecules, nucleic acids, proteins, macromolecular complexes, sub-cellular components, viruses, and cells can all be effectively processed. Every aspect of the electrophoresis cell design, including fluidic chambers, ports, and separation channels, can be tailored to suit various applications.
  • Prototypical electrophoresis cells are designed using Adobe Illustrator or Xara Photo & Graphics Designer 2D Graphics Software. Designs are exported as DXF or SVG files and cut as layers from either clear or opaque acrylic sheets using a laser cutter. The designs typically comprise three layers; the first layer of fluidic chambers, followed by the layer housing fluidic ports, and finally, the layer dedicated to the separation channel. The electrophoresis cell is fabricated by aligning and fusing the three layers together using acrylic cement.
  • the base of the electrophoresis cell is sealed using MicroAmpTM Optical Adhesive Film for 96-well plates, procured from Thermo Fisher Scientific (Catalog number: 4360954).
  • Separation matrices are suspended in standard electrophoresis buffers such as sodium phosphate buffer pH 7.2, Tris borate EDTA pH 8, Tris acetate ETDA pH 8, or sodium borate buffer pH 7.4, and may be supplemented with dyes for the staining of DNA, RNA, or proteins.
  • the separation channel is filled through the output port of the electrophoresis cell, and allowed to polymerize or solidify. Fluidic chambers are filled with electrophoresis buffers. Finally, the top of the cells are sealed utilizing the optical adhesive film and stored till used.
  • Electrophoretic concentration of dyes Fig. 20 shows an image of three separate electrophoresis cells in which the process for the concentration of three dyes was performed.
  • the separation channels 510 within the cells are filled with a 1% agarose suspension in 25 mM sodium phosphate buffer at pH 7.2, and solidified at 4°C.
  • the input chamber 515, output chamber 520, and side chambers 525 are filled with 25 mM sodium phosphate buffer at pH 7.2.
  • a cylindrical graphite electrode of 0.7 mm diameter is placed in the input chamber 515, away the input port 610.
  • the concentration electrode a cylindrical graphite rod of 0.7 mm diameter, is enveloped in an electrically insulating sleeve, leaving approximately 1 mm exposed at one end. This concentration electrode is situated within the input port 610, with the exposed 1 mm of graphite penetrating into the separation matrix at the origin of the separation channel 510.
  • a direct current (DC) voltage of 50V is applied between the input electrode set at the negative potential and the concentration electrode set at the positive potential, with the constant current limit set at 5 mA.
  • the color intensities of the food dyes decrease within the input chamber, accompanied by a proportional enhancement in color intensity at the tip of the concentration electrode.
  • Cells are photographed under ambient lighting conditions, after removing the electrodes, revealing the concentrated dyes at the juncture of the input port and the beginning of the separation channel 2001 , as depicted in Fig. 20 showing successful concentration, an advantage of the electrophoresis cell architecture.
  • Fig. 21A shows an image of the electrophoresis of dyes in a spiral separation channel 510 containing a sample well 505.
  • the electrophoresis cell measuring 36 mm by 36 mm, was made from clear acrylic sheets for the chambers and separation channel layers, and from opaque white acrylic for the fluidic ports layer, as described previously.
  • the separation channel 510 is filled with a 1 % agarose solution in 25 mM sodium phosphate buffer at pH 7.2.
  • the input chamber 515, output chamber 520, and side chambers 525 are filled with 100 pl of 25 mM sodium phosphate buffer at pH 7.2.
  • a mixture comprising five dyes suspended in a sample buffer containing 3% glycerol in 25mM sodium phosphate buffer is deposited within the sample well 505.
  • the input electrode, placed in the input chamber 515, is connected to a negative voltage, while the output electrode, placed in the output chamber 520, is connected to a positive voltage of 100VDC with the constant current limit set at 10 mA.
  • Both electrodes are made of a platinum wire. Passage of electric current results in the migration of the dyes through the spiral separation channel. At the end of the run, the electrodes and buffers are removed, the cell is inverted and photographed under ambient lighting conditions, with the results depicted in Fig. 21A.
  • Fig. 21 B displays an image depicting the electrophoretic separation of a DNA size ladder obtained from New England Biolabs in a straight separation channel 510 with a sample well 505 positioned within it.
  • the electrophoresis cell measuring 36 mm by 36 mm, was made with acrylic, following the previously outlined methodology.
  • the separation channel 510 is filled with a 1 % agarose suspension in 25 mM sodium phosphate buffer at pH 7.2, and solidified at 4°C.
  • the output chamber 520 is filled with an electrophoresis Buffer comprised of a AmberChromTM 50WX2 200-400 Mesh (H+) cation exchange tesin from Millipore-Sigma (Cat. No. 217476-100G) saturated with 25mM sodium phosphate buffer at pH 7.2 suspended in agarose to a final concentration of 0.5%.
  • a SYBR Green staining dye is added to the saturated resin to a final concentration of 1 :10,000 of the commercial solution.
  • the input chamber 515 is filled with an electrophoresis buffer comprised of AmberTecTM UP550 OH anion exchange resin from Millipore-Sigma (Cat. No. 1015-U) saturated in 25 mM sodium phosphate buffer at pH 7.2 and suspended in agarose to a final concentration of 0.5%.
  • an electrophoresis buffer comprised of AmberTecTM UP550 OH anion exchange resin from Millipore-Sigma (Cat. No. 1015-U) saturated in 25 mM sodium phosphate buffer at pH 7.2 and suspended in agarose to a final concentration of 0.5%.
  • the input electrode made of a platinum wire and placed in the input chamber 515, is connected to the negative voltage terminal of a power supply, while the output electrode, also made of a platinum wire and placed in the output chamber 520, is connected to the positive terminal of the power supply.
  • a direct current (DC) voltage of 100V is applied, with the constant current limit set at 10 mA.
  • Separation of the DNA fragments is monitored by illuminating the electrophoresis cell with 465 nm wavelength light from high-power LEDs, covered with a blue light filter, to excite the SYBR Green DNA stain. Fluorescent emission is captured with a camera through an amber filter that blocks the 465 nm light but passes the 550 nm fluorescent emission.
  • Electrophoresis proceeds until adequate separation of DNA fragments is achieved within the separation channel. Subsequent to the removal of electrodes and buffers from the fluidic chambers, the cell is inverted, placed on the blue light source, and photographed using the amber filter to obstruct the exciting blue light and detect the fluorescent emission. The obtained results are depicted in Fig. 21 B.
  • RNA analysis is performed in the assembled electrophoresis cell with the separation channel filled with 1 % agarose dissolved in RNAse free 25mM Sodium phosphate buffer at pH 7.2 to which a fluorescent RNA staining dye, SYBR Green II from Thermo Fisher (Cat. No. S7568), and an RNAse inhibitor from Thermo Fisher (Cat. No. N8080119) is added at manufacturer recommended concentrations.
  • RNAse free 25mM Sodium phosphate buffer at pH 7.2 to which a fluorescent RNA staining dye, SYBR Green II from Thermo Fisher (Cat. No. S7568), and an RNAse inhibitor from Thermo Fisher (Cat. No. N8080119) is added at manufacturer recommended concentrations.
  • the agarose is allowed to solidify at room temperature.
  • the input and output chambers are filled with the same ion exchange electrophoresis buffer as described for the electrophoretic separation of DNA.
  • RNA Century Plus size standard from Thermo Fisher Scientific (Cat. No. AM7145) is denatured by heating a mixture of 1 pL of the RNA preparation mixed with 10 pL of a sample loading buffer (sodium phosphate, pH 7.2, 2 mM EDTA, pH 8.0, 4 M formaldehyde, 60% formamide, 5% glycerol, 0.025% bromophenol blue) at 75°C for 5 min and chilling immediately in an ice bath.
  • the denatured samples are placed into the sample well of the electrophoresis cell and electrophoretic separation is carried out at 50V.
  • the separated fragments are visualized with blue light excitation and photographed with an amber filter that allows passage of the fluorescent wavelengths emitted by the SYBR Green II dye bound to RNA fragments while blocking the exciting blue light.
  • Proteins from 6 kilodaltons to over 200 kilodaltons can be resolved in an electrophoresis cell with a separation matrix prepared from MetaPhor XR agarose, available from FMC BioProducts, dissolved in an electrophoresis buffer composed of 0.5M Tris, 0.2 M boric acid, 1 mM EDTA, adjusted to pH 7.8 with HCI at a 7% agarose concentration (Wu M, 1998). Urea is added to the molten agarose to a final concentration of 1 M and the solution is reheated to maintain the molten state.
  • the separation channel is filled with the separation matrix to about 80% of the length of the channel with a 20% gap being retained in the separation channel adjacent to the input chamber.
  • a stacking gel composed of molten 1 % agarose in a stacking buffer composed of 0.125 M Tris-HCI, pH 6.4 is layered on top of the solidified separation matrix in the separation channel filling the remaining 20% length of the separation channel.
  • the electrophoresis cells are chilled to 4°C for 30 min to complete gelation of the separation matrix.
  • Samples are mixed with a sample buffer composed of final concentrations of 10% glycerol, 0.125 M Tris-HCI, pH 6.8, 2% SDS, 1% 2-mercaptoethanol, 0.0005% bromophenol blue, and Thermo Scientific Krypton Protein Stain (cat no. 46628).
  • the protein samples are separated by electrophoresis in an electrophoresis buffer composed of 89 mM Tris, 89 mM boric acid, 2 mM EDTA, 0.1% SDS.
  • Fig. 22 shows the purification of a food dye from a mixture comprising two distinct food dyes.
  • the prototypical electrophoresis cell 110 measuring 36 mm by 36 mm, is assembled from layers of transparent acrylic, as previously outlined.
  • the separation channels 510 within this cell are filled with a 1 % agarose solution in 25 mM sodium phosphate buffer at pH 7.2.
  • the input chamber 515, output chamber 520, and the two purification chambers 530, 535 are filled with 100 pl of 25 mM sodium phosphate buffer at pH 7.2.
  • the input electrode a cylindrical graphite rod of diameter 0.7 mm, is positioned within the input chamber 515, farthest from the input port 610.
  • the concentration electrode made from a similar graphite rod, is covered with an electrically insulating sleeve but leaving 1 mm exposed at the end of the electrode that is inserted in the input port 610.
  • the output electrode made from a similar graphite rod of 0.7 mm diameter, positioned within the output chamber 520, is connected to the positive terminal of the power supply and the negative terminal is connected to the concentration electrode for 10 sec to release the dyes bound to the concentration electrode.
  • the concentration electrode is disconnected from the power supply, and the negative terminal of the power supply is now connected to the input electrode in the input chamber, and current is applied until the dye mixture migrates into the separation channel, as shown in Fig. 22A and 22B. Electrophoresis is continued till the red dye advances beyond the intersection of the separation channel and the purification channels 540, 542, as shown in Fig. 22C at which point the input and output electrodes are disconnected from the power supply.
  • the blue dye is moved into the first purification chamber 530, as shown in Fig. 22D, by setting the first purification electrode in the first purification chamber 530 at a positive voltage and the second purification electrode in the second purification chamber 535 at a negative voltage with the same voltage settings as before (75V DC with a constant current limit of 10 mA).
  • the electrical current is applied till the dye has migrated into the first purification port 530, as shown in Fig. 22E. This completes the purification process of one dye.
  • An additional sample constituent can be isolated by providing an electrical current between the input electrode (-) and the second purification electrode (+) resulting in the constituent migrating into the second purification chamber.
  • Fig. 23 illustrates the results of a simulation of electrophoretic migration conducted by a finite-element based method which can be run with open-source software tools, such as SfePy or FEniCS.
  • the simulation aimed to scrutinize the impact of DNA-fragment migration across the cross junction formed by the intersection of purification channels with the primary separation channel.
  • the simulation adopted an architecture similar to the electrophoresis cell featuring a straight separation channel 510 originating from the input chamber 515, intersecting two opposing purification channels: the first purification channel 540 terminating into the first purification chamber 530, and the second purification channel 542 terminating into the second purification chamber 535.
  • the two opposing purification channels formed a right angle to the separation channel, with the separation channel culminating into the output chamber 520.
  • the simulation accurately replicated the observed band distortion observed at the fluidic junctions 2301 in similarly structured electrophoresis cells.
  • the distortion can be mitigated by applying counter voltages to the purification chambers, preventing material leakage from the separation channel into the purification channel 2302 during migration across this junction, as depicted in Fig. 23C.
  • the dynamic imaging method employed to monitor the electrophoretic process offers an advantage wherein an automated process can regulate the counter voltage potentials based on images captured from the electrophoresis cell to minimize distortions.
  • the current disclosure describes a system for performing various forms of analytical, preparative and affinity electrophoresis in a microplate format, wherein each electrophoresis microplate comprises a multitude of electrophoresis cells.
  • the autonomous electrophoresis cell architecture allows diverse electrophoresis applications with assorted samples such as cells, organelles, viruses, macromolecules such as DNA, RNA, proteins, and molecules to be processed at up to 96 samples, in parallel, at the same time on a single instrument.
  • the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under.
  • the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • a system for performing electrophoresis comprising: an electrophoresis microplate, comprising;
  • electrophoresis cells 6, 12, 24, 48, or 96 electrophoresis cells, arranged in a planar rectangular grid with a ratio of 2:3 (rows:columns); wherein the electrophoresis microplate conforms to the ANSI SLAS 1-2004 (R2012) laboratory microplate standard dimensions; wherein each electrophoresis cell in said microplate operates independently of the other cells.
  • a system for performing electrophoresis comprising: an electrophoresis strip, comprising;
  • each electrophoresis cell in said strip operates independently of the other cells.
  • a system for performing electrophoresis comprising: an electrophoresis cell, comprising; an upper cover, comprising; a top surface, comprising features to guide entry of pipettes; a bottom surface; that attaches to the top surface of the electrophoresis cell, to shield said cell’s contents from contamination, oxidation, evaporative loss, and unwanted mixing of reagents during storage, transport, and handling, while allowing the introduction of samples and electrodes; a fluidic chambers layer, comprising;
  • fluidic chambers serve as containers for samples, buffers and reagents; wherein the top surface of said fluidic chambers is attached to the bottom surface of the top cover; a fluidic ports layer, comprising; a multitude of fluidic ports, that provide fluidic connections within the electrophoresis cell; wherein the top surface of the fluidic ports layer is attached to the bottom surface of the fluidics chamber layer; a fluidic channels layer, comprising;
  • the electrophoresis cell of clause 3 or 4 comprising; 1 , 2, 3, or 4 sample wells placed in the separation channels; serving to hold a sample to be subjected to electrophoresis.
  • electrodes such as conductive carbon compositions, conductive ceramics, conductive polymers, or metals and metal alloys
  • An electrophoresis instrument comprising: an electrophoresis microplate compartment, comprising; mechanisms to facilitate loading and unloading of the electrophoresis strips and electrophoresis plate; mechanisms to securely hold said strips and plate during the electrophoresis process; devices to maintain pre-determined temperatures in said compartment; and sensors to sense presence and position of strips or plate, temperature, and incident illumination intensities; an electrode interface module, comprising; an array of electrodes that penetrate through the top cover of the electrophoresis cell to establish electrical contact with the fluidic chambers, fluidic ports and fluidic channels within the electrophoresis cell; an illumination module, comprising; light sources configured to illuminate the electrophoresis microplate when said electrophoresis strips and microplate is positioned within the electrophoresis microplate compartment; light wavelength selection filters arranged between the light element and the microplate holder; an imaging module, comprising; a camera or an array of cameras to facilitate imaging of the said electrophoresis microplate in the said electrophoresis microplate holder; light wavelength
  • a method for performing electrophoresis on a sample comprising the steps of: mixing a sample with an electrically conductive fluid and a fluorescent stain; loading the mixed sample into an input chamber of the electrophoresis cell, said cell comprising: an upper cover, comprising; a top surface comprising features to guide entry of pipettes; a bottom surface; that attaches to the top surface of the electrophoresis cell to shield said cell’s contents from external contaminants, oxidation, and evaporation; a fluidic chambers layer comprising 0, 1 , 2, 3, 4, 5, 6, 7, or 8 fluidic chambers; wherein said fluidic chambers serve as containers for samples, buffers and reagents; wherein the top surface of said fluidic chambers is attached to the bottom surface of the top cover; a fluidic ports layer; said fluidic ports forming apertures to provide fluidic connections within the electrophoresis cell; wherein the top surface of the fluidic ports layer is attached to the bottom surface of the fluidics chamber layer; a fluidic channels layer;
  • An electrophoresis kit comprising: a plurality of electrophoresis strips; an electrophoresis strip holder; a sample suspension buffer comprising; staining reagents to stain sample constituents; electrophoresis buffers; thickening agents; dyes; molecular weight fiducials; control samples; instructions.

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Abstract

L'invention concerne un système pour effectuer une électrophorèse dans des formats de microplaque. La microplaque d'électrophorèse, également fournie sous la forme de bandes d'électrophorèse, comprend des cellules d'électrophorèse indépendantes disposées dans une grille rectangulaire. Chaque cellule comprend des canaux de séparation, des électrodes et des réactifs scellés à l'intérieur d'éléments de recouvrement. Chaque cellule d'électrophorèse fonctionne de manière autonome, permettant une électrophorèse analytique, préparative et d'affinité parallèles sur divers types d'échantillons. L'invention permet une séparation électrophorétique compatible avec l'automatisation et une purification efficaces sur une large gamme de types d'échantillons.
PCT/IB2025/050665 2024-05-08 2025-01-22 Système d'électrophorèse à base de microplaque Pending WO2025233688A1 (fr)

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