Electrotransfer and electrophoresis devices, systems and methods

Abstract

This disclosure provides apparatus and systems for gel electrophoresis and / or electrotransfer and / or detection of biomolecules. An apparatus for electrotransfer comprises: one or more containers containing: a biomolecule receiving material (BMR); a first electrode and a second electrode; wherein the second electrode is located after and / or distal to the region receiving the biomolecule, or wherein the two electrodes are arranged to allow current to flow in at least two directions, wherein a second direction of current flow is along the plane of the BMR. The apparatus and system are adapted to perform electrophoresis, wherein the container further comprises a matrix for electrophoretic separation of biomolecules. The apparatus and system are further adapted to perform detection, wherein the container further comprises reagent dispensing channels or ports and / or reagents for detecting the electrotransferred biomolecules. The apparatus and systems of this disclosure may be microfluidic devices. Methods of using the systems and apparatus of this disclosure are also provided.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 057,784, filed July 28, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to systems and apparatuses capable of performing electrotransfer of biomolecules. This disclosure relates to systems and apparatuses capable of performing both electrophoresis and electrotransfer of biomolecules. This disclosure further relates to electrophoresis and electrotransfer apparatuses and systems capable of performing biomolecule detection. Methods for electrophoresis, electrotransfer, and / or detection using the systems and apparatuses of this disclosure are also described. Background Technology

[0004] Electrophoresis is a common method for separating biomolecules (such as nucleic acids, DNA, RNA, peptides, and proteins) based on their size and charge. In gel electrophoresis, biomolecules are separated into bands by an electric field that causes them to migrate through a filter matrix. A typical filter matrix consists of a gel. A conventional electrophoresis chamber contains a filter matrix (such as a gel) sandwiched between two glass or plastic plates. The gel has an open molecular network structure that defines pores saturated with a conductive buffer solution containing salts. These pores are large enough to allow biomolecules to migrate through the gel in response to an electric field. Several types of gels can be used for electrophoresis, such as, but not limited to, polyacrylamide gels, agarose gels, and starch gels.

[0005] During conventional electrophoresis, a gel in a pre-cast or self-cast electrophoresis chamber is typically loaded with a sample containing biomolecules and tracer dyes and placed in a chamber with a cathode and an anode in contact with a buffer solution. When connected to a power source, the buffer solution generates an electric field on the gel. This generated electric field, consisting of a negative charge at one end and a positive charge at the other, is applied to the gel, causing the sample biomolecules and tracer dyes to separate from each other and migrate towards the bottom of the gel. Electrophoresis is stopped before the biomolecules of interest reach the ends of the gel.

[0006] The electrophoretically separated biomolecules are then transferred from the separation gel to another material for further analysis, such as, but not limited to, immunological characterization, chemical reactions, and quantification. wait Electroimprinting or electrotransfer is a method known in the art for transferring analyzed or separated biomolecules from a gel onto another material.

[0007] In electrotransfer methods, after the electrophoretic separation of biomolecules, the electrophoretic gel containing the separated biomolecules is brought into contact with a relatively thin material or support. This material is typically porous, such as, but not limited to, nitrocellulose-based membranes, PVDF-based membranes, activated paper, activated nylon membranes, etc. A buffer is added to the electrotransfer apparatus, allowing an electric current to pass through the sandwich gel and the imprinted membrane in a direction typically perpendicular to the membrane surface. This causes some or most of the biomolecules to transfer from the gel electrophoretically to the porous material.

[0008] The biomolecules electrotransferred to the aforementioned materials and carriers are then further analyzed using methods including immunoassay. Several immunoassay methods are known in the art, in which labeled antibodies bind to the biomolecules of interest, thereby detecting and identifying the biomolecules. Typically, chemiluminescent labeling and reagents are used for detection.

[0009] Most existing electrophoresis and electrotransfer systems and apparatus require separate systems and apparatus for each procedure. Each procedure requires separate setup and experimental time, as well as multiple pieces of equipment and accessories. Electrotransfer systems also generate significant amounts of hazardous waste because they use transfer buffers that typically contain methanol, acetic acid, or other hazardous materials.

[0010] Several automated solutions for electrophoresis, electrotransfer, and immunoassay have been described, such as those disclosed in U.S. Patents 7,846,676, 7,935,308, 7,935,489, 9,400,277, 7,935,479, 9,304,133, 9,108,195, 9,523,684, 9,766,206, and U.S. Patent Publication 20180321189. However, these prior art techniques involve separating proteins from a matrix and then crosslinking the separated proteins to a capillary or to a component in the matrix prior to immunoassay. A major drawback of these systems and methods is that the crosslinking step is known to mask specific epitopes on protein biomolecules, resulting in poor antibody recognition during immunoassay. Therefore, these systems and methods require re-optimization and validation of antibodies for immunoassay to suit each new biomolecule and / or antibody used.

[0011] Furthermore, the data output from capillary-based instruments is a “virtual” blot-like image generated by electrophoretic traces of immunoassay signals from inside the capillary. Traditional Western blot users have not yet fully embraced this kind of protein blot-like reading.

[0012] Woodham describes another combined electrophoresis / electrotransfer system in U.S. Patent 9,702,851, which describes a polyacrylamide gel matrix for electrophoresis placed adjacent to a blot membrane, both sandwiched between two plates containing a semi-conductive polymer. To facilitate the transfer of proteins separated on the polyacrylamide gel matrix to the blot membrane, an electric current must be applied perpendicular to the direction of protein migration in the polyacrylamide gel. For immunoassay or further processing of the biomolecules transferred to the blot membrane, the entire system must be disassembled to access the blot membrane. Furthermore, in practice, this system does not work well because no semiconductor polymer with the required conductivity has yet been found.

[0013] In her paper "Microfluidic integration for automated targeted proteomic assays," Proceedings of the National Academy of Sciences (PNAS) (2012) 109:5972-5977, Amy Herr describes the direct detection of proteins cross-linked to a polyacrylamide gel matrix. However, these devices and methods are ineffective due to limitations in antibody diffusion kinetics within the polyacrylamide gel matrix and the antigen-polyacrylamide gel matrix cross-linking step, both of which can lead to antigen masking and detection problems.

[0014] Therefore, there is a need in the art for better electrotransfer systems and devices, as well as systems that can combine gel electrophoresis and electrotransfer with immunoassay. Summary of the Invention

[0015] The systems, apparatuses, and methods described herein address several problems in the art. In some embodiments, the systems, apparatuses, and methods described herein address problems in the art by providing a single system usable for both gel electrophoresis and electrotransfer. In some embodiments, the systems, apparatuses, and methods described herein address problems in the art by providing electrophoresis and electrotransfer apparatuses and systems that provide a convenient pathway to obtain electroblotting for further detection / processing / analysis of biomolecules. In some embodiments, the systems, apparatuses, and methods described herein address problems in the art by providing a single system usable for gel electrophoresis, electrotransfer, and further detection / processing / analysis of biomolecules (e.g., a system for immunoassay). In some embodiments, the systems, apparatuses, and methods described herein address problems in the art by providing increased throughput, lower buffer volume requirements, and reduced volumes of hazardous liquid waste for electrophoresis and / or electrotransfer.

[0016] In one embodiment, an apparatus for electrotransfer of biomolecules includes: at least one container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the second electrode is in electrical contact with the material and is located behind and / or distal to and / or posterior to the region receiving the biomolecules. In one embodiment, in an apparatus for electrotransfer, the second electrode is positioned above the material. In one embodiment, in an apparatus for electrotransfer, the second electrode is positioned along the same plane as the material. In one embodiment, in an apparatus for electrotransfer, the second electrode is positioned below the material.

[0017] In one embodiment, an apparatus for electrotransfer of biomolecules includes: at least one container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the electrodes are arranged to allow current to flow in at least two directions, wherein a second direction of the current is along the plane of the membrane. In one embodiment, in an apparatus for electrotransfer, the first direction of the current is transverse to the plane of the membrane. In one embodiment, in an apparatus for electrotransfer, the first direction of the current is diagonal to the plane of the membrane. In one embodiment, in an apparatus for electrotransfer, the first direction of the current is perpendicular to the plane of the membrane.

[0018] In some embodiments, in an apparatus for electrotransfer, the container is a channel, microfluidic channel, capillary, conduit, flow path, surface, slide, plastic slide, chamber, vessel, or container, and includes channels, microfluidic channels, capillary, conduit, flow path, surface, slide, or plastic slide disposed within the chamber, vessel, container, or container. In some embodiments, an apparatus for electrotransfer includes multiple containers. In some embodiments, the containers are made of a transparent or translucent material. In some embodiments, the containers are completely closed. In some embodiments, the containers have an opening at the top. In some embodiments, the containers may be closed or sealed by a removable cap.

[0019] In some embodiments, an apparatus for electrotransfer according to the present disclosure is a microfluidic device.

[0020] In some embodiments, a biomolecule receiving material is placed or coated onto a container. The biomolecule receiving material can be composed of any porous material capable of receiving biomolecules and may include one or more of the following non-limiting examples: membranes, nitrocellulose, PVDF (polyvinylidene fluoride), cellulose acetate, anodized aluminum oxide, nylon, glass fiber, and polyester. The biomolecule receiving material may be composed of one or more conductive materials embedded in any of the foregoing materials. Some non-limiting examples of conductive materials that can be embedded in the biomolecule receiving material include tantalum, copper, and indium tin oxide. waitIn some embodiments, the biomolecule receiving material comprises one or more ions.

[0021] In some embodiments, the biomolecule receiving material has a pore size of about 0.05 µM to 10 µM. Non-limiting pore sizes may include, but are not limited to, 0.05 µM, 0.1 µM, 0.15 µM, 0.2 µM, 0.25 µM, 0.3 µM, 0.4 µM, 0.45 µM, 0.5 µM, 0.55 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5.0 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, and numbers between these values. In some embodiments, the pore size of the biomolecule receiving material may differ in different regions of the biomolecule receiving material. In some embodiments, the biomolecule receiving material has at least two layers. In some embodiments, each layer of the biomolecule receiving material has a different pore size.

[0022] In some embodiments of the apparatus disclosed herein, the first electrode is positioned above at least a portion of the biomolecule receiving material. In some embodiments, the first electrode is further positioned before and / or in front of the region where the biomolecule is received on the biomolecule receiving material.

[0023] In some embodiments of the device disclosed herein, the second electrode is located in the same plane as the biomolecule receiving material, or below or above the biomolecule receiving material.

[0024] In some embodiments of the device disclosed herein, the second electrode is in direct contact with the biomolecule receiving material. In other embodiments of the device disclosed herein, the second electrode is not in direct contact with the biomolecule receiving material.

[0025] In some embodiments of the apparatus disclosed herein, at least one of the two electrodes may be physically movable within the container. In some embodiments, the electrodes are positioned such that they do not obscure the observation of the area where biomolecules are received on the membrane.

[0026] In some embodiments, the apparatus of this disclosure further includes an ion reservoir. Non-limiting examples of ion reservoirs include: buffer cartridges, buffer solutions, filter paper containing ions, solid matrices containing ions, or liquid matrices containing ions.

[0027] In some embodiments of the apparatus disclosed herein, the container further includes a matrix in which biomolecules can be electrophoretically separated. In some embodiments, the matrix is ​​positioned above at least a portion of the biomolecule receiving material. In some other embodiments, the matrix is ​​positioned above at least a portion of the biomolecule receiving material and in a plane parallel to the biomolecule receiving material. In some other embodiments, the matrix has a uniform thickness. In some other embodiments, the matrix has a non-uniform thickness. In some other embodiments, the matrix has a non-uniform thickness, being thinner at a rear end and gradually increasing in thickness towards its front end. In some other embodiments, the matrix has a non-uniform thickness, and the matrix is ​​positioned above at least a portion of the biomolecule receiving material. Non-limiting examples of the matrix include polymeric materials, gels, agarose gels, acrylamide gels, polyacrylamide gels, dextran, or polyethylene glycol.

[0028] In some embodiments, the apparatus of this disclosure further includes a third electrode. In some embodiments, the apparatus of this disclosure further includes a fourth electrode.

[0029] The apparatus of this disclosure may have electrical connections to independently control each electrode. In some embodiments, the apparatus of this disclosure includes at least one electrode that is physically movable. In some embodiments, the electrode may be movable through a distal end of a matrix.

[0030] In some embodiments of the apparatus of this disclosure, at least one electrode is positioned beyond the distal end of the substrate and separated by an insulating region. In some embodiments of the apparatus of this disclosure, at least one electrode is located above at least a portion of the substrate.

[0031] In some embodiments of the apparatus of this disclosure, at least one ion reservoir is positioned above at least a portion of the substrate. In some embodiments, the apparatus of this disclosure includes a second ion reservoir. The second ion reservoir is located at or behind the distal end of the substrate. The first or second ion reservoir of the apparatus of this disclosure is physically movable.

[0032] In some embodiments of the apparatus disclosed herein, the matrix is ​​a liquid separation matrix. In non-limiting examples, the liquid separation matrix may comprise a liquid polymer material, liquid polyacrylamide, a polyacrylamide solution, polyethylene glycol, a mixture of one or more polyethylene glycols, or dextran, or a certain amount of agarose.

[0033] In some embodiments, the liquid separation matrix further comprises at least one ion source. Non-limiting examples of ion sources include one or more ions selected from glycine, chloride, sodium, sulfate, acetate, and tris(hydroxymethyl)aminomethane.

[0034] In some embodiments of the apparatus disclosed herein, the matrix includes at least one sample container operable to receive a sample containing the biomolecule, the sample container being positioned proximal to the matrix. Non-limiting examples of the sample container include pores, ports, etc. In non-limiting examples, the depth of the matrix is ​​from about 2 µM to about 2 mm.

[0035] In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occur in a horizontal direction.

[0036] In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occur in a vertical direction.

[0037] In some embodiments, the apparatus of this disclosure further includes at least one port. The port may additionally include at least one seal or sealing mechanism, such as, but not limited to, a resealable plastic seal, a rubber stopper, or an automatically resealable seal. In some embodiments, the apparatus of this disclosure includes at least one puncture-resistant and automatically resealable port.

[0038] In some embodiments, a port of the device disclosed herein includes at least one removable plug. The port in the device disclosed herein may include at least one support structure to prevent one or more ports from collapsing. The support structure may comprise or be made of a porous structure that supports one or more ports from collapsing. In some embodiments, the support structure, which comprises or is made of a porous support structure, may comprise a gel matrix, such as, but not limited to, agarose.

[0039] In some embodiments, the apparatus of this disclosure has at least one port operable to receive a sample. In some embodiments, at least one or more ports of the apparatus of this disclosure may be used for one or more of the following, including but not limited to: receiving or accommodating an electrode; adding or removing a matrix; adding or moving an electrode; adding a sample to a matrix; adding or removing one or more components selected from solutions containing one or more antibodies, solutions containing one or more biomolecular detection reagents, reagents that bind and / or detect biomolecules transferred to biomolecular receiving materials; adding or removing one or more buffer solutions and any combination thereof.

[0040] In some embodiments, in the apparatus of this disclosure, at least one electrode is located in a port. In some embodiments, a first electrode is located in a sample loading port. In some embodiments, a second electrode is located in a port at the distal end of the matrix. In some embodiments, a third electrode is positioned beyond the distal end of the matrix and in physical contact with the membrane. In some embodiments, the third electrode is located after / behind an insulator adjacent to the rear end of the matrix. In some embodiments, the third electrode is located behind an insulator encapsulating the matrix. In some embodiments, the insulator is air or a non-conductive material. In some embodiments, the insulator is a non-conductive material such as polycarbonate, nylon, or high-density polyethylene (HTPE). In some embodiments, a fourth electrode is located above the matrix. In some embodiments, the fourth electrode is further positioned along the edge of the container such that it does not obstruct observation of the biomolecule receiving material. In some embodiments, the fourth electrode is positioned along the edge of the container. In some embodiments, the fourth electrode can be separated from the liquid matrix separation medium by a thin layer of porous material containing high resistance or low conductivity. In a non-limiting example, the thin layer may have a thickness of 0.1 mm to 0.5 mm. The porous material may include, for example, but not limited to, agarose.

[0041] In some embodiments, the apparatus of this disclosure is further adapted to detect biomolecules by methods such as, but not limited to, immunoassay, colorimetry, chemiluminescence, fluorescence, radiolabeling, or heavy isotope labeling. In some embodiments, the apparatus of this disclosure is further adapted to perform immunoassay, wherein the container further includes a reagent dispensing channel and / or reagents for immunoassay of electrotransferred biomolecules.

[0042] In some embodiments, an apparatus includes: one or more containers, each container including: a biomolecule receiving material; a first electrode; a second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside a region where one or more biomolecules are received on the biomolecule receiving material; and at least a first ion reservoir; means for moving or positioning the first electrode and the first ion reservoir over at least a portion of a matrix in which biomolecules are present; and means for activating and deactivating the first electrode and the second electrode.

[0043] In some embodiments, an apparatus includes: at least one container comprising: a matrix operable to separate one or more biomolecules contained in a sample along the length of the matrix, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside the region where biomolecules are received on the biomolecule receiving material; and at least one ion reservoir wherein the first electrode and the second electrode are activating and deactivating, and wherein the first electrode and the first ion reservoir are movable or positioned on at least a portion of the top of the matrix, and wherein the second electrode and the second ion reservoir are movable or positioned past a distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material.

[0044] In some embodiments, an apparatus includes: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, and a third electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned above at least a portion of the matrix and near the rear end of the matrix; the third electrode being in electrical contact with the biomolecule receiving material and positioned behind the rear end of the matrix; and at least one ion reservoir. In some embodiments, the first, second, and third electrodes can be activated and deactivated.

[0045] In some embodiments, an apparatus includes: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, a third electrode, and a fourth electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned toward the rear end of the matrix; the third electrode being in electrical contact with the biomolecule receiving material and positioned behind the rear end of the matrix; the fourth electrode being positioned above the matrix; and at least one ion reservoir. In some embodiments, the first and second electrodes, as well as the third and fourth electrodes, can be activated and deactivated.

[0046] Some embodiments describe devices having a liquid matrix and a vertical orientation. One exemplary device includes a container with two ports, closed on at least three sides, and a biomolecule receiving material placed on one side (inner side) of the container. At least one port is sealed with a removable stopper (the seal forming part of the container's housing). At least one port is a puncture-resistant and self-sealing port, which can be used to fill the container with a liquid matrix. The container of the device further includes electrodes adjacent to an insulator for separating and / or forming the housing of the liquid matrix, and at least one electrode adjacent to the resealable port.

[0047] Another embodiment of the vertically oriented device disclosed herein has a container with two ports and a biomolecule receiving material placed on one side (inner side) of the container. The first port is operatively sealed by a removable stopper (which forms part of the housing of the container) and is vertically positioned above the second port. A first electrode may be positioned on top of the removable stopper and may have a buffer core below it for contact with the container. The second port is a puncture-resistant and self-sealing port and contains an electrode (second electrode). This port can be used to fill the container with a liquid matrix. The liquid matrix is ​​filled into the first port, and the stopper is used to seal the first port located at the top. The container of the device further includes an electrode (third electrode) placed adjacent to an insulator and another electrode (fourth electrode) located on or within the sidewall of the container opposite the biomolecule receiving material.

[0048] This disclosure describes an apparatus for electrophoresis, electrotransfer, and detection of biomolecules, wherein the apparatus comprises: at least one container including: a matrix layer operable to separate biomolecules along its length, the matrix having at least one port; a biomolecule receiving material disposed in a parallel plane beneath the matrix; a first electrode electrically contacting the matrix and located before or within the port; a second electrode located after the matrix and electrically contacting the biomolecule receiving material; a third electrode electrically contacting the matrix and located after the biomolecule receiving region; and a fourth electrode located above at least a portion of the matrix. In some embodiments, the port may receive a sample and may (later) receive one or more reagents for detecting biomolecules. Alternatively, the detection reagent may be laminated on the matrix layer and may migrate through the matrix to reach biomolecules on the biomolecule receiving material. In some embodiments, the apparatus has a single port. In some embodiments, the matrix layer is a thin layer. In some embodiments, the thin layer may be from about 20 µM to 200 µm, and in some embodiments about 100 µm.

[0049] In some embodiments, this disclosure provides systems for electrophoresis and electrotransfer. In some embodiments, this disclosure provides systems for electrophoresis, electrotransfer, and detection (e.g., but not limited to immunoassay). The systems of this disclosure may be microfluidic systems or include microfluidic components. The apparatuses and systems of this disclosure may also have detection and imaging capabilities to perform detection and imaging during (in real time) and after completion of electrophoresis, electrotransfer, and / or immunoassay.

[0050] Some embodiments relate to systems comprising: any apparatus described herein, further comprising one or more of the following: an instrument or base capable of providing power; means for activating and / or deactivating electrodes; a capillary; a conduit system; a channel; a microchannel; a pump; a valve; a robotic arm or motor; means for regulating electrode movement / repositioning; means for dispensing and / or regulating reagent flow rates; and software components for regulating reagent release / timed release and / or regulating electrode activation / deactivation and / or regulating electrode movement / repositioning during operation.

[0051] Embodiments of this disclosure relate to methods of electrotransfer or gel electrophoresis and electrotransfer using the apparatus and systems described herein. Biomolecules that can be electrophoresed and / or electrotransferred by the apparatus and methods described herein include proteins, peptides, glycoproteins, phosphoproteins, nucleic acids, DNA, or RNA.

[0052] In one embodiment, a method for electrotransferring biomolecules comprises: 1) positioning a matrix containing one or more biomolecules in or on top of a container of a device, the biomolecules having been separated based on their molecular weight or charge, the device comprising: one or more containers, each container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside the region where the biomolecules are received on the biomolecule receiving material; 2) performing electrotransferring biomolecules from the matrix to the biomolecule receiving material, comprising: (i) positioning the first electrode and a first ion reservoir above at least a portion of the matrix in which the biomolecules are present; and (ii) activating the first electrode and the second electrode to transfer the biomolecules onto the biomolecule receiving material.

[0053] In one embodiment, a method for electrotransferring biomolecules comprises: 1) positioning a matrix containing one or more biomolecules in or on top of a container of a device, the biomolecules having been separated based on their molecular weight or charge, the device comprising: one or more containers, each container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the electrodes are arranged to allow current to flow in at least two directions, wherein a second direction of the current is along a plane of the biomolecule receiving material; 2) performing electrotransferring biomolecules from the matrix to the biomolecule receiving material, comprising: (i) positioning the first electrode and a first ion reservoir above at least a portion of the matrix in which the biomolecules are present; and (ii) activating the first electrode and the second electrode to transfer the biomolecules onto the biomolecule receiving material.

[0054] In one embodiment, a method for performing electrophoresis and electrotransfer of biomolecules comprises: 1) loading a sample containing one or more biomolecules into a sample loading region of a device comprising (i.e., a device having two electrodes, wherein optionally the electrodes are mechanically movable) at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region having at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode, wherein the second electrode is in electrical contact with a biomolecule receiving material and is located outside the region where the biomolecules are received on the biomolecule receiving material; and at least one ion reservoir; 2 1) Electrophoresis of biomolecules in the matrix by activating two electrodes; 2) Deactivating the first and second electrodes to stop electrophoresis; and 3) Electrotransfer of biomolecules from the matrix to the biomolecule receiving material by: (i) moving / positioning the first electrode and the first ion reservoir onto at least a portion of the top of the matrix; (ii) moving / positioning the second electrode and the second ion reservoir past the distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material; (iii) activating the first and second electrodes such that biomolecules can be transferred from the matrix to the biomolecule receiving material; and (iv) deactivating the first and second electrodes once completed to stop the electrotransfer.

[0055] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample containing one or more biomolecules into a sample loading region of a device comprising (i.e., a device having two electrodes, wherein the electrodes are optionally mechanically movable) at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region having at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode, wherein the second electrode is electrically contacted with a biomolecule receiving material and located behind a distal end of the matrix; and at least one ion reservoir; 2) by activating the two electrodes 3) Electrophoresis of biomolecules in the matrix; 4) deactivating the first and second electrodes to stop electrophoresis; and 5) performing electrotransfer of biomolecules from the matrix to the biomolecule receiving material by: (i) moving / positioning the first electrode and the first ion reservoir onto at least a portion of the top of the matrix; (ii) moving / positioning the second electrode and the second ion reservoir past the distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material; (iii) activating the first and second electrodes such that biomolecules can be transferred from the matrix to the biomolecule receiving material; and (iv) once completed, deactivating the first and second electrodes to stop the electrotransfer.

[0056] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample containing biomolecules into a sample loading region in a matrix of a device of the present disclosure (such as a device having three electrodes), the device comprising: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, and a third electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; the second electrode being located above at least a portion of the matrix and... And located near the rear end of the matrix; the third electrode is electrically contacted with the biomolecule receiving material and positioned behind the rear end of the matrix that is insulated from and separated from the third electrode; at least one ion reservoir of at least one of the adjacent electrodes; 2) electrophoresis of the sample in the matrix by activating the first and second electrodes; 3) deactivating the first and second electrodes to stop electrophoresis after the biomolecules are resolved; 4) electrotransfer of biomolecules from the matrix to the biomolecule receiving material by reactivating the first electrode and activating the third electrode, wherein the third electrode is electrically contacted with the biomolecule receiving material; and 5) deactivating the first and third electrodes to stop electrotransfer once completed.

[0057] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample or multiple samples containing biomolecules into a sample loading port of a device (e.g., a device having four electrodes): at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, a third electrode, and a fourth electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned toward the rear end of the matrix; the third electrode being aligned with the biomolecule receiving material. The biomolecule receiving material is electrically contacted and positioned at the rear end of a matrix that is insulated from and separated from the third electrode; a fourth electrode is positioned above the matrix; the fourth electrode is separated from the matrix by a transparent material having a higher resistance compared to the electrode material; and at least one ion reservoir; 2) electrophoresis of a sample in the matrix by activating the first and second electrodes; 3) when complete, deactivating the first and second electrodes to stop the electrophoresis; 4) electrotransfer of biomolecules from the matrix to the biomolecule receiving material by (i) activating the third and fourth electrodes; and 5) when the electrotransfer is complete. In a non-limiting example, a material separating the matrix and the fourth electrode may comprise agarose or another gel or agarose-like material about 0.1 mm to 5 mm thick. In some embodiments of the method described herein, the third electrode is further positioned along the edge of the container such that it does not obstruct observation of the biomolecule receiving material from either side of the device.

[0058] In some embodiments of the methods disclosed herein, as described differently in the preceding sections, the apparatus used comprises multiple containers, and multiple samples are loaded into the sample loading region of each container. In some embodiments, the biomolecules to be electrophoresed and / or electrotransferred are stained prior to loading. The biomolecules are pre-stained.

[0059] In some embodiments of the methods disclosed herein, as described differently in the preceding sections, biomolecules may be visualized during electrophoresis and electrotransfer. In some embodiments, the methods of this disclosure may include visualizing biomolecules from a top observation device during or at the end of the electrophoresis and / or electrotransfer steps. In some embodiments, the methods of this disclosure may further include imaging biomolecules during or at the end of the electrophoresis and / or electrotransfer steps.

[0060] In various embodiments of the methods described herein, the sample loading region is a sample port on the apparatus of this disclosure. In some embodiments, the sample loading port is supported by a molecularly permeable physical barrier, such as a barrier made of agarose. In some embodiments, the sample loading port leads to a microfluidic insert that receives biomolecules. In some embodiments, a sample is loaded into the sample port after a plug located therein is removed.

[0061] The systems, apparatus, boxes, and methods disclosed herein advantageously result in one or more of the foregoing, including: an apparatus / system for electrophoresis and electrotransfer capable of simultaneously performing high-throughput electrophoresis and / or electrotransfer of biomolecules, which, compared to existing systems and apparatuses for electrophoresis or electrotransfer, reduces the number of devices or components, lowers costs, reduces accessories, reduces the footprint of required equipment, reduces storage space for the system and apparatus, eliminates buffer spills and leaks, reduces the amount of gels, biomolecule receiving materials, samples, buffers, and other components used, and reduces liquid hazardous waste.

[0062] These and other features of this teaching will become more apparent from the detailed descriptions in the following sections. Attached Figure Description

[0063] One or more embodiments of this disclosure can be better understood with reference to the following one or more accompanying drawings. Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0064] Figure 1 It is a schematic representation of the arrangement of components that can be used to perform electrotransfer and / or electrophoresis in existing technology systems;

[0065] Figures 2A to 2D This is a schematic diagram of four exemplary arrangements of components in the apparatus / system of this disclosure that can be used to perform electrophoresis and / or electrotransfer and / or detection, wherein... Figure 2A The arrangement of various components and the direction of current flow are shown according to one embodiment, wherein Figure 2B The arrangement of various components and the direction of current flow according to another embodiment are shown, wherein Figure 2C The arrangement of various components and the direction of current flow are shown according to yet another embodiment; and wherein Figure 2D The arrangement of various components and the direction of current flow are shown according to yet another embodiment;

[0066] Figures 3A to 3C This is a schematic diagram of three exemplary arrangements of components in the apparatus / system of this disclosure that can be used to perform electrophoresis and / or electrotransfer and / or detection, wherein... Figure 3A The arrangement of various components and the direction of current flow are shown according to one embodiment, wherein Figure 3B The arrangement of various components and the direction of current flow according to another embodiment are shown, and in which Figure 3C The arrangement of various components and the direction of current flow are shown according to yet another embodiment;

[0067] Figures 4A and 4B show a side view of an exemplary electrical transfer apparatus of the present disclosure according to one embodiment and an electrical transfer result using the apparatus;

[0068] Figures 5A to 5E are side views of an exemplary electrophoresis and electrotransfer system of the present disclosure according to one embodiment;

[0069] Figure 6A These are side views of two exemplary electrophoresis and / or electrotransfer systems of this disclosure according to one embodiment;

[0070] Figure 6B These are side views of two exemplary electrophoresis and / or electrotransfer systems of this disclosure according to one embodiment;

[0071] Figure 7A This is a side view of an exemplary system and method of the present disclosure according to one embodiment, illustrating electrophoresis and electrotransfer of biomolecules;

[0072] Figure 7B This is according to one embodiment, following electrophoresis and electrotransfer of biomolecules. Figure 7A Top view;

[0073] Figure 8A This is a side view of an exemplary system and method of the present disclosure according to one embodiment, illustrating electrophoresis and electrotransfer of biomolecules;

[0074] Figure 8B This is according to one embodiment, following electrophoresis and electrotransfer of biomolecules. Figure 8A Top view;

[0075] Figure 9A This is a side view of an exemplary system and method of the present disclosure according to one embodiment, illustrating electrophoresis and electrotransfer of biomolecules;

[0076] Figure 9B This is according to one embodiment, following electrophoresis and electrotransfer of biomolecules. Figure 9A Top view;

[0077] Figure 10A This is a side view of an exemplary system of the present disclosure according to one embodiment, illustrating the electrophoresis and electrotransfer of biomolecules;

[0078] Figure 10B This is according to one embodiment, following electrophoresis and electrotransfer of biomolecules. Figure 10B Top view;

[0079] Figure 11A This is a side view of an exemplary device of the present disclosure according to one embodiment, showing an exemplary port operable to fill a liquid matrix into the device;

[0080] Figure 11B This is a side view of an exemplary device of the present disclosure according to one embodiment, showing an exemplary port operable to load a sample into the device;

[0081] Figure 11C This is a side view of an exemplary system of the present disclosure according to one embodiment, illustrating the electrophoresis and electrotransfer of biomolecules;

[0082] Figure 11D According to one embodiment, after electrophoresis and electrotransfer of biomolecules, multiple such... Figure 11C A top view of the apparatus of the container shown;

[0083] Figure 12A This is a side view of an exemplary device of the present disclosure according to one embodiment, showing an exemplary port operable to load a substrate into the device;

[0084] Figure 12B This is a side view of an exemplary device of the present disclosure according to one embodiment, showing an exemplary port operable to load a substrate into the device;

[0085] Figure 12C This is a side view of an exemplary system of the present disclosure according to one embodiment, illustrating the electrophoresis and electrotransfer of biomolecules;

[0086] Figure 12D According to one embodiment, after electrophoresis and electrotransfer of biomolecules, multiple such... Figure 12C A top view of the apparatus of the container shown;

[0087] Figure 13 Example components and example dimensions of some components of an exemplary electrophoresis and / or electrotransfer system of the present disclosure according to one embodiment are shown;

[0088] Figure 14A This is a side view of an exemplary system of the present disclosure according to one embodiment, illustrating the electrophoresis and electrotransfer of biomolecules;

[0089] Figure 14B This is a side view of an exemplary system of the present disclosure according to one embodiment, illustrating electrophoresis, electrotransfer, and immunoassay of biomolecules; and

[0090] Figure 14C According to one embodiment, after electrophoresis and electrotransfer of biomolecules, multiple such... Figure 14A A top view of the apparatus of the container shown. Detailed Implementation

[0091] It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory, and are not intended to limit the scope of the present teaching. In this application, unless otherwise specified, the use of the singular includes the plural. For example, unless the context otherwise requires, the singular forms “a,” “an,” and “the” used in the specification also include the plural aspect. Similarly, unless the context otherwise requires, any singular term used in the specification also implies the plural, and vice versa.

[0092] Furthermore, the use of “comprises,” “containing,” and “including,” or modifications of these roots, such as but not limited to “comprises,” “contained,” and “including,” is not intended to be restrictive. Unless otherwise stated, the use of “or” means “and / or.” The term “and / or” means that the preceding and following terms can be used together or separately. For illustrative purposes, but not as a limitation, “X and / or Y” can mean “X” or “Y” or “X and Y.”

[0093] Whenever a range of values ​​is provided in this document, the range is intended to include the starting value and the ending value, as well as any values ​​or ranges of values ​​in between, unless otherwise specified. For example, "0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; the ranges between them are such as 0.2 to 0.3, 0.3 to 0.4, 0.2 to 0.4; the increments between them are such as 0.25, 0.35, 0.225, 0.335, 0.49; the increment ranges between them are such as 0.26 to 0.39; and so on.

[0094] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing this example, what is explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that the number of items or terms in any combination is generally not limited unless otherwise apparent from the context.

[0095] This document describes systems, apparatus, and methods for electrophoresis and / or electrotransfer and / or detection of biomolecules. Some embodiments describe systems for the transfer / blotting (protein blotting, RNA blotting (northern blotting), DNA blotting (southern blotting)) and detection of biomolecules. The systems, apparatus, and methods of this disclosure overcome several problems in the art. In some embodiments, systems and / or apparatuses that can be used for electrotransfer are described. In some embodiments, systems and / or apparatuses that can be used for gel electrophoresis and electrotransfer are described. In some embodiments, systems and / or apparatuses that can be used for gel electrophoresis and electrotransfer, as well as for the detection of transferred biomolecules (e.g., by immunoassay), are described. Thus, the systems of this disclosure provide a single instrument platform for performing two different biomolecular analysis methods.

[0096] Therefore, compared to existing systems and apparatuses for electrophoresis and / or electrotransfer, the systems, apparatuses, cartridges, and methods of this disclosure advantageously produce at least one or more of the aforementioned benefits and / or include: a system or platform for electrophoresis, electrotransfer, and detection of biomolecules; the ability to perform multiplex electrophoresis and / or electrotransfer and / or procedures in a single device by simultaneously processing biomolecules in two or more gels or transfer membranes, thereby providing increased throughput for electrophoresis and electrotransfer and / or immunoassay; providing a simple device with a small footprint; eliminating the need for complex robotic instruments; reducing the number of devices or parts or components, reducing costs, reducing the footprint of equipment storage, reducing spills, reducing leaks, reducing decontamination, reducing the amount of buffers and reagents used, and reducing liquid hazardous waste (such as methanol in transfer buffer waste compared to existing systems and apparatuses for electrophoresis and / or electrotransfer).

[0097] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. All references and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and internet web pages, regardless of their format, are expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more incorporated references and similar materials define or use a term in a manner that contradicts the definitions in this application, this application shall prevail. While the teachings of this invention have been described in conjunction with various embodiments, the teachings are not intended to be limited to such embodiments. Rather, the teachings encompass various alternatives, modifications, and equivalents, as will be understood by those skilled in the art based on these teachings.

[0098] The accompanying drawings and illustrations provided in this specification are intended to describe exemplary embodiments. Those skilled in the art will recognize that the drawings and examples are for illustrative purposes only and are not intended to limit the scope of this teaching in any way. Not all components are labeled in every drawing, and similar components may have similar designations that are not described in every drawing or every part of the specification unless otherwise indicated.

[0099] I. Systems and devices:

[0100] Embodiments of this disclosure describe systems and apparatuses that can be used for at least one or more of the following: gel electrophoresis, electrotransfer, and detection. Apparatuses of this disclosure include one or more containers in which electrophoresis, electrotransfer, and / or detection of biomolecules can be performed. Containers of this apparatus / system include, but are not limited to: channels, microfluidic channels, capillaries, conduits, flow paths, surfaces, glass slides, plastic glass slides, chambers, dishes, or containers, and also include containers / vessels / dishes / slides, wherein or thereon may contain one or more of multiple channels, or microfluidic channels, or capillaries, or conduits, or flow paths. The terms container, conduit, flow path, channel, microfluidic channel, surface, glass slide, plastic glass slide, chamber, dish, or container are used interchangeably in this specification. In some embodiments, the flow path may contain a gel. In some embodiments, an apparatus for electrotransfer includes multiple containers. In some embodiments, the containers are made of a transparent material. In some embodiments, the containers are completely closed. In some embodiments, the containers have an opening at the top. In some embodiments, the containers may be closed or sealed by a removable cap.

[0101] The container, including but not limited to channels, conduits, flow paths, microfluidic channels, surface slides, plastic slides, chambers, vessels, or containers, can be of any suitable shape, size, or configuration and can be formed of any suitable material (e.g., glass, plastic, silicon, fused silica, gel, PYEX™ (amorphous glass), etc.). For example, in some embodiments, the container may define an inner cavity with a diameter of about 30 micrometers (μm) to about 3000 μm. In other embodiments, the container may include a capillary, channel, or microchannel defining an inner cavity with a diameter of about 25 μm to about 400 μm. The size of the inner cavity diameter may be at least partially based on the sample and / or sample volume. For example, an inner cavity with a relatively small diameter uses a relatively small sample volume, which may be suitable for expensive samples or reagents, while an inner cavity with a relatively large diameter uses a relatively large sample volume and may result in improved signal detection. In some embodiments, any capillary, channel, etc., may be included. waitThe length of the container can be based at least in part on factors such as sample size and the degree of sample separation required to resolve the biomolecules of interest. In some embodiments, the container may have a length of about 2 cm to 20 cm. In some embodiments, the container / channel may have a length of less than 2 cm. In some embodiments, the container / channel may have a length of about 3 cm, 4 cm, 5 cm, or 6 cm or greater. In some embodiments, using a longer container / channel may result in better sample separation and improved resolution of complex mixtures and / or when resolving low-abundance biomolecules.

[0102] The apparatus disclosed herein is not limited to any size and can be scaled up or down to suit any need for electrophoresis, electrotransfer, or detection.

[0103] In some embodiments, the device disclosed herein is a microfluidic device. A microfluidic device generally refers to a device that can contain and / or process and / or handle reagents in microvolumes, such as introducing samples or reagents less than 1 μl, less than 500 nl, and / or between 1 nl and 10 nl into the device. Fluids include liquids, gels, matrices, semi-solids, and suspensions of microparticles in liquids. In various embodiments, the microfluidic device of this disclosure comprises one or more of the following: a biomaterial receiving material, a matrix, an ion reservoir, one or more electrodes, a buffer solution, a washing solution, and / or a detection reagent.

[0104] Microfluidic devices typically include channels that form the boundaries of fluid flow. As used herein, "channel" refers to a feature on or within a substrate that at least partially guides fluid flow. In some cases, channels may be at least partially formed by a single component, such as an etched substrate or a molded unit. Channels can have any cross-sectional shape, such as circular, elliptical, triangular, irregular, square, or rectangular (with any aspect ratio), and may or may not be covered. Right now (Opening to the external environment surrounding the channel). A removable cover allows access to certain components (e.g., biomolecule receiving material with transferred biomolecules can be removed from the microfluidic device and processed separately). A removable cover allows dispensing of reagents or components. In embodiments where the channel is completely covered, at least a portion of the channel may have a completely closed cross-section, and / or the entire channel may be completely closed along its entire length, except for its inlet and outlet. Additional features of the microfluidic device may include one or more chambers for reagents (pre-filled or user-filled), an inlet, a microchannel, a detection chamber, a fluid-flowable outlet, a waste container, valves, pumps, electrodes, electrical connections, etc.

[0105] In some embodiments, the microfluidic device of this disclosure is transparent on top. The substrate on which the microfluidic device is fabricated may be transparent and may be covered with a material having transparent properties, such as a glass coverslip, to allow for detection of the report, for example, by an optical device such as an optical microscope. This material may be perforated for functional interconnects, such as fluid interconnects, electrical interconnects, and / or optical interconnects, and is sealed to the rear interface of the device such that the junctions to the device are leak-proof. This device can allow high pressure to be applied to the fluid channels without leakage. In some embodiments, the microfluidic device of this disclosure may have dimensions ranging from about 0.5 cm to about 15 cm on each side and a thickness of about 1 micrometer to about 1 cm. In some embodiments, the microfluidic device of this disclosure may have dimensions of about 6 cm long to about 6 cm wide and about 2 mm thick.

[0106] Biomolecular receiving materials (such as nitrocellulose, PVDF) wait This material can be coated or adhered to the channels or containers of the microfluidic device disclosed herein. In some embodiments, a multilayer biomolecule receiving material can be coated. In a non-limiting example, a first layer of the biomolecule receiving material can bind to biomolecules (such as proteins), and a second layer of the biomolecule receiving material can facilitate the passage of electric current. In one exemplary embodiment, the first layer (located on top) can be a biomolecule receiving material about 10 µm thick and having a pore size of 0.2 µm or 0.45 µm to allow protein binding. A second layer, about 10 µm thick and having a pore size of 5 µm, directly below the first layer, can enhance the conductivity of the nitrocellulose to facilitate current flow for electrotransfer (and / or electrophoresis).

[0107] 1) Apparatus for electrophoresis and / or electrotransfer and / or detection:

[0108] Figure 1 This is a schematic diagram of a typical prior art device for electrotransfer, in which two electrodes, a cathode and an anode surround a matrix (typically a gel, shown as a polyacrylamide gel) and a biomolecule receiving material (such as a nitrocellulose / PVDF membrane). Electrophoretic biomolecules are present on the matrix (either in the same device or in different devices), and the electrophoretic biomolecules are transferred onto the biomolecule receiving material. Figure 1 As shown, in existing technology devices, the anode is typically located below the cathode, with the substrate and membrane sandwiched between them.

[0109] In one embodiment, this disclosure describes an apparatus for biomolecular electrotransfer, comprising: at least one container including: a biomolecular receiving material; a first electrode; and a second electrode, wherein the second electrode is in electrical contact with the biomolecular receiving material and is located after and / or distal to and / or rear of the region receiving the biomolecular molecules. Figure 2A , 2BFigures 2C and 2D depict some exemplary components of the electric transfer device of this disclosure placed in one or more containers, and depict the arrangement of various components and the current flow in these exemplary devices.

[0110] While the description and figures relate to an exemplary type of biomolecule receiving material, matrix, specific conductive material for electrodes, etc., it should be understood that the apparatus of this disclosure is not limited to these examples and may be replaced by other materials.

[0111] Figure 2A An embodiment of the arrangement of some components of an electrical transfer device is depicted, wherein a first electrode (depicted as being for use with...) Figure 1 The cathode (for comparison) is located on top of the matrix and the biomolecule receiving material, and the second electrode (described as being used for...) Figure 1 The anode (for comparison) is located above and in electrical contact with the biomaterial receiving material.

[0112] Figure 2B Another embodiment of the arrangement of some components is depicted, wherein a first electrode (e.g., a cathode) is located on top of the matrix and the biomolecule receiving material, and wherein a second electrode (e.g., an anode) is positioned along the same plane as the biomolecule receiving material and is in electrical contact with the biomolecule receiving material.

[0113] Figure 2C Another embodiment of the arrangement of some components is depicted, wherein a first electrode (e.g., a cathode) is located on top of the matrix and the biomolecule receiving material, and wherein a second electrode (e.g., an anode) is located below the biomolecule receiving material and is in electrical contact with the biomolecule receiving material.

[0114] Figure 2D Another embodiment of component placement is depicted, wherein the biomolecule receiving material is not placed in a parallel plane beneath the matrix. In some embodiments, the matrix is ​​thicker on one side. While not limited to any particular theory, in some embodiments, one side (e.g.) Figure 2D The thicker matrix (shown on the right) generates substantially uniform transfer of proteins of all molecular weights because smaller proteins, which are further electrophoresed along the gel, will be electrotransferred more quickly to the biomolecular receiving material. Therefore, relative to... Figure 2D The high-MW proteins on the left side of the diagram migrate more slowly; a thicker gel is used for transfer to normalize the transfer rate. (As shown...) Figure 2D As shown, a first electrode (e.g., a cathode) is located on top of a matrix having a non-uniform thickness, below which is a biomolecule receiving material, and wherein a second electrode (e.g., an anode) is positioned along the same plane as the biomolecule receiving material and is in electrical contact with the biomolecule receiving material.

[0115] In another embodiment of this disclosure, the apparatus for biomolecular electrotransfer includes: at least one container comprising: a biomolecular receiving material; a first electrode; and a second electrode, wherein the electrodes are arranged to allow current to flow in at least two directions, wherein the second direction of the current is along the plane of the biomolecular receiving material. Figure 2A , 2B Figures 2C and 2D show a set of arrows along the plane of the biomolecular receiving material (membrane), depicting a second direction of the current. Figure 2A , 2B In the embodiments shown in 2C and 2D, the first direction of the current is perpendicular to the plane of the biomolecule receiving material and is depicted by an arrow starting from the cathode. Therefore, Figure 2A , 2B The arrangement of electrodes (and other components) depicted in Figures 2C and 2D shows a first direction of current perpendicular to the plane of the biomolecule receiving material (from cathode to anode in the figures) depicted by a vertical arrow from the cathode toward the biomolecule receiving material, and a second direction of current depicted by an arrow in the biomolecule receiving material. As described above, in some embodiments, the apparatus for electrotransfer of this disclosure includes one or more of these exemplary component arrangements to facilitate said direction of current in one or more containers.

[0116] In one embodiment, in the electrotransfer apparatus of this disclosure, a first direction of the current is transverse to the plane of the biomolecule receiving material. In one embodiment, in the apparatus for electrotransfer, the first direction of the current is diagonal to the plane of the biomolecule receiving material. In these embodiments, a second direction of the current is along the plane of the biomolecule receiving material. Figure 3A , 3B Several exemplary arrangements of components for assembling the electrotransfer device of this disclosure having these current directions are depicted in 3C. These exemplary assemblies indicate a first direction of current by transverse / diagonal arrows from the cathode through the matrix toward the biomolecule receiving material, and a second direction of current along the plane of the biomolecule receiving material is depicted by arrows along the plane of the biomolecule receiving material.

[0117] Although not in Figures 2A to 3CWhile explicitly depicted herein, electrical contact between the electrodes and other components of the devices / systems described herein can be achieved by any number of means. Some non-limiting examples of achieving electrical contact may include direct contact with a matrix or biomolecular receiving material (as depicted); or contact via a conductive material (such as an ion reservoir, buffer core, buffer solution, ion-containing matrix, conductive material embedded in (or in contact with) a component of the matrix or biomolecular receiving material, electrical interface, etc.). In some embodiments, the devices of this disclosure further include an ion reservoir. Non-limiting examples of ion reservoirs include: buffer cores, buffer solutions, ion-containing filter paper, ion-containing solid matrices, or ion-containing liquid matrices.

[0118] Biomolecule receiving materials can be composed of any porous material capable of receiving biomolecules, and may include one or more of the following non-limiting examples: membranes, nitrocellulose, PVDF (polyvinylidene fluoride), cellulose acetate, anodized aluminum oxide, nylon, glass fiber, and polyester. Biomolecule receiving materials can also be composed of one or more conductive materials embedded in any of the aforementioned materials. Some non-limiting examples of conductive materials that can be embedded in biomolecule receiving materials include tantalum, copper, indium tin oxide, etc.

[0119] As described in the preceding sections, the containers include, but are not limited to, channels, capillaries, microfluidic channels, surfaces, glass slides, plastic glass slides, chambers, vessels, or containers, and also include one or more channels, microfluidic channels, surfaces, glass slides, or plastic glass slides disposed within the containers, vessels, chambers, or containers. In some embodiments, an apparatus for electrotransfer includes multiple containers. In some embodiments, the containers are made of a transparent material. In some embodiments, an apparatus for electrotransfer according to this disclosure is a microfluidic device.

[0120] In some embodiments, a biomolecule receiving material is placed on a container. The biomolecule receiving material can be composed of any porous material capable of receiving biomolecules and may include one or more of the following non-limiting examples: membranes, nitrocellulose, PVDF (polyvinylidene fluoride), cellulose acetate, anodized aluminum oxide, nylon, glass fiber, and polyester. The biomolecule receiving material may be composed of one or more conductive materials embedded in any of the foregoing materials. Some non-limiting examples of conductive materials that can be embedded in the biomolecule receiving material include tantalum, copper, and / or indium tin oxide.

[0121] In some embodiments, the biomolecule receiving material has a pore size of about 0.2 µM to 10 µM, including all values ​​and ranges therebetween. In some embodiments, the pore size of the biomolecule receiving material may vary in different regions of the biomolecule receiving material. In some embodiments, the biomolecule receiving material has at least two layers. In some embodiments, each layer of the biomolecule receiving material has a different pore size.

[0122] In some embodiments, Figure 2A , 2B and 2C and Figure 3A , 3B The arrangement of the components depicted in 3C can be placed in a container to form an apparatus / system further configured to perform biomolecular electrophoresis. In such embodiments of the apparatus disclosed herein, the container further includes a biomolecular matrix in which electrophoretic separation can be performed. In some embodiments, the matrix is ​​positioned parallel to or above at least a portion of the biomolecular receiving material. Non-limiting examples of the matrix include polymeric materials, gels, agarose gels, acrylamide gels, polyacrylamide gels, dextran, or polyethylene glycol. Some of these embodiments will be described below.

[0123] In some embodiments, the matrix is ​​a thin matrix or a thin gel comprising, for example, an SDS-PAGE gel. In some embodiments, the thin gel has a thickness of about 20 µm to about 200 µm, including values ​​such as 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 120 µm, 130 µm, 140 µm, 150 µm, 160 µm, 170 µm, 180 µm, 190 µm, and 200 µm, and in some embodiments about 100 µm. In some embodiments, the thickness of the biomolecule receiving material in the device of this disclosure may be about half the thickness of the thin matrix layer.

[0124] Exemplary matrices can be prepared and cast as described in the examples herein. Three basic types of buffers are commonly used in SDS-PAGE electrophoresis: gel casting buffer, sample buffer, and electrophoresis buffer for filling electrode reservoirs. Discontinuous gel systems are often used for efficient separation of protein biomolecules. Discontinuous buffer systems use different gel buffers and electrophoresis buffers. These systems also use two gel layers (stacked and separating gels) with different pore sizes and different buffer compositions. Using discontinuous buffer systems for electrophoresis allows for sample concentration and higher resolution. Various commonly used discontinuous gel buffer systems include A) the classic protein separation buffer system using a tris(hydroxymethyl)aminomethane-glycine buffer system. The pH and ionic strength of the buffer used for the electrophoresis gel (tris(hydroxymethyl)aminomethane, pH 8.3) differ from the pH and ionic strength of the buffers used for the stacked gel (tris(hydroxymethyl)aminomethane, pH 6.8) and the separating gel (tris(hydroxymethyl)aminomethane, pH 8.8). B) Bis-Tris System: In a Bis-Tris gel, chloride acts as the leader ion, and MES or MOPS as the trailing ion. The Bis-Tris buffer forms a common cation. Electrophoresis using MES or MOPS denaturing buffer on the Bis-Tris gel produces significantly different protein migration patterns: MES buffer is used for smaller proteins, while MOPS buffer is used for medium-sized proteins. C) Tris(hydroxymethyl)aminomethane-acetate. Tris(hydroxymethyl)aminomethane-acetate gel chemistry enables optimal separation of high molecular weight proteins. Tris(hydroxymethyl)aminomethane-acetate gels use a discontinuous buffer system comprising tris(hydroxymethyl)aminomethane, trimethylglycine, and tris(hydroxymethyl)aminomethane. Compared to tris(hydroxymethyl)aminomethane-glycine gels, tris(hydroxymethyl)aminomethane-acetate gels have a lower pH, which enhances the stability of these gels and minimizes protein modification, resulting in clearer bands.

[0125] In some embodiments, Figure 2A , 2B and 2C and Figure 3A , 3B The arrangement of the components depicted in 3C can be placed in a container to form an apparatus / system further configured for electrophoresis and biomolecular detection. In such embodiments, the container further includes a matrix in which biomolecules can be separated by electrophoresis, and the apparatus further includes at least one or more chambers / channels / kits containing detection solutions, such as, but not limited to, one or more of the following: one or more antibodies, one or more antigens, chemiluminescent reagents, fluorescent reagents, colorimetric reagents, solutions containing other biomolecular detection reagents, one or more buffer / wash solutions, and any combination thereof. Some of these embodiments will be described below.

[0126] The apparatus and system disclosed herein may have several different electrode configurations and numbers. In some embodiments of the apparatus of this disclosure, a first electrode is positioned above at least a portion of the biomolecule receiving material. In some embodiments, the first electrode is further positioned before and / or in front of the region where biomolecules are received on the biomolecule receiving material. In some embodiments, a second electrode is positioned distally / after / behind the biomolecule receiving region in the biomolecule receiving material and may be further positioned above the biomolecule receiving material. In some embodiments, at least one of the two electrodes may be physically movable within a container. In some embodiments, the electrode may be movable past the distal end of the matrix. In some embodiments of the apparatus of this disclosure, at least one electrode is positioned past the distal end of the matrix and separated by an insulating region. In some embodiments of the apparatus of this disclosure, at least one electrode is positioned above at least a portion of the matrix. In some embodiments, the electrodes are positioned such that they do not obscure the observation of the region where biomolecules are received on the membrane. The apparatus of this disclosure may have electrical connections to independently control each electrode. In some embodiments of the apparatus of this disclosure, at least one ion reservoir is placed above at least a portion of the matrix. In some embodiments, the apparatus of this disclosure includes a second ion reservoir. The second ion reservoir is located at the distal end of the matrix or behind the distal end of the matrix. The first or second ion reservoir of the device disclosed herein is physically movable. As described in the preceding sections, electrodes may contact other components directly or via the ion reservoir. Examples of these embodiments are shown in Figures 4A and 4B, and Figures 5A-5E.

[0127] Figures 4A and 4B are side views of two exemplary electrotransfer devices of this disclosure, shown arranged in / on a container (shown as a surface or slide in a non-limiting example). Figures 4A and 4B depict an electrode, for example, a copper wire cathode electrode (-) (depicted as an orange circle and indicated by a negative sign as the cathode) and a longer cathode, such as a copper plate cathode electrode (-) (in Figure 4B), which is placed on top of a buffer core (shown in gray) for electrical contact with the gel matrix (shown in blue), and an anode is also placed on top of the buffer core for electrical contact with the biomolecule receiving material (shown in this example as a nitrocellulose membrane). Red arrows depict the direction of current flow from the electrodes through the various components. The images of the nitrocellulose membrane on the right side of Figures 4A and 4B show that near-infrared (NIR) protein labels separated on a solid polyacrylamide gel (PAG) are directly transferred to the nitrocellulose surface placed on a glass slide as a container. In both devices, the proteins (biomolecules) are successfully transferred to the nitrocellulose membrane coated on the glass plate container.

[0128] As mentioned above, Figure 2A , 2B and 2C and Figure 3A , 3BThe arrangement of the components depicted in 3C can be placed in a container to form an apparatus / system further configured for electrophoresis of biomolecules and further configured for detection. In such embodiments of the apparatus of this disclosure, the container further includes a biomolecular matrix in which biomolecules can be electrophoretically separated. In some embodiments, the matrix is ​​positioned parallel to or above at least a portion of the biomolecular receiving material. Non-limiting examples of the matrix include polymeric materials, gels, agarose gels, acrylamide gels, polyacrylamide gels, dextran, or polyethylene glycol. Figures 5A-5E are side views of another exemplary electrophoresis and electrotransfer system of this disclosure according to one embodiment. In this embodiment, 3 μL of pre-stained near-infrared (NIR) labeled protein is loaded into the sample wells in a PAG, as shown in Figures 5A-5E. Buffer cores, represented herein by two paper cores, each 2 sq. cm, pre-soaked in 1X electrophoresis buffer, are placed at both ends of the PAG gel, just overlapping sufficiently to maintain electrocontinuity. Two electrodes, - and +, as shown in Figures 5A and 5B, are placed on top of these cores, and electrophoresis is performed at 100V for 15 minutes to separate the proteins (as shown in Figures 5A and 5B). After the proteins are separated by electrophoresis, another negative electrode and a core of equal size soaked in 1X electrophoresis buffer are placed on top of the PAG, completely covering it. The positive electrode and its ion reservoir (buffered from a 2 sq. cm core) are then removed from the PAG or placed (at a distance of about one centimeter or less) so that they remain in electrical contact with the nitrocellulose membrane. 0.2 ml of 1X electrophoresis buffer is added to the nitrocellulose exposed between the electrodes to keep it moist and conductive (Figure 5C). A current of 150V is applied for 15 minutes. As the electrode position changes, the current now flows through the PAG and nitrocellulose to the positive electrode, and in this process, the proteins are transferred from the PAG to the surface of the nitrocellulose. The proteins are then immobilized in the nitrocellulose membrane, as shown in Figure 5D. Following electrotransfer, the PAG and associated core are gently removed, leaving the exposed nitrocellulose surface with transferred biomolecules ready for the next step of Western blotting. As shown in Figure 5E, the exposed nitrocellulose surface can be washed or exposed with assay reagents and can be processed for Western blotting analysis.

[0129] In some embodiments, an apparatus includes: one or more containers, each container including: a biomolecule receiving material; a first electrode; a second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside a region where one or more biomolecules are received on the biomolecule receiving material; and at least a first ion reservoir; means for moving or positioning the first electrode and the first ion reservoir over at least a portion of a matrix in which biomolecules are present; and means for activating and deactivating the first electrode and the second electrode.

[0130] In some embodiments, an apparatus includes: at least one container comprising: a matrix operable to separate one or more biomolecules contained in a sample along the length of the matrix, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside the region where biomolecules are received on the biomolecule receiving material; and at least one ion reservoir wherein the first electrode and the second electrode are activating and deactivating, and wherein the first electrode and the first ion reservoir are movable or positioned on at least a portion of the top of the matrix, and wherein the second electrode and the second ion reservoir are movable or positioned past a distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material.

[0131] In some embodiments, the apparatus of this disclosure further includes a third electrode. In some embodiments, the apparatus of this disclosure further includes a fourth electrode. These embodiments are in... Figures 6A to 12D and Figure 14A The following is shown and described in section C.

[0132] In some embodiments, the apparatus for electrophoresis and electrotransfer may comprise a liquid matrix and may be configured as a microfluidic device as described in the examples below. Several embodiments of such apparatus. In one embodiment, an apparatus for electrophoresis and electrotransfer of biomolecules comprises: at least one container comprising: a biomolecule receiving material; a matrix; a first electrode; and a second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located after and / or distal to and / or rear of the region receiving the biomolecule. In some embodiments of the apparatus disclosed herein, the first electrode is located above at least a portion of the biomolecule receiving material. In some embodiments, the first electrode is further located before and / or in front of the region where the biomolecule is received on the biomolecule receiving material. The matrix is ​​a liquid separation matrix, which, in non-limiting examples, may comprise a liquid polymer material, liquid polyacrylamide, a polyacrylamide solution, polyethylene glycol, a mixture of one or more polyethylene glycols, or dextran, or a certain amount of agarose. In some embodiments, the liquid separation matrix further comprises at least one ion source. Non-limiting examples of ion sources comprise one or more ions selected from glycine, chloride, sodium, sulfate, acetate, tris(hydroxymethyl)aminomethane, etc. Some of these embodiments are in Figures 6A to 12D and Figure 14A The following is shown and described in section C.

[0133] In some embodiments, an apparatus includes: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, and a third electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned above at least a portion of the matrix and near the rear end of the matrix; the third electrode being in electrical contact with the biomolecule receiving material and positioned behind the rear end of the matrix; and at least one ion reservoir. In some embodiments, the first, second, and third electrodes can be activated and deactivated.

[0134] In some embodiments, the device of this disclosure further includes at least one port. The port may additionally include at least one seal or sealing mechanism, such as, but not limited to, a resealable plastic seal, a rubber stopper, or an automatically resealable seal. In some embodiments, the device of this disclosure includes at least one puncture-resistant and automatically resealable port. In some embodiments, the device of this disclosure has at least one port operable to receive a sample. In some embodiments, at least one or more ports of the device of this disclosure can be used for one or more of the following, including but not limited to: receiving or accommodating an electrode; adding or removing a matrix; adding or moving an electrode; adding a sample to a matrix; adding or removing one or more components selected from solutions containing one or more antibodies, solutions containing one or more biomolecular detection reagents, reagents that bind and / or detect biomolecules transferred to a biomolecular receiving material; adding or removing one or more buffer solutions and any combination thereof. Some of these embodiments are... Figures 6A to 12D and Figure 14A The following is shown and described in section C.

[0135] In some embodiments, one port of the device of this disclosure includes at least one removable plug. The port in the device of this disclosure may include at least one support structure to prevent one or more ports from collapsing. The support structure may comprise or be made of a porous structure that supports one or more ports from collapse. In some embodiments, the support structure, composed of or made of a porous support structure, may comprise a gel matrix, such as, but not limited to, agarose. In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occurs in a horizontal direction. In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occurs in a vertical direction. These embodiments are described in... Figures 6A to 12D and Figure 14A The following is shown and described in section C.

[0136] In some embodiments, in the apparatus of this disclosure, at least one electrode is located in a port. In some embodiments, a first electrode is located in a sample loading port. In some embodiments, a second electrode is located in a port at the distal end of the matrix. In some embodiments, a third electrode is positioned beyond the distal end of the matrix and in physical contact with the membrane. In some embodiments, the third electrode is located after / behind an insulator adjacent to the rear end of the matrix. In some embodiments, the third electrode is located behind an insulator encapsulating the matrix. In some embodiments, the insulator is air or a non-conductive material. In some embodiments, the insulator is a non-conductive material such as polycarbonate, nylon, or HTPE. In some embodiments, a fourth electrode is located above the matrix. In some embodiments, the fourth electrode is further positioned along the edge of the container such that it does not obstruct observation of the biomolecule receiving material. In some embodiments, the fourth electrode is positioned along the edge of the container. In some embodiments, the fourth electrode can be separated from the liquid matrix separation medium by a thin layer of porous material containing high resistance or low conductivity. In a non-limiting example, the thin layer may have a thickness of 0.1 mm to 0.5 mm. The porous material may include, for example, but not limited to, agarose. These embodiments are in... Figures 6A to 12D and Figure 14A One or more of C are shown and described.

[0137] In some embodiments, the apparatus / system of this disclosure comprises three electrodes. In one embodiment, the three-electrode apparatus comprises: at least one container (or multiple containers) comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, and a third electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned above at least a portion of the matrix and near the rear end of the matrix; the third electrode being electrically contacted with the biomolecule receiving material and positioned behind the rear end of the matrix; and at least one ion reservoir. In some embodiments, electrophoresis of a sample in the matrix of the apparatus can be performed by activating the first and second electrodes and deactivating the first and second electrodes to stop electrophoresis after biomolecule resolution. In some embodiments, electrotransfer of biomolecules in the apparatus can be performed by reactivating the first electrode and activating the third electrode to transfer biomolecules from the matrix to the biomolecule receiving material, wherein the third electrode is electrically contacted with the biomolecule receiving material; then, once completed, the first and third electrodes are deactivated to stop electrotransfer.

[0138] Figure 6AAn embodiment of the device illustrated in the figure above is depicted, which includes three electrodes and two ports in a container. As shown herein, the three electrodes are depicted as a first electrode B, a second electrode C, and a third electrode E. Electrode B is located in the first port (also denoted as B, port B). Port B is also the sample port. Port B has a stopper that can be removed by the user before a sample is added to port B. The second port C has electrode C, and electrode E is positioned behind or after the biomolecule receiving region on the biomolecule receiving material (shown in blue at the bottom of the container), and the gray area depicts the liquid / solid matrix used for electrophoresis. In this two-port device, if a liquid matrix is ​​used, port C can be used to fill the liquid matrix while port B remains sealed with the stopper. Once a sample is added to the unblocked port B, electrodes B and C are activated to electrophoretically capture biomolecules in the sample within the matrix. Once the biomolecules are resolved, electrodes B and C are closed, and electrodes B and E are opened to initiate electrophoretic transfer of the electrophoretically separated biomolecules from the matrix to the biomolecule receiving material at the bottom of the container. The separated biomolecules are now driven by an electric current into the biomolecule receiving material, and once the transfer is complete, electrodes B and E can be turned off.

[0139] In some embodiments, the apparatus / system of this disclosure comprises four electrodes. In one embodiment, the apparatus / system has at least one (or more) container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, a third electrode, and a fourth electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned toward the rear end of the matrix; the third electrode being in electrical contact with the biomolecule receiving material and positioned behind the rear end of the matrix; the fourth electrode being positioned above the matrix; and at least one ion reservoir. To perform electrophoresis on biomolecules in a sample within the matrix, the first and second electrodes are activated. These first and second electrodes are deactivated to stop electrophoresis upon completion. To perform electrotransfer of biomolecules from the matrix to the biomolecule receiving material, the third and fourth electrodes are activated and deactivated after electrotransfer is complete.

[0140] Figure 6A Depicting in Figure 6AAn embodiment of the device illustrated in the figure below has four electrodes and four ports in a container. As shown herein, the four electrodes are depicted as a first electrode B, a second electrode C, a third electrode E, and a fourth electrode T. The first port A can be used to add reagents for detection, such as, but not limited to, Western blotting reagents, such as antibodies, washing solutions, and other buffers. Electrode B is located in the second port (also denoted as B, port B). Port B is also the sample port. Port B has a stopper that can be removed by the user before adding a sample to port B. The third port C has electrode C, and electrode E is positioned behind or after the biomolecule receiving area on the biomolecule receiving material (shown in blue at the bottom of the container), and the gray area depicts the liquid / solid matrix used for electrophoresis. In this 4-port device, if a liquid matrix is ​​used, port C can be used to fill the liquid matrix while ports A, B, and D are kept sealed with stoppers or self-sealing and / or puncture-resistant port mechanisms. Once a sample is added to the unblocked port B, electrodes B and C are activated to perform electrophoresis on biomolecules in the matrix. Once the biomolecules have been resolved, electrodes B and C are turned off, and electrodes T and E are turned on to initiate the electrophoretic transfer of the separated biomolecules from the matrix to the biomolecule receiving material at the bottom of the container. The current now drives the separated biomolecules into the biomolecule receiving material, and once the transfer is complete, electrodes T and E can be turned off.

[0141] The ports in the apparatus of this disclosure may include at least one support structure to prevent one or more ports from collapsing. The support structure may comprise or be made of a porous structure that supports one or more ports from collapsing. In some embodiments, the support structure, which comprises or is made of a porous support structure, may comprise a gel matrix, such as, but not limited to, agarose. Figure 6B The image above depicts a 2-port, three-electrode setup, where the black area below port B is a support structure that prevents port B from collapsing after the sample is added to the liquid matrix. Figure 6B The figure below depicts a 4-port, four-electrode device, where the black area below port B is a support structure that prevents port B from collapsing after the sample is added to the liquid matrix.

[0142] Figure 7A and Figure 9A A side view of an exemplary device / system with four ports and three electrodes for electrophoresis and electrotransfer of biomolecules is depicted. Figure 7B and Figure 9B A top view of the same system after the electrophoresis and electrotransfer of biomolecules have been completed is depicted. Figure 8A and Figure 10A A side view of an exemplary system with three ports and three electrodes is depicted, and the method steps for electrophoresis and electrotransfer of biomolecules are shown. Figure 8B and Figure 10B A top view of the same system is depicted after the electrophoresis and electrotransfer of biomolecules have been completed. These components are similar to the four-port and three-port, as well as the four-electrode and three-electrode embodiments described above. The methodological steps of these figures are described in detail in the following sections.

[0143] In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occur in a horizontal direction. This is illustrated in Figures 4A to 10B and Figure 14A -C is shown.

[0144] In some embodiments, the separation and transfer of biomolecules in the matrix and biomolecule receiving material occur in a vertical direction. This is in Figures 11A to 1 The exemplary device depicted in 2-D is shown.

[0145] Figure 11A An embodiment of the device of this disclosure in a vertical orientation is depicted, having a container with two ports (port B and port C) closed on all sides, and biomolecule receiving material (shown in blue) disposed on the sides of the container. Port B is sealed with a removable plug (this seal forms part of the outer shell of the container). In this embodiment, as... Figure 11A As shown in the right figure, port C is a puncture-resistant and self-sealing port used to fill the container with a liquid matrix. Once the liquid matrix is ​​filled to the beginning of port B, a stopper (shown in red) is used to seal port B at the top. The container of the device further includes an electrode E (shown in yellow) adjacent to an insulator (shown in black lines) and an electrode C (shown in yellow) located in port C.

[0146] Before use, such as Figure 11B As shown, remove the plug; this area will be used as a sample well. Add the sample to this sample well and use a cover containing the other electrode (see [link]). Figure 11B The yellow line in the image covers port B and the hole area of ​​the container. (For example...) Figure 11C Electrophoresis is performed followed by electrotransfer, as shown. The top electrode, cathode A, can be placed on the buffer core (ion reservoir), and electrodes A and C are activated for electrophoresis. Electrode C is then turned off, and electrodes A and E are activated for electrotransfer. Once electrotransfer is complete, biomolecules are transferred to the biomolecule receiving material on one side of the container. Figure 11C As shown in the last figure, the liquid matrix can be drained from the container via port C, and the device can now perform detection by adding test reagents via port B. Figure 11D A front view of the apparatus is shown after the separation and / or detection of biomolecules. Figure 12AAnother embodiment of the device of this disclosure, in a vertical orientation, is depicted, having a container with two ports (port B and port C) closed on all sides, and biomolecule receiving material (shown in blue) disposed on the sides of the container. Port B is sealed with a removable plug (this seal forms part of the outer shell of the container). In this embodiment, as... Figure 12A As shown in the right figure, port C is a puncture-resistant and self-sealing port used to fill the container with a liquid matrix. Once the liquid matrix is ​​filled to the beginning of port B, a stopper (shown in red) is used to seal port B at the top. The container of the device further includes an electrode E (shown in yellow) adjacent to an insulator (shown in black line), an electrode C located in port C (shown in yellow), and an electrode T located on or within the sidewall of the container on the side opposite the biomolecule receiving material. Before use, as... Figure 12B As shown, remove the plug; this area will be used as a sample well. Add the sample to this sample well and use a cover containing the other electrode (see [link]). Figure 12B The yellow line in the image covers port B and the sample port area of ​​the container. (e.g., ...) Figure 12C Electrophoresis is performed followed by electrotransfer. The top electrode, cathode A, can be placed on the buffer core (ion reservoir), and electrodes A and C are activated for electrophoresis. Electrode C is then turned off, and electrodes A and E are activated for electrotransfer. Once electrotransfer is complete, biomolecules are transferred to the biomolecule receiving material on one side of the container.

[0147] like Figure 12C As shown in the last figure, the liquid matrix can be drained from the container via port C, and the device can now perform detection by adding test reagents via port B. Figure 12D A front view of a device after biomolecule separation and / or detection is shown. In some embodiments, a device includes: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, a third electrode, and a fourth electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned toward the rear end of the matrix; the third electrode being in electrical contact with the biomolecule receiving material and positioned behind the rear end of the matrix; the fourth electrode being positioned above the matrix; and at least one ion reservoir. In some embodiments, the first and second electrodes, as well as the third and fourth electrodes, can be activated and deactivated.

[0148] Figure 13An exemplary embodiment of the components of the device disclosed herein is shown, wherein the container is a flat surface such as a glass slide or a plastic glass slide. The container of the device comprises two layers of nitrocellulose coating as biomolecule receiving regions. Above this is a matrix (shown herein as PAG) having sample wells in a PAG (see the white area in the top view and the gray area in the 3-D view for the matrix). The top view and 3D view show an example container (shown herein as a glass slide, but could also be a plastic glass slide) with a length of 8 cm, a width of 2.5 cm, and a thickness of 1 mm. Top figures numbered 1, 2, 3, and 4 represent multiple electrodes ( Figure 13 The diagram shows four electrodes arranged on top of an ion reservoir (shown here as a buffer core). The top view diagram below shows the layers from top to bottom, including the bottom glass slide, a 6 cm layer of nitrocellulose (shown in blue), and a 3 cm layer of PAG. The electrodes with buffer cores are positioned on top of the device.

[0149] Figure 13 The side view shows an exemplary size of the sample well in the PAG, which in this embodiment has a pore depth of 0.4 mm for sample addition, while the total height of the PAG is 0.8 mm. An exemplary biomolecular receiving material is shown below, here depicted as nitrocellulose with a thickness of 20 µM, consisting of two layers with different pore sizes of 0.2 µM and 5 µM, respectively. As described in the previous sections, the first layer is used for protein binding, and the second layer is used to facilitate current flow. The first layer can be approximately 10 µm thick, with pore sizes of 0.2 µm or 0.45 µm. The second layer, approximately 10 µm thick, with pore sizes of 5 µm, is located directly below the first layer. A 3 cm long, 2 cm wide, and 0.8 mm thick polyacrylamide gel (PAG) is cast onto the top of the nitrocellulose, as shown below. Figure 13 As shown. For sample addition purposes, several sample wells approximately 0.25 mm deep can be formed in the PAG approximately 10 mm from one end. The well depth can be designed to provide sufficient separation from the bottom of the PAG to allow proteins to migrate along a straight path above the nitrocellulose layer for electrophoretic resolution. For example, wells too close to the bottom surface may cause protein channels to come into premature contact with the nitrocellulose, resulting in a streaking effect in the latter. Electrodes 1, 3, and 4 shown in this example embodiment are 2 cm long × 4 mm wide, while electrode 2 has the same dimensions as the PAG. Electrodes 1 and 3 are used for electrophoretic separation, and electrodes 2 and 4 are used to transfer current from the PAG via the nitrocellulose to the positive electrode. All cores are pre-soaked in 2 ml of 1X electrophoresis buffer.

[0150] Figure 14A , 14BFigures 14C and 14C illustrate one embodiment of a device of the present disclosure, comprising a thin layer of a matrix (such as PAG) and a thin layer of a biomolecule receiving material (such as NC). In some embodiments, these devices are capable of penetrating gel detection, allowing detection reagents and components (e.g., but not limited to antibodies, antigens, and detectable labels and reagents) to penetrate the thin layer of the matrix to reach biomolecules on the biomolecule receiving material electrically transferred thereon. In some embodiments, the thin layer of the matrix gel has a thickness of about 20 µm to about 200 µm, including thicknesses between 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 120 µm, 130 µm, 140 µm, 150 µm, 160 µm, 170 µm, 180 µm, 190 µm, and 200 µm, and in some embodiments about 100 µm. In some non-limiting embodiments, the thin layer of the matrix may comprise an SDS-PAGE gel layer. In some embodiments, the thickness of the biomolecule receiving material in the device disclosed herein may be about half the thickness of the thin matrix layer.

[0151] like Figure 14B As shown in the side view of the device, the detection reagents can be easily layered or washed on the device, and the detection of biomolecules can be achieved. As described in the previous embodiments, Figure 14A The device shown in -C has one port and a four-electrode arrangement of electrodes B, C, E and T.

[0152] Therefore, as Figure 14A As shown in -C, this disclosure describes an apparatus for electrophoresis, electrotransfer, and detection of biomolecules, wherein the apparatus comprises: at least one container comprising: a matrix layer operable to separate biomolecules along its length, the matrix having at least one port (e.g., Figure 14A Port B in the middle); biomolecule receiving material, which is placed below at least a portion of the matrix (in a plane parallel to the matrix if the matrix has a uniform thickness, or inclined to the matrix if the matrix has a non-uniform thickness); first electrode (e.g. Figure 14A Electrode B), which is in electrical contact with the matrix and is positioned before or within the port; a second electrode (such as...) positioned after the matrix... Figure 14A The first electrode (E) is in contact with the second electrode, which is electrically connected to the biomolecule receiving material; the second electrode (e.g., the third electrode) is in contact with the third electrode. Figure 14A Electrode C), which is electrically contacted with the matrix and positioned after the biomolecule receiving region; a fourth electrode (e.g., located above at least a portion of the matrix) Figure 14A The electrode T in the middle, where port (port B) can receive a sample and (later) receive one or more reagents for detecting biomolecules.

[0153] 2) Systems for electrophoresis and / or electrotransfer and / or detection:

[0154] In some embodiments, this disclosure provides systems for performing gel electrophoresis, electrotransfer, or detection, wherein one of the devices described in the preceding sections may be placed in or on an instrument capable of providing power and / or means to activate and / or inactivate an electrode. In some embodiments, this disclosure provides systems for performing gel electrophoresis, electrotransfer, and / or detection, wherein one of the devices described in the preceding sections may be connected to an electrical interface or power interface to activate and / or inactivate an electrode. In some embodiments, this disclosure provides systems for performing gel electrophoresis, electrotransfer, or detection, wherein one of the devices described in the preceding sections may be placed in an instrument or additionally have proximity or functionality to a source of reagents for biomolecular detection. In other non-limiting embodiments, such functionality may include capillary, conduit, channel, or microchannel pumps and valves, timers, and / or software components for dispensing and / or regulating reagent flow rates to regulate reagent release / timed release and / or regulate electrode activation / inactivation and / or regulate electrode movement / repositioning during operation.

[0155] Some embodiments relate to systems comprising: any apparatus described herein, further comprising one or more of the following: an instrument or base capable of providing power; means for activating and / or deactivating electrodes; a capillary; a conduit system; a channel; a microchannel; a pump; a valve; a robotic arm or motor; means for regulating electrode movement / repositioning; means for dispensing and / or regulating reagent flow rates; and software components for regulating reagent release / timed release and / or regulating electrode activation / deactivation and / or regulating electrode movement / repositioning during operation.

[0156] II. method:

[0157] Embodiments of this disclosure relate to methods for performing gel electrophoresis and / or electrotransfer and / or detection of biomolecules contained in a sample using the apparatus and systems described herein. Non-limiting examples of biomolecules that can be electrophoretically or transferred or detected include, but are not limited to, nucleic acids, DNA, RNA, peptides, and proteins.

[0158] In one embodiment, a method for electrotransferring biomolecules comprises: 1) positioning a matrix containing one or more biomolecules in or on top of a container of a device, the biomolecules having been separated based on their molecular weight or charge, the device comprising: one or more containers, each container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located outside the region where the biomolecules are received onto the biomolecule receiving material; 2) performing electrotransfer of biomolecules from the matrix to the biomolecule receiving material, comprising: (i) positioning the first electrode and a first ion reservoir above at least a portion of the matrix in which the biomolecules are present; and (ii) activating the first and second electrodes to transfer the biomolecules onto the biomolecule receiving material. Examples of such methods and results are illustrated in Figures 4A and 4B (described in more detail above).

[0159] In one embodiment, a method for electrotransferring biomolecules comprises: 1) positioning a matrix containing one or more biomolecules in or on top of a container of a device, the biomolecules having been separated based on their molecular weight or charge, the device comprising: one or more containers, each container comprising: a biomolecule receiving material; a first electrode; and a second electrode, wherein the electrodes are arranged to allow current to flow in at least two directions, wherein a second direction of the current is along a plane of the biomolecule receiving material; 2) performing electrotransfer of biomolecules from the matrix to the biomolecule receiving material, comprising: (i) positioning the first electrode and a first ion reservoir above at least a portion of the matrix in which the biomolecules are present; and (ii) activating the first and second electrodes to transfer the biomolecules onto the biomolecule receiving material. Examples of such methods and results are illustrated in Figures 4A and 4B (described in more detail above).

[0160] In one embodiment, a method for performing electrophoresis and electrotransfer of biomolecules comprises: 1) loading a sample containing one or more biomolecules into a sample loading region of a device comprising (i.e., a device having two electrodes, wherein optionally the electrodes are mechanically movable) at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region having at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode, wherein the second electrode is in electrical contact with a biomolecule receiving material and is located outside the region where the biomolecules are received on the biomolecule receiving material; and at least one ion reservoir; 2 The electrophoresis of biomolecules in the matrix is ​​performed by activating two electrodes; 3) deactivating the first and second electrodes to stop the electrophoresis; and 4) performing the electrotransfer of biomolecules from the matrix to the biomolecule receiving material by: (i) moving / positioning the first electrode and the first ion reservoir onto at least a portion of the top of the matrix; (ii) moving / positioning the second electrode and the second ion reservoir past the distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material; (iii) activating the first and second electrodes such that biomolecules can be transferred from the matrix to the biomolecule receiving material; and (iv) once completed, deactivating the first and second electrodes to stop the electrotransfer. Examples of such methods and results are illustrated in Figures 5A and 5B (described in more detail above).

[0161] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample containing one or more biomolecules into a sample loading region of a device comprising (i.e., a device having two electrodes, wherein the electrodes are optionally mechanically movable) at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region having at least a first electrode and a second electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; a second electrode, wherein the second electrode is electrically contacted with a biomolecule receiving material and located behind a distal end of the matrix; and at least one ion reservoir; 2) by activating the two electrodes The process involves: 3) electrophoresis of biomolecules in the matrix; 4) deactivating the first and second electrodes to stop the electrophoresis; and 5) performing electrotransfer of biomolecules from the matrix to the biomolecule receiving material by: (i) moving / positioning the first electrode and the first ion reservoir onto at least a portion of the top of the matrix; (ii) moving / positioning the second electrode and the second ion reservoir beyond the distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material; (iii) activating the first and second electrodes such that biomolecules can be transferred from the matrix to the biomolecule receiving material; and (iv) deactivating the first and second electrodes once this is complete to stop the electrotransfer. Examples of such methods and results are illustrated in Figures 5A and 5B (described in more detail above).

[0162] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample containing biomolecules into a sample loading region in a matrix of a device of the present disclosure (such as a device having three electrodes), the device comprising: at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, and a third electrode; the first electrode being located above at least a portion of the matrix and in front of the sample loading region; the second electrode being located above at least a portion of the matrix and... And located near the rear end of the matrix; the third electrode is electrically contacted with the biomolecule receiving material and positioned behind the rear end of the matrix that is insulated from and separated from the third electrode; at least one ion reservoir of at least one of the adjacent electrodes; 2) electrophoresis of the sample in the matrix by activating the first and second electrodes; 3) deactivating the first and second electrodes to stop electrophoresis after the biomolecules are resolved; 4) electrotransfer of biomolecules from the matrix to the biomolecule receiving material by reactivating the first electrode and activating the third electrode, wherein the third electrode is electrically contacted with the biomolecule receiving material; and 5) deactivating the first and third electrodes to stop electrotransfer once completed.

[0163] Figure 7AA side view of an exemplary system with four ports and three electrodes is depicted, and the method steps for electrophoresis and electrotransfer of biomolecules are shown. Figure 7B A top view of the same system after the electrophoresis and electrotransfer of biomolecules have been completed is depicted. Figure 8A A side view of an exemplary system with three ports and three electrodes is depicted, and methodological steps for electrophoresis and electrotransfer of biomolecules are illustrated. Figure 8B A top view of the same system after the electrophoresis and electrotransfer of biomolecules have been completed is depicted.

[0164] In another embodiment, a method for performing electrophoresis and electrotransfer of biomolecules includes: 1) loading a sample or multiple samples containing biomolecules into a sample loading port of a device (e.g., a device having four electrodes): at least one container comprising: a matrix operable to separate biomolecules along its length, the matrix having a sample loading region at its front end; a biomolecule receiving material disposed in a parallel plane below the matrix; at least a first electrode, a second electrode, a third electrode, and a fourth electrode; the first electrode being positioned above at least a portion of the matrix and in front of the sample loading region; the second electrode being positioned toward the rear end of the matrix; the third electrode being aligned with the biomolecule receiving material. The biomolecule receiving material is electrically contacted and positioned at the rear end of a matrix that is insulated from and separated from the third electrode; a fourth electrode is positioned above the matrix; the fourth electrode is separated from the matrix by a transparent material having a higher resistance compared to the electrode material; and at least one ion reservoir; 2) electrophoresis of a sample in the matrix by activating the first and second electrodes; 3) when complete, deactivating the first and second electrodes to stop the electrophoresis; 4) electrotransfer of biomolecules from the matrix to the biomolecule receiving material by (i) activating the third and fourth electrodes; and 5) when the electrotransfer is complete. In a non-limiting example, a material separating the matrix and the fourth electrode may comprise agarose or another gel or agarose-like material about 0.1 mm to 5 mm thick. In some embodiments of the method described herein, one of the electrodes (e.g., T) is positioned along the edge of the container such that it does not obstruct observation of the biomolecule receiving material from either side of the device.

[0165] Figure 9A A side view of an exemplary system with four ports and three electrodes is depicted, and the method steps for electrophoresis and electrotransfer of biomolecules are shown. Figure 9B A top view of the same system is described after the electrophoresis and electrotransfer of biomolecules have been completed. As depicted here, electrode T is along the edge of the container (the edge of the channel in the container – see...). Figure 9B The positioning is such that it does not obstruct the observation of biomolecule-receiving materials when viewed from above to monitor the progress of electrophoresis and / or electrotransfer and / or immunoassay.

[0166] Figure 10A A side view of an exemplary system with three ports and three electrodes is depicted, and the method steps for electrophoresis and electrotransfer of biomolecules are shown. Figure 10B A top view of the same system is described after electrophoresis and electrotransfer of biomolecules have been completed. As depicted here, electrode T is along the edge of the container (channels / ducts / capillaries / paths in the container or multiple channels in the container, see...). Figure 10B The positioning is such that it does not obstruct the observation of biomolecule-receiving materials when viewed from above to monitor the progress of electrophoresis and / or electrotransfer and / or immunoassay.

[0167] Figure 11A An embodiment of the device of this disclosure in a vertical orientation is depicted, having a container with two ports (port B and port C) closed on all sides, and biomolecule receiving material (shown in blue) disposed on the sides of the container. Port B is sealed with a removable plug (this seal forms part of the outer shell of the container). In this embodiment, as... Figure 11A As shown in the right figure, port C is a puncture-resistant and self-sealing port used to fill the container with a liquid matrix. Once the liquid matrix is ​​filled to the beginning of port B, a stopper (shown in red) is used to seal port B at the top. The container of the device further includes an electrode E (shown in yellow) adjacent to an insulator (shown in black lines) and an electrode C (shown in yellow) located in port C.

[0168] In use, such as Figure 11B As shown, remove the plug; this area will be used as a sample well. Add the sample to this sample well and use a cover containing the other electrode (see [link]). Figure 11B The yellow line in the image covers port B and the hole area of ​​the container. (For example...) Figure 11C Electrophoresis is performed followed by electrotransfer, as shown. The top electrode, cathode A, can be placed on the buffer core (ion reservoir), and electrodes A and C are activated for electrophoresis. Electrode C is then turned off, and electrodes A and E are activated for electrotransfer. Once electrotransfer is complete, biomolecules are transferred to the biomolecule receiving material on one side of the container. Figure 11C As shown in the last figure, the liquid matrix can be drained from the container via port C, and the device can now perform detection by adding test reagents via port B. Figure 11D A front view of the apparatus is shown after the separation and / or detection of biomolecules.

[0169] Figure 12AAnother embodiment of the device of this disclosure, in a vertical orientation, is depicted, having a container with two ports (port B and port C) closed on all sides, and biomolecule receiving material (shown in blue) disposed on the sides of the container. Port B is sealed with a removable plug (this seal forms part of the outer shell of the container). In this embodiment, as... Figure 12A As shown in the right figure, port C is a puncture-resistant and self-sealing port used to fill the container with a liquid matrix. Once the liquid matrix is ​​filled to the beginning of port B, a stopper (shown in red) is used to seal port B at the top. The container of the device further includes an electrode E (shown in yellow) adjacent to an insulator (shown in black line), an electrode C located in port C (shown in yellow), and an electrode T located on or within the sidewall of the container on the side opposite the biomolecule receiving material.

[0170] Before use, such as Figure 12B As shown, remove the plug; this area will be used as a sample well. Add the sample to this sample well and use a cover containing the other electrode (see [link]). Figure 12B The yellow line in the image covers port B and the sample port area of ​​the container. (e.g., ...) Figure 12C Electrophoresis is performed followed by electrotransfer, as shown. The top electrode, cathode A, can be placed on the buffer core (ion reservoir), and electrodes A and C are activated for electrophoresis. Electrode C is then turned off, and electrodes A and E are activated for electrotransfer. Once electrotransfer is complete, biomolecules are transferred to the biomolecule receiving material on one side of the container. Figure 12C As shown in the last figure, the liquid matrix can be drained from the container via port C, and the device can now perform detection by adding test reagents via port B. Figure 12D A front view of the apparatus is shown after the separation and / or detection of biomolecules.

[0171] Figure 14A -C, This disclosure describes an apparatus for electrophoresis, electrotransfer, and detection of biomolecules, wherein the apparatus comprises: at least one container comprising: a matrix layer operable to separate biomolecules along its length, the matrix having at least one port (e.g. Figure 14A Port B in the matrix); biomolecule receiving material, which is placed in a parallel plane below the matrix; first electrode (e.g., port B); Figure 14A Electrode B), which is in electrical contact with the matrix and is positioned before or within the port; a second electrode (such as...) positioned after the matrix... Figure 14A The first electrode (E) is in contact with the second electrode, which is electrically connected to the biomolecule receiving material; the second electrode (e.g., the third electrode) is in contact with the third electrode. Figure 14A Electrode C), which is electrically contacted with the matrix and positioned after the biomolecule receiving region; a fourth electrode (e.g., located above at least a portion of the matrix) Figure 14AThe electrode (T) in the matrix can receive a sample and (later) receive one or more reagents for detecting biomolecules. In some embodiments, the matrix layer is a thin layer.

[0172] like Figure 14A As shown in -C, this disclosure describes a method for electrophoresis, electrotransfer, and detection of biomolecules, comprising: 1) loading a sample or plurality of samples containing biomolecules into a port of a device, the device comprising: wherein the device includes: at least one container comprising: a matrix layer operable to separate biomolecules along its length, the matrix having at least one port (e.g. Figure 14A Port B in the matrix); biomolecule receiving material, which is placed in a parallel plane below the matrix; first electrode (e.g., port B); Figure 14A Electrode B), which is in electrical contact with the matrix and is positioned before or within the port; a second electrode (such as...) positioned after the matrix... Figure 14A The first electrode (E) is in contact with the second electrode, which is electrically connected to the biomolecule receiving material; the second electrode (e.g., the third electrode) is in contact with the third electrode. Figure 14A Electrode C), which is electrically contacted with the matrix and positioned after the biomolecule receiving region; a fourth electrode (e.g., located above at least a portion of the matrix) Figure 14A 1) Electrophoresis of the sample in the thin matrix by activating the first electrode (electrode B) and the third electrode (electrode C); 2) Electrophoresis of the sample in the thin matrix by activating the first electrode (electrode B) and the third electrode (electrode C); 3) Electrophoresis of the first electrode and the third electrode by deactivating the first electrode (electrode E) and the fourth electrode (electrode T) to stop the electrophoresis; 4) Electrotransfer of the biomolecule from the thin matrix to the biomolecule receiving material by activating the second electrode (electrode E) and the fourth electrode (electrode T); 5) Electrotransfer of the second electrode and the fourth electrode by deactivating the second electrode (electrode C) when the electrotransfer is complete; and 6) Adding one or more detection reagents to a single port (port B), or adding one or more detection reagents to the matrix layer, and allowing the detection reagents to pass through the matrix into the biomolecule receiving material to contact and detect the biomolecules. In some embodiments, the matrix layer is a thin matrix layer with a thickness of about 20 µm to about 200 µm, including thicknesses such as 20 µm, 30 µm, 40 µm, 50 µm, 60 µm, 70 µm, 80 µm, 90 µm, 100 µm, 120 µm, 130 µm, 140 µm, 150 µm, 160 µm, 170 µm, 180 µm, 190 µm, and 200 µm. In some embodiments, the thickness of the thin layer is about 100 µm. In some non-limiting embodiments, the thin matrix layer may comprise an SDS-PAGE gel layer. In some embodiments, the thickness of the biomolecule receiving material in the device of this disclosure may be about half the thickness of the thin matrix layer.

[0173] In some embodiments, one or more ports, or any pair or combination of ports, such as ports AC, AD, BC, or BD, may be used to facilitate the addition and removal of buffer solutions, antibody solutions, and detection reagents (such as chemiluminescent reagents). In some embodiments, ports may be used in various ways if protein blotting is automated by evacuating the liquid matrix from a container (such as a channel) after electrotransfer and allowing the detection reagent to flow into the same container / channel that now contains biomolecule-receiving material having the biomolecules electrotransferred therein.

[0174] In some embodiments of the methods disclosed herein, as described differently in the preceding sections, the apparatus used comprises multiple containers, and multiple samples are loaded into the sample loading region of each container. In some embodiments, the biomolecules to be electrophoresed and / or electrotransferred are stained prior to loading. The biomolecules are pre-stained.

[0175] In some embodiments of the methods disclosed herein, as described differently in the preceding sections, biomolecules may be visualized during electrophoresis and electrotransfer. In some embodiments, the methods of this disclosure may include visualizing biomolecules from a top observation device during or at the end of the electrophoresis and / or electrotransfer steps. In some embodiments, the methods of this disclosure may further include imaging biomolecules during or at the end of the electrophoresis and / or electrotransfer steps.

[0176] The systems, apparatus, boxes, and methods disclosed herein advantageously result in one or more of the foregoing, including: an apparatus / system for electrophoresis and electrotransfer capable of simultaneously performing high-throughput electrophoresis and / or electrotransfer of biomolecules, which, compared to existing systems and apparatuses for electrophoresis or electrotransfer, reduces the number of devices or components, lowers costs, reduces accessories, reduces the footprint of required equipment, reduces storage space for the system and apparatus, eliminates buffer spills and leaks, reduces the amount of gels, biomolecule receiving materials, samples, buffers, and other components used, and reduces liquid hazardous waste.

[0177]

[0178] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference.

[0179] While embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used to practice the present disclosure. It is contemplated that the following claims define the scope of the present disclosure and thus cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A device for the electrotransfer of biomolecules, comprising: At least one container comprising: Biomolecular receiving materials; First electrode; and The second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located at the end of the region receiving biomolecules, in, The first direction of current flow between the first electrode and the second electrode is perpendicular to the plane of the biomolecule receiving material or diagonally opposite to the plane of the biomolecule receiving material. The biomolecule receiving material is placed on the container.

2. The apparatus according to claim 1, characterized in that, The biomolecule receiving material is located in a plane, and The second electrode is located above the biomolecule receiving material, or the second electrode is positioned along the same plane as the biomolecule receiving material, or the second electrode is located below the biomolecule receiving material.

3. The apparatus according to claim 1, characterized in that, The first electrode and the second electrode are arranged to allow current to flow in at least two directions, wherein the second direction of current flow is along the plane of the biomolecule receiving material.

4. The apparatus according to claim 1, characterized in that, The container is a channel, a glass slide, or a vessel.

5. The apparatus according to claim 1, characterized in that, It contains multiple containers.

6. The apparatus according to claim 1, characterized in that, The container is made of a transparent material.

7. The apparatus according to claim 1, characterized in that, The device is a microfluidic device.

8. The apparatus according to claim 1, characterized in that, The biomolecule receiving material comprises a membrane, which includes nitrocellulose, PVDF, cellulose acetate, anodic aluminum oxide, or one or more conductive materials embedded in any of the aforementioned materials.

9. The apparatus according to claim 8, characterized in that, The conductive material embedded in the biomolecule receiving material comprises tantalum, copper, indium, or tin oxide.

10. The apparatus according to claim 1, characterized in that, The biomolecule receiving material contains one or more ions.

11. The apparatus according to claim 1, characterized in that, The biomolecule receiving material has a pore size of 0.2µm to 10µm.

12. The apparatus according to claim 1, characterized in that, The biomolecule receiving material has different pore sizes in different regions of the same layer of the biomolecule receiving material.

13. The apparatus according to claim 1, characterized in that, The biomolecule receiving material has one or more layers.

14. The apparatus according to claim 13, characterized in that, Each layer of the biomolecule receiving material has a different pore size.

15. The apparatus according to claim 1, characterized in that, The first electrode is located above at least a portion of the biomolecule receiving material.

16. The apparatus according to claim 15, characterized in that, The first electrode is further located before the region where biomolecules are received on the biomolecule receiving material.

17. A system for the electrotransfer of biomolecules, comprising the apparatus according to claim 1.

18. A method for electrophoresis and electrotransfer of biomolecules, comprising: 1) Loading a sample containing one or more biomolecules into a sample loading region of a device, the device comprising: At least one container comprising: A matrix operable to separate the biomolecules along its length, the matrix having the sample loading region at its front end; A biomolecule receiving material is placed below the matrix and on the container; At least the first electrode and the second electrode; The first electrode is located above at least a portion of the matrix and in front of the sample loading region; The second electrode is in electrical contact with the biomolecule receiving material and is located at the end of the region where the biomolecule is received by the biomolecule receiving material; as well as At least one ion storage device; 2) Electrophoresis of the biomolecules in the matrix is ​​performed by activating the two electrodes; 3) Deactivate the first and second electrodes to stop the electrophoresis; as well as 4) The electrotransfer of the biomolecule from the matrix to the biomolecule receiving material is performed in the following manner: (i) Positioning the first electrode and the first ion reservoir onto at least a portion of the top of the matrix; (ii) Position the second electrode and the second ion reservoir beyond the distal end of the matrix such that the second electrode and the second ion reservoir are electrically connected to the biomolecule receiving material; (iii) Activate the first electrode and the second electrode so that the biomolecule can be transferred from the matrix to the biomolecule receiving material; as well as (iv) Once completed, deactivate the first and second electrodes to stop the electrotransfer.

19. An apparatus for the electrotransfer of biomolecules, comprising: At least one container comprising: Biomolecular receiving materials; First electrode; and The second electrode, wherein the second electrode is in electrical contact with the biomolecule receiving material and is located at the end of the region receiving biomolecules, in, The biomolecule receiving material has different pore sizes in different regions of the same layer of the biomolecule receiving material. The biomolecule receiving material is placed on the container.

20. The apparatus according to claim 19, characterized in that, The biomolecule receiving material is located in a plane; and wherein the second electrode is located above the biomolecule receiving material, or wherein the second electrode is positioned along the same plane as the biomolecule receiving material, or wherein the second electrode is located below the biomolecule receiving material.

21. The apparatus according to claim 19, characterized in that, The first electrode and the second electrode are arranged to allow current to flow in at least two directions; wherein the first direction of current flow is diagonal to or perpendicular to the plane of the biomolecule receiving material; and wherein the second direction of current flow is along the plane of the biomolecule receiving material.

22. The apparatus according to claim 19, characterized in that, The container is a channel, a glass slide, or a vessel.

23. The apparatus according to claim 19, characterized in that, It contains multiple containers.

24. The apparatus according to claim 19, characterized in that, The container is made of a transparent material.

25. The apparatus according to claim 19, characterized in that, The device is a microfluidic device.

26. The apparatus according to claim 19, characterized in that, The biomolecule receiving material comprises a membrane, which includes nitrocellulose, PVDF, cellulose acetate, anodic aluminum oxide, or one or more conductive materials embedded in any of the aforementioned materials.

27. The apparatus according to claim 26, characterized in that, The conductive material embedded in the biomolecule receiving material comprises tantalum, copper, indium, or tin oxide.

28. The apparatus according to claim 19, characterized in that, The biomolecule receiving material contains one or more ions.

29. The apparatus according to claim 19, characterized in that, The biomolecule receiving material has a pore size of 0.2µm to 10µm.

30. The apparatus according to claim 19, characterized in that, The biomolecule receiving material has one or more layers.

31. The apparatus according to claim 30, characterized in that, Each layer of the biomolecule receiving material has a different pore size.

32. The apparatus according to claim 19, characterized in that, The first electrode is located above at least a portion of the biomolecule receiving material.

33. The apparatus according to claim 32, characterized in that, The first electrode is further located before the region where biomolecules are received on the biomolecule receiving material.

34. A system for the electrotransfer of biomolecules, comprising the apparatus according to claim 19.

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