Patent Description:
Understanding cell-to-cell variation in protein expression is a large part of understanding the pathogenesis of tumors, characterizing the differentiation states of stem cells, and developing well-controlled and functionally-validated in vitro human 'disease in a dish' models for drug development, target screening, and toxicity studies. Single-cell analysis is of growing importance, but has been largely limited to transcriptome (e.g., RNA) and genome (e.g., DNA) technologies. Importantly, these measurements do not always correlate with the protein levels that dictate phenotype. Single-cell protein measurements represent an unsurmounted hurdle in stem cell, cancer, and immunology research.

Methods to determine protein level expression of a biological sample include electrophoresis followed by probe analysis. Electrophoresis is a technique that typically applies an electric field to a biological sample in a separation medium, such that individual molecules of the biological sample will disperse throughout the separation medium based on molecular size and charge. Cell lysis prior to electrophoresis, may reduce the biological sample to a set of individual molecules that can effectively separate throughout the separation medium. Cell lysis, electrophoresis, and subsequent probe analysis are typically executed on separate platforms with extensive user manipulation. <CIT> discloses an electrophoresis device comprising a holder defining a recess area containing a cassette with gel such that the top surface of the cassette is flush with a non-recessed surface of the holder, and an electrode for producing an electric field across the recess area.

<CIT> discloses methods and apparatus for reference lab diagnostics, wherein target analytes are resolved by isoelectric focusing in channels of a microfabricated silicon device which is located in a recess area of a holder provided with electrodes producing an electric field across the recess area.

<CIT> discloses a transilluminator base and scanner for imaging fluorescent gels, charging devices and portable electrophoresis systems. <CIT> discloses a cartridge for conducting electrophoresis in view of separation and subsequent transfer of macromolecules to a membrane to facilitate detection thereof. The electrophoresis cartridge comprises a tray with a bottom surface defining a recess area containing a support covered by a fluid-permeable membrane which binds nucleic acids, an electrode for producing an electric field across the recess area, and a dam disposed at the end of the tray for controlling the flow of solution below the dam and across the recess area.

Thus, a need exists for devices, systems, and methods for assaying for a presence of various types of analytes using a single, self-contained system.

The scope of protection of the present invention is defined in the appended claims.

The novel features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present invention are utilized, and the accompanying drawings (also "figure" and "FIG. " herein), of which:.

While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments described herein may be employed.

As used in this specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "a member" is intended to mean a single member or a combination of members, "a material" is intended to mean one or more materials, or a combination thereof.

As used herein, the term "protein" refers to proteins, oligopeptides, peptides, and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs, and peptidomimetic structures.

As used herein, the term "analyte" refer to any molecule or compound to be detected, as described herein. Suitable analytes can include but are not limited to, small chemical molecules such as, for example, environmental molecules, clinical molecules, chemicals, pollutants, and/or biomolecules. More specifically, such chemical molecules can include but are not limited to pesticides, insecticides, toxins, therapeutic and/or abused drugs, hormones, antibiotics, antibodies, organic materials, proteins (e.g., enzymes, immunoglobulins, and/or glycoproteins), nucleic acids (e.g., DNA and/or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and/or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, nutrients, electrolytes, growth factors and other biomolecules and/or non-biomolecules, as well as fragments and combinations thereof. Some analytes described herein can be proteins such as enzymes, drugs, cells, antibodies, antigens, cellular membrane antigens, and/or receptors or their ligands (e.g., neural receptors or their ligands, hormonal receptors or their ligands, nutrient receptors or their ligands, and/or cell surface receptors or their ligands).

As used herein, the term "sample" refers to a composition that contains an analyte or analytes to be detected. A sample can be heterogeneous, containing a variety of components (e.g., different proteins) or homogenous, containing one component. In some instances, a sample can be naturally occurring, a biological material, and/or a man-made material. Furthermore, a sample can be in a native or denatured form. In some instances, a sample can be a single cell (or contents of a single cell) or multiple cells (or contents of multiple cells), a blood sample, a tissue sample, a skin sample, a urine sample, a water sample, and/or a soil sample. In some instances, a sample can be from a living organism, such as a eukaryote, prokaryote, mammal, human, yeast, and/or bacterium or the sample can be from a virus. In some instances, a sample can be one or more stem cells (e.g., any cell that has the ability to divide for indefinite periods of time and to give rise to specialized cells). Suitable examples of stem cells can include but are not limited to embryonic stem cells (e.g., human embryonic stem cells (hES)), and non-embryonic stems cells (e.g., mesenchymal, hematopoietic, induced pluripotent stem cells (iPS cells), or adult stem cells (MSC)).

The term "level," as used herein, generally refers to variation from a plane that is orthogonal to the gravitational acceleration vector.

The present disclosure is directed to automated systems for performing analyte detection, such as western blotting. Systems provided herein can be used to detect analytes in samples of substantially low volume, such as single cell analysis. In some examples, such analysis can be performed using single cell western blotting (scWB), which employs microfluidic design to introduce tried-and-true microarray formats that combine: (i) protein molecular weight determination via electrophoresis with (ii) protein identity determination via subsequent antibody-based probing of resolved protein bands. This two-stage assay can include western blotting. This two-stage analysis can offer ultra-high specificity performance at the bench, without the need for costly infrastructure or core facilities. The scWB brings high-specificity protein assays to single-cells, while taking advantage of the vast reagent infrastructure already available for conventional western blotting.

Widespread implementation of scWB can impact biomedicine through: a) elucidating intratumor cell-to-cell heterogeneity, thus advancing therapeutic efficacy of cancer drugs; b) assessing patient-specific cell-level response for companion diagnostics to targeted therapy drug cocktails; c) quantifying the purity and safety of cell-based therapies before implantation, which may accelerate regulatory approval and lead to the creation of safer, more effective cell-based therapies; d) enabling analysis of rare cell populations from patients (e.g., circulating tumor cells, CTCs) for targeted therapies; and e) enabling characterization of rare cell therapies such as iPSC-derived somatic cells to accelerate creation of functional tissues and in vitro disease models for drug development. Single-cell western-based protein-level information may provide a transformative leap towards realizing personalized and regenerative medicine.

To achieve microarray-like density of single-cell resolution western blots, the scWB can use a microscope slide coated with a thin photoactive polymer stippled with thousands of microwells having a diameter that is about <NUM> microns or less. In some embodiments, each microwell can have a diameter in a range of about <NUM> microns to about <NUM> microns in diameter (see e.g., <FIG>). The chip can be fabricated using soft lithography. The chip can be removable, consumable, disposable, or combinations thereof. The chip can be a scWB chip. The scWB workflow can initiate when a cell suspension is dispensed on top of the microwell array having a gel, with single cells settling into individual microwells passively under the action of gravity. Microwell occupancy can be controlled by limiting dilution and microwell design (e.g., microwells sized to accommodate single cells). After cell lysis and application of an electric field across the gel, the photo-active gel, which can comprise benzophenone-methacrylamide, can act as a sieving matrix that separates molecules (such as proteins) according to their molecular weight over short separation distances (<<NUM>). With a brief UV exposure, the benzophenone-methacrylamide co-monomer can covalently immobilize proteins via hydrogen abstraction, which can yield capture efficiencies approaching <NUM>%. Thus, protein bands can be fixed in position in the gel after the size-based separation and subsequently the gel can be probed with antibodies which can obviate the transfer and blocking steps typically used in traditional western blotting approaches. In some instances, a scWB can be performed without pumps, valves, precise alignment or complex electrical or pneumatic interfaces typically found in scWB systems (e.g., mass spectrometers, mass cytometry, flow cytometers, etc.). Multiplexing of targets can be achieved by size separation, multispectral detection, and/or repeated stripping and re-probing of the captured proteins.

On one microscope slide, thousands or more of single cells can be assayed in parallel and as little as about <NUM>-<NUM> femtograms or less of target protein per cell can be detected which, in some instances, can impart higher throughput and analytical sensitivity than mass spectrometry. Given the widespread use of conventional western blotting and less strict antibody specificity requirements, about <NUM> times more commercially-available antibodies can be validated for western blotting as compared to flow cytometry. Cell lysis can allow for measurement of diverse targets such as intracellular proteins, protein complexes, and post-translational modifications that may not accessible using flow cytometry.

Flow cytometry is an established single-cell proteomic tool with high throughput. Yet flow cytometry can require high antibody specificity and intracellular proteins can be more challenging to detect. Mass cytometry can make single-cell proteomic measurements but may use custom heavy metal-tagged antibodies, expensive instrumentation (e.g., $<NUM>,<NUM> per instrument) and high antibody specificity. Mass spectrometry is emerging as a single-cell analysis tool, yet it may require expert spectra interpretation, and can have low throughput and low analytical sensitivity. Automated western blotting instrumentation exists but is unable to reach single-cell resolution. In some instances, this is attributable in part to capture efficiencies of about <NUM>% versus about <NUM>% for the scWB.

In some embodiments, a system for scWB can include instrumentation that can integrate the electrophoresis and UV capture steps. The present disclosure described here also provides for safety interlocking (to prevent user exposure to hazardous high voltage and UV light) and enables integrated electrophoresis and UV exposure on a microscope slide format. While the initial application of the instrument can perform the scWB assay, the same present disclosure can enable related assays such as small volume (but not single-cell) western blotting or small volume, parallel electrophoresis assays.

The present disclosure presents a system for performing electrophoretic separations and photocapture of separated molecules within a thin gel attached to a planar substrate. The system can perform single-cell resolution western blotting (scWB) in a thin (<<NUM> micron thick) gel layer on a microscope slide. It can be a system comprising an instrument, software, associated accessories, and assay methods.

The system is shown in <FIG>. The main unit <NUM> (also referred to herein as "instrument" and/or "device") can be comprised of a base <NUM>, a lid <NUM>, an integrated computer (such as a tablet computer with a touch screen, <NUM>), a high voltage power supply (that can be contained in a compartment <NUM>) and a source of activation energy (e.g., radiation energy) such as an ultraviolet (UV) light source <NUM>. The main unit <NUM> is configured to receive an electrophoresis cell <NUM> and can be used to perform an assay on a biological sample included in and/or otherwise contained by the electrophoresis cell <NUM>, as described in further detail herein. The system can be controlled by a computer, microprocessor or microcontroller which can either be integrated into the main unit <NUM> or separate from but in communication with the main instrument body or housing (e.g., through a wired or wireless data connection). Integration of a computer with the instrument body can allow for flexible integration of instrument control and data management in a compact form. For example, the tablet computer <NUM> can have a built-in sensor (e.g., camera) that can be used to read barcodes printed on a chip, for example, a scWB consumable chip. The chip can be adapted, configured, or otherwise suitable for use in scWB. The sensor can be used to read or identify one or more identification members, such as identification numbers, on the removable electrophoresis cell. Each of the one or more identification members can be unique. The one or more identification members can include one-dimensional or two-dimensional identification barcodes. The one or more identification members can include radio frequency identification (RFID) units. The sensor can store the one or more identification members, such as identification numbers, in memory coupled to the computer processor. The memory can be part of the system or a remote computer system, such as a remote server in network communication with the system (e.g., the "cloud"). A first identification member of the one or more identification members can determine a first set of assay parameters for the assaying. The computer processor can be programmed to upload the first unique identification member, the first set of assay parameters, or a combination thereof to a computer network. In some cases, the instrument can include detection of the presence of one or more target analytes and the computer processor can be programed to upload the presence of one or more target analytes. The barcode can then be used to determine a set of assay parameters to be used for running the barcoded chip. Once the run is complete, the tablet can upload run information along with the barcode to a computer network, such as a network server. The server can later be accessed by a separate reader instrument so that imaging data and electrophoresis run data can be associated with the same chip through the barcode.

The main unit <NUM> can include a frame <NUM> which can position and hold the touch screen or tablet computer <NUM> and can hide any cable connections to the tablet computer or screen. The source of activation energy, such as the UV light source <NUM>, can be a fluorescent bulb, LED, an Hg-Xe source, or other common sources. The instrument body can include a curved surface <NUM> to accommodate a cylindrical-shaped UV source (such as a fluorescent lamp) while maintaining a compact and aesthetically pleasing profile. The exterior face of the UV light source <NUM> can be an optical filter or a window that can reduce the light intensity (e.g., a neutral density filter) or that can control the wavelengths of light that pass through such that optimal wavelengths can reach the scWB consumable chip. The filter or window can be heated or treated with a coating to reduce condensation of buffer during electrophoresis.

In some embodiments, sensitive and high voltage components can be sealed within a separate compartment <NUM> so that any liquid (e.g., buffer solution) spilled inside the instrument <NUM> cannot come in contact with the electronics. In such an example, air cooling and venting can occur from the bottom of the instrument. The lid <NUM> can rotate open about axis <NUM> and can be supported by a friction hinge <NUM> which can allow the lid <NUM> to stay open at various angles. The lid <NUM> can close if opened less than a threshold angle (e.g., <NUM> degrees or any other suitable angle). The lid <NUM> may include dampening mechanisms to slow the lid <NUM> closure, and can latch closed using one of various methods known to one skilled in the art (e.g., magnetic or mechanical latches). Leveling feet <NUM> can be used to level the instrument base <NUM> such that liquid (e.g., buffer solution) in the electrophoresis cell <NUM> can be level. Leveling can be accomplished by placing a standard bubble level in the electrophoresis cell <NUM> while adjusting the leveling feet <NUM> or the instrument <NUM> can include a permanently mounted bubble level or an electronic leveler that can interface to the computer <NUM> which in turn can inform the user if the instrument <NUM> is level. In some embodiments, the instrument <NUM> can include any suitable device and/or mechanism configured to automatically or at least semi-automatically level the instrument.

In an example, a chip <NUM> (<FIG>), such as a disposable or consumable chip that is <NUM> long, being <NUM> degree "off level" can have a fluid height that can vary by <NUM> across the top of the chip. If the nominal height of the fluid <NUM> (<FIG>) is only <NUM>, then being off level can result in about a <NUM>% variation in fluid height which can result in a <NUM>% variation in the electric field.

Interlock switches <NUM> can ensure that the lid <NUM> is closed before hazardous UV and high voltage (HV) are enabled. The instrument <NUM> can be configured to disconnect the HV power supply from the pogo pins <NUM> when the lid <NUM> is opened and/or the HV supply can be disabled and the residual charge can be dissipated to ground. The main body of the instrument <NUM> may include vents which can prevent accumulation of explosive electrolysis gases inside the instrument body. Light blocking features (such as a raised rim around the base <NUM> of the instrument <NUM>) may be included, which can minimize any leakage of UV light when the lid <NUM> is closed.

As shown in <FIG>, the main instrument body or housing can include a receptacle <NUM> that can hold an electrophoresis cell <NUM>. In some instances, the electrophoresis cell <NUM> can be modular, removable, or a combination thereof. In an example, an electrophoresis cell, such as the modular removable electrophoresis cell <NUM> can be aligned using one or more alignment pins <NUM> which can interface to corresponding slots and/or holes on the modular removable electrophoresis cell <NUM>. This modular design can allow for any number of removable electrophoresis cell designs to be used in the main instrument unit <NUM>. Some such designs can accommodate different chips which can fit into a recess area <NUM> of the electrophoresis cell <NUM>, different electrode configurations, or electrical resistance that can simulate a filled removable electrophoresis cell to allow for convenient dry testing of the system. In some embodiments, any number of slots can be formed in the main instrument body or housing to accommodate the one or more pins. An electrical connection can be formed when one or more pins are adjacent to the one or more conductive pads of the removable electrophoresis cell <NUM>. In an example, the slots can be configured to accommodate various removable electrophoresis cell form factors and/or various electrode configurations. In an example, the slots can remain spatially fixed and the pin configuration can be modular. In another example, the slots and pin configuration of the housing can remain spatially fixed and the configuration of the contact pads of the modular removable electrophoresis cell can also remain spatially fixed. In this example, the electrical connections and mechanical features within the modular electrophoresis cell can be reconfigured for different functions, such as a plurality of removable electrophoresis cell designs.

Electrical connection can be made to the removable electrophoresis cell <NUM> via spring-loaded pins, i.e., "pogo pins," <NUM> which can connect to conductive contact pads <NUM> on the electrophoresis cell <NUM>. This interface can allow for dry contact to the HV pogo pins <NUM>. The electrical contact pads <NUM> can be connected to conductors (for example, platinum or carbon conductors) which can contact the liquid (e.g., buffer solution) in the removable electrophoresis cell <NUM>. The instrument <NUM> can control the voltage difference between the pogo pins <NUM> such that the electric field strength in the removable electrophoresis cell can be programmatically controlled. In an example, the instrument <NUM> can programmatically change the polarity of the pogo pin voltages such that the direction of the electric field within the removable electrophoresis cell can also be controlled via software. Additional pins and contact pads may be added to accommodate different removable electrophoresis cell designs, if more than two voltages are needed. Although not shown, in some embodiments, the receptacle <NUM> is surrounded by a grounding ring such that any fluid that spills outside of the electrophoresis cell <NUM> during operation will be electrically grounded once it crosses over the grounding ring. The grounding ring can be any suitable shape, size, and/or configuration and can be formed from any suitable grounding material.

The receptacle <NUM> and removable electrophoresis cells can be transparent such that an additional UV light source can be included below the receptacle <NUM>. The transparent surfaces can include lens elements to help focus light from a UV source. The imaging instrumentation (e.g., lenses, cameras, photodetectors, etc.) can be included below the receptacle <NUM>. The imaging instrumentation can enable brightfield or fluorescence imaging of the chip, before, during, or after electrophoresis. In an example, the surface of the receptacle <NUM> can be reflective such that UV light from the UV source <NUM> can be reflected back to the chip to increase the UV flux. In another example, the surface of the receptacle <NUM> can include ultrasonic transducers (or the like) which can transmit energy to a chip seated in the recess area <NUM> and can assist in lysis of cells contained within the chip. In a further example, the receptacle <NUM> can incorporate temperature control (heaters and/or coolers) to assist with lysis, to control heating during electrophoresis, or to provide temperature cycling or control for desired chemical reactions (e.g., for melting or annealing of nucleic acid-tagged detection probes).

<FIG> shows an example of an electrophoresis cell, such as the removable electrophoresis cell <NUM>. The removable electrophoresis cell <NUM> can have a body 120A that defines the recess area <NUM> designed to accommodate a chip <NUM>, an example of which can be a scWB consumable chip having outer dimensions equal to a standard microscope slide. Conductive contact pads <NUM> are coupled to the body 120A and can provide an electrical interface to the instrument. Platinum wire <NUM> can connect from the contact pads <NUM> into the body 120A of the removable electrophoresis cell <NUM>. The platinum wire <NUM> can be inserted into nonconductive tubing such that the portion of the wire <NUM> outside of the removable electrophoresis cell <NUM> can be electrically insulated while at least of portion of the tubing can be removed so that the platinum wire <NUM> inside of the removable electrophoresis cell <NUM> can be exposed to facilitate contact with the buffer. To maximize activation energy flux onto the scWB consumable chip <NUM>, the removable electrophoresis cell <NUM> can be shallow to minimize the distance between the top of the slide and the source of activation energy, such as a UV source. The removable electrophoresis cell <NUM> can incorporate features to avoid wicking of the electrophoresis buffer outside of a shallow removable electrophoresis cell <NUM>. For example, internal corners <NUM> may be radiused to reduce wicking. In an example, the removable electrophoresis cell <NUM> can include pouring or spout features to aid in emptying the removable electrophoresis cell when the assay is complete.

<FIG> shows a cross-sectional view of an electrophoresis cell, such as a modular removable electrophoresis cell <NUM>. The chip <NUM> can sit flush inside of the recess area <NUM> such that the thin gel layer on the top surface of the chip <NUM> can be level with the bottom of the removable electrophoresis cell <NUM>. In an example, the chip <NUM> can sit flush within the recess area <NUM> to minimize or eliminate changes in height that can cause electric field distortions or perturbations near the edge of the chip <NUM>. Electric field distortions or perturbations can cause variations in the migration distance and in the direction of protein bands that are separated in the chip <NUM>. Electrical field distortions can be spatial distortions or perturbations, such as distortions near the edge of the chip <NUM>. Electrical field distortions can be temporal distortions or perturbations, such as bubbles passing over the chip <NUM>. Electrical current can be proportional to a fluid height <NUM> and the fluid can absorb some of the UV light from the UV light source <NUM>. In some instances, the fluid or liquid height (e.g., buffer solution height) can be minimized. Selection of the electrophoresis cell dimensions can minimize liquid height. The fluid height <NUM> may be less than about <NUM> millimeters (mm), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In some instances, a distance <NUM> between the chip <NUM> and the top of the removable electrophoresis cell <NUM> can be reduced to increase the flux of UV light from the UV light source <NUM> onto the chip <NUM>. The electrophoresis cell dimensions may be selected so as to reduce the fluid height <NUM> and increase the flux or power of UV light.

The distance <NUM> may be less than or equal to about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In some instances, the distance <NUM> is between about <NUM> and <NUM>, or <NUM> and <NUM>.

The removable electrophoresis cell <NUM> can be designed so that the depth of the electrophoresis buffer <NUM> is deeper near the electrodes <NUM> such that the electrodes can be covered by the electrophoresis buffer while allowing for a smaller fluid height <NUM> above the chip <NUM>. In some instances, the electric field strength and migration distances can vary across the chip <NUM> unless the liquid (e.g., buffer solution) height <NUM> is uniform across the chip <NUM>. In the example shown in <FIG>, a uniform liquid height <NUM> can be achieved by leveling the removable electrophoresis cell <NUM>, e.g., using a bubble level or electronic level while adjusting the instrument feet <NUM>, while the fluid height <NUM> can be controlled by the volume of electrophoresis buffer <NUM> that is added to the removable electrophoresis cell <NUM>. Leveling features can be attached to the modular removable electrophoresis cell <NUM> or be integrated into the receptacle <NUM> such that the removable electrophoresis cell <NUM> can be leveled independent of the main instrument body or housing <NUM>.

In an example, the removable electrophoresis cell <NUM> can be configured such that the lid <NUM> defines the fluid height <NUM> by creating a fixed distance between the lid <NUM> and surface of the chip <NUM>. Excess liquid (e.g., buffer solution) can be displaced such that the fluid height <NUM> is no longer a function of the volume of electrophoresis buffer <NUM> or whether or not the removable electrophoresis cell <NUM> is level. The fluid height <NUM> can be controlled by spillover features so that once the target fluid level is reached, excess buffer can flow over the spillover feature into a reservoir such that the excess buffer addition does not change the fluid height <NUM>.

The modular removable electrophoresis cell <NUM> incorporates one or more weir structures <NUM>. These weir structures <NUM> can prevent electrolysis bubbles, which can be generated at the electrodes <NUM>, from drifting over the chip <NUM>. Bubbles above the chip <NUM> can distort or perturb the electric field and may block activation energy (e.g., UV light) from the source of activation energy (e.g., UV light source) <NUM>. When the electrophoresis buffer is poured into the removable electrophoresis cell in the region above the electrodes <NUM>, the weir structures <NUM> can prevent bubbles already present in the buffer from flowing across the chip <NUM>. The weir structures <NUM> can help dampen fluid motion after pouring such that the buffer <NUM> can become quiescent more rapidly than in a removable electrophoresis cell <NUM> without one or more weir structures. In some instances, the dampening of fluid motion can be important to reduce loss of proteins from the microwells during lysis. The removable electrophoresis cell <NUM> can further incorporate a sieve or one or more other features that filter out bubbles already present in the electrophoresis buffer or generated by electrolysis.

The modular removable electrophoresis cell <NUM> can comprise one or more structures (126A, 126B) to capture and restrain the electrode wire <NUM>. A hole <NUM> in the wall of the removable electrophoresis cell <NUM> can allow the electrode wire <NUM> to pass through the wall and to connect electrically to the contact pads <NUM>. The wire <NUM> can be sheathed in a nonconductive tube (such as polytetrafluoroethylene (PTFE)). The wire <NUM> can be sheathed in a nonconductive tube at the point where it passes through the hole <NUM> such that the wire <NUM> can be electrically insulated outside of the cell <NUM>.

In some instances, when loading the chip <NUM>, such as a disposable chip, into the modular removable electrophoresis cell <NUM>, air can be trapped under the chip <NUM>. Air may remain trapped when the electrophoresis buffer is added to the removable electrophoresis cell <NUM> and can result in the chip <NUM> floating up or bubbles escaping along the edges of the chip <NUM> during electrophoresis, which can distort or perturb the electric field. A droplet of buffer (e.g., <NUM>-<NUM> microliters) can be added to the recess area <NUM>. In some instances, a droplet of buffer can be added to one side of the recess area <NUM> or can be added prior to loading the chip <NUM> into the recess area <NUM> after which the chip <NUM> can be lowered into the recess area <NUM>, allowing the liquid drop to wick underneath the chip <NUM> to exclude any trapped air. The surface tension of the liquid (e.g., buffer solution) under the slide (e.g., chip) can hold the slide in the recess area <NUM> until the electrophoresis buffer can be added to the cell <NUM>. The recess area <NUM> can be opaque or dark color which can increase the visual contrast between the air and liquid under the chip <NUM>.

<FIG> shows how a dual-purpose electrophoresis/lysis buffer can be added to an electrophoresis cell, such as the modular removable electrophoresis cell <NUM>. In some examples, the dual-purpose buffer <NUM> can be added manually wherein the user can pour the buffer <NUM> into the modular removable electrophoresis cell <NUM> in the region above the electrode <NUM>, can close the instrument lid <NUM>, and can initiate the assay through the integrated computer <NUM>. Cell lysis can occur when the dual-purpose buffer <NUM> is poured into the removable electrophoresis cell <NUM>. The user can initiate the run after adding the dual-purpose buffer <NUM>.

The instrument <NUM> can include a reservoir <NUM> which can contain the dual-purpose buffer <NUM> (<FIG>). The reservoir can have an initially closed valve <NUM> which can prevent the buffer <NUM> from exiting the reservoir. The software can programmatically control the release of the buffer <NUM> by opening a valve <NUM> allowing the buffer <NUM> to drain into the modular removable electrophoresis cell <NUM> (<FIG>). The computer processor may separately introduce a first solution and a second solution from the source of solution to the chip. The first and second solutions may be different, such as a lysis buffer and an electrophoresis buffer. Alternatively, the first and second solutions may be the same, such as washing buffers.

The computer processor may be programmed to separately introduce two or more sequential solutions from the source of solution to the chip. The two or more sequential solutions may be the same. As an alternative, the two or more sequential solutions may be different. In some instances, one or more solutions are manually introduced to the chip. As an alternative, one or more solutions are manually introduced and one or more solutions are programmatically introduced when the computer processor opens the reservoir valve <NUM>.

Programmatic control of buffer release can provide consistent timing of the lysis step. The buffer <NUM> can be released rapidly and can fill the removable electrophoresis cell <NUM> in less than about <NUM> second. In some instances, the buffer <NUM> can fill the removable electrophoresis cell <NUM> in less than about <NUM> seconds. In some instances, the buffer <NUM> can fill the removable electrophoresis cell <NUM> in less than about <NUM> seconds. The instrument <NUM> can include one or more active pumps. The instrument <NUM> can utilize gravity release to fill the reservoir <NUM> with buffer <NUM>. The reservoir <NUM> can be configured to contain a single aliquot of buffer <NUM>. In some instances, the entire volume of a single aliquot of buffer <NUM> can be released when the valve <NUM> is opened. In some instances, the reservoir <NUM> can contain larger volumes of buffer <NUM>, and a subvolume of the larger volume can be released for one assay. The release of a subvolume of buffer <NUM> can be controlled by the instrument <NUM>. In some instances, the instrument <NUM> can comprise multiple reservoirs and/or the buffer <NUM> can be introduced into the removable electrophoresis cell <NUM> at multiple locations.

Many lysis buffers can contain high concentrations of surfactants and salts. These additives can assist in cell lysis. However, such additives can also increase the conductivity of the buffer making such buffers less suitable for electrophoresis as excessive electrical current can lead to joule heating and loss of separation resolution. The dual-purpose electrophoresis/lysis buffer <NUM> can therefore strike a balance between low conductivity for electrophoresis and the ability to lyse cells. In some instances, separately-optimized electrophoresis and lysis buffers can be used. For example, <FIG> shows a scWB assay using a separate electrophoresis buffer <NUM> and lysis buffer <NUM>. Approximately <NUM> of lysis buffer <NUM> can be added directly to the top surface of a chip, such as the disposable chip <NUM>, to initiate lysis (<FIG>). After a lysis time (such as between about <NUM> to about <NUM> seconds), a volume (such as about <NUM>) of electrophoresis buffer <NUM> can be released from a reservoir <NUM>, as in <FIG>. The electrophoresis buffer <NUM> can wash over the surface of the chip <NUM> and can displace the lysis buffer <NUM> and can wash it to the side of the removable electrophoresis cell <NUM> (<FIG>) where it can be diluted by the larger volume of electrophoresis buffer <NUM>. This dilution can significantly mitigate the impact of the higher conductivity lysis buffer <NUM> during electrophoresis. In addition, the electrical resistance of the removable electrophoresis cell <NUM> can be largely determined by the thin layer of fluid directly above the chip <NUM> (defining the liquid height <NUM>), therefore removing the lysis buffer <NUM> from the region above the chip <NUM> can greatly reduce the current during electrophoresis, independent of effect of dilution. A holding area below the electrode <NUM> can be created such that the lysis buffer <NUM> washes into the holding region thereby removing it from the electrical circuit entirely.

The system can also include tools and fixtures for conducting a scWB assay. <FIG> depict a device and method for performing antibody probing using a chip, which may be the scWB chip <NUM>. The chip <NUM> may be consumable. In some cases, the chip <NUM> is disposable or reusable. To ensure uniform antibody staining and facilitate the recovery and reuse of primary antibodies, the device can comprise an incubation chamber, such as an antibody probing fixture <NUM>. The probing fixture <NUM> can contain a rectangular region with a defined depth or recession <NUM> with a length and width slightly smaller than the chip <NUM>, as in <FIG>. During the antibody probing step, the gel layer <NUM> containing immobilized proteins (see <FIG>) can be exposed to a uniform concentration of detection antibodies, such as a) labeled primary antibodies or b) unlabeled primary antibodies followed by incubation with labeled secondary antibodies. A non-uniform gap size <NUM> may create a non-uniform concentration of antibody across the chip <NUM> during the incubation. The antibody fixtures can ensure a uniform gap that can be filled with antibody solution <NUM>. The fixture <NUM> can be constructed of a hydrophobic material (such as PTFE) or can incorporate a hydrophobic coating such that the antibody solution cannot spread out from underneath the more hydrophilic gel layer <NUM>. Keeping the antibody solution <NUM> contained under the chip <NUM> can reduce evaporation of the antibody solution <NUM>.

Loading of the fixture is shown in <FIG>. A small volume of antibody solution <NUM> can be placed on the surface of the fixture. In some instances, the small volume can be less than about <NUM> microliters. The chip <NUM> with gel layer <NUM> facing down, can then be rotated onto the fixture <NUM> as shown in <FIG>. Once the chip <NUM> has been lowered onto the fixture <NUM>, the gap between the gel layer <NUM> and the bottom of the rectangular depression (recession) <NUM> can be filled with antibody solution <NUM>. In some instances, the gap size <NUM> can be about <NUM> microns or less. Once in place, the scWB consumable chip <NUM> can be allowed to incubate with the antibody solution <NUM> (see e.g., <FIG>). In some instances, the incubation is for about <NUM> to about <NUM> minutes. Extended incubation periods can be possible. Incubations may occur at reduced temperatures. During this incubation period, the entire fixture <NUM> may be covered to reduce evaporative losses. A small portion of wet cloth or paper may also be placed adjacent to the fixture <NUM> so as to further limit evaporative losses. At the end of the incubation step, the chip <NUM> can be removed from the fixture <NUM> by rotating the chip <NUM> away from the rectangular depression <NUM>. In addition to helping to contain the antibody solution <NUM> under the chip <NUM> during incubation, a hydrophobic fixture can facilitate recovery of the used antibody solution <NUM> as the solution can tend to reform into a contiguous droplet when the chip <NUM> is rotated away from the fixture <NUM>. Primary antibody solutions <NUM> may therefore be reused several times to reduce cost.

The fixture <NUM> shown in <FIG> may be constructed using many common techniques such as machining, injection molding, or hot or cold embossing. <FIG> depicts a cross-sectional view of one construction for an antibody incubation fixture <NUM>. In this example construction, a rectangular gap <NUM> can be formed on both sides of the fixture <NUM> using laminated layers. The core <NUM> of the fixture <NUM> may be constructed of any suitable, flat material such as for example, <NUM> inch thick poly(methyl methacrylate) (PMMA). Thin hydrophobic layers <NUM>, such as <NUM> inch thick PTFE layers are then attached to the core layer <NUM>. Finally, an additional hydrophobic layer having a rectangular region <NUM> removed can be attached to the first hydrophobic layer <NUM>. The advantage of using laminated layers to define the gap size is that the laminated layers can be very flat over distances comparable to the size of the chip. The gap size of such a laminated device can be very uniform and can be defined by the thickness of the layer. The layers may be attached to each other using many techniques known to one skilled in the art, such as thermal bonding, solvent bonding, or use of intermediate pressure-sensitive adhesive layers.

As described with reference to <FIG>, the first step of the scWB assay can be to settle cells into the array of the microwells formed into the gel layer <NUM>. Once the cells are settled into wells, the user can gently rinse the chip <NUM> to remove cells that have not settled into wells substantially without washing cells out of microwells. <FIG> depicts another fixture <NUM> included with the system that can aid in rinsing of the chip <NUM> after cell settling. The rinsing fixture <NUM> can place the chip <NUM> at a shallow angle <NUM> which has a typical value of approximately <NUM> degrees. In other embodiments, the angle <NUM> can be greater than <NUM> degrees or less than <NUM> degrees. Wash buffer <NUM> can be ejected slowly from a pipette tip <NUM>. The angle <NUM> of the rinsing fixture <NUM> can allow the wash buffer <NUM> to flow across the gel layer <NUM> at a controlled velocity thereby minimizing perturbation of cells that are trapped inside microwells while rinsing away cells that remain on the surface of the gel layer <NUM>.

The present disclosure also presents methods for obtaining molecular weight sizing of endogenous proteins contained in the cells. Separately optimized lysis and electrophoresis buffers can allow for the convenient introduction of protein standards. Preferential partitioning of proteins to open wells versus gel can allow for facile loading of protein markers that separate with the expected linear log molecular weight (MW) vs. mobility relationship. Thus, it may be not necessary to load distinct sub-nanoliter volumes of protein marker solutions into the thousands of microwells contained within the chip. Protein markers can be fluorescently tagged using a distinct wavelength such that they do not interfere with later detection of endogenous proteins. The protein markers can be tagged with a cleavable or quenchable fluorophore which can be detected and then removed or quenched prior to detection of endogenous proteins. Using the two-buffer method described with reference to <FIG>, one can add protein markers to the lysis buffer such that they can be introduced simultaneously as cell lysis occurs. Alternately, protein markers can be loaded into lysosomes or agarose (or other polymer) beads. The lysosomes and beads can then be settled simultaneously along with the cells resulting in some microwells containing marker proteins. Proteins can leave the lysosomes or beads when an external electric field is supplied to commence electrophoresis. An AC electric field can be applied to release the protein markers in a manner similar to electroporation of cells. The lysis buffer can be modified to release the protein markers from the lysosomes or beads.

Protein sizing standards can be spotted onto a chip at various locations during manufacturing and rehydrated during use of the chip. Separate regions on a chip, such as alternating separate regions, may be designated for protein sizing ladders where the wells for the sizing ladders can be increased in at least one dimension to allow for more convenient loading of sizing markers.

Another aspect of the present disclosure refers to the introduction of fluorescence standards such that detected endogenous proteins can be compared to a known fluorescence signal and measurements can more readily be compared between chips. In an example, the fluorescence standard can be introduced into the chip at discreet locations during manufacturing. Standards can be, for example, incorporated throughout the gel, or can be spotted onto regions of the chip before cross-linking of the gel has completed, or the reactive groups within the gel (e.g., benzophenone) may be activated to covalently bind the fluorescence standards. Fluorescence standards can be introduced in beads which co-settle into the wells along with cells. The beads can remain in the wells and the fluorescence signal from these beads can be compared to the signal from labeled, endogenous proteins that separate away from the wells. The beads can be commercially available silica beads (or the like), or they may be beads formed from a hydrogel. An example of a hydrogel bead includes polyacrylamide beads containing a benzophenone-methacrylamide co-monomer. Fluorescent dyes can be incorporated into the polyacrylamide bead by incubation with a desired dye followed by photo-initiated binding of the dye to benzophenone functional group incorporated into the hydrogel bead. Polyacrylamide beads can allow a user to conveniently add a desired marker to the beads. Beads can also incorporate magnetic nanoparticles to assist in manipulation of the beads.

The present disclosure presents computer control systems that are programmed to implement methods of the disclosure. <FIG> shows a computer system <NUM> that is programmed or otherwise configured to implement systems and methods provided herein. The computer system <NUM> can regulate various aspects of analyte detection of the present disclosure, such as, for example, automated single cell western blogging. The computer system <NUM> can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

The computer system <NUM> includes a central processing unit (CPU, also "processor" and "computer processor" herein) <NUM>, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system <NUM> also includes memory or memory location <NUM> (e.g., random-access memory, read-only memory, flash memory), electronic storage unit <NUM> (e.g., hard disk), communication interface <NUM> (e.g., network adapter) for communicating with one or more other systems, and peripheral devices <NUM>, such as cache, other memory, data storage and/or electronic display adapters. The memory <NUM>, storage unit <NUM>, interface <NUM>, and peripheral devices <NUM> are in communication with the CPU <NUM> through a communication bus (solid lines), such as a motherboard. The storage unit <NUM> can be a data storage unit (or data repository) for storing data. The computer system <NUM> can be operatively coupled to a computer network ("network") <NUM> with the aid of the communication interface <NUM> via a wired or wireless connection. The network <NUM> can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network <NUM> in some cases is a telecommunication and/or data network. The network <NUM> can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network <NUM>, in some cases with the aid of the computer system <NUM>, can implement a peer-to-peer network, which may enable devices coupled to the computer system <NUM> to behave as a client or a server.

The CPU <NUM> can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory <NUM>. The instructions can be directed to the CPU <NUM>, which can subsequently program or otherwise configure the CPU <NUM> to implement methods of the present disclosure. Examples of operations performed by the CPU <NUM> can include fetch, decode, execute, and writeback, and/or any other suitable operation.

The CPU <NUM> can be part of a circuit, such as an integrated circuit. One or more other components of the computer system <NUM> can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC). In some embodiments, the CPU <NUM> and/or integrated circuit can include and/or can execute one or more modules associated with controlling the systems described herein. As used herein the term "module" refers to any assembly and/or set of operatively-coupled electrical components that can include, for example, a memory, a processor, electrical traces, optical connectors, software (executing in hardware), and/or the like. For example, a module executed in the processor can be any combination of hardware-based module (e.g., a field-programmable gate array (FPGA), an ASIC, a digital signal processor (DSP)) and/or software-based module (e.g., a module of computer code stored in memory and/or executed at the processor) capable of performing one or more specific functions associated with that module.

The storage unit <NUM> can store files, such as drivers, libraries, profiles, saved programs, etc. The storage unit <NUM> can store user data, e.g., user preferences and user programs. The computer system <NUM> in some cases can include one or more additional data storage units that are external to the computer system <NUM>, such as located on a remote server that is in communication with the computer system <NUM> through an intranet or the Internet (e.g., network attached storage (NAS) device).

The computer system <NUM> can communicate with one or more remote computer systems through the network <NUM>. For instance, the computer system <NUM> can communicate with a remote computer system of a user (e.g., service provider). Examples of remote computer systems include servers, host devices, personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system <NUM> via the network <NUM>.

Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system <NUM>, such as, for example, on the memory <NUM> or electronic storage unit <NUM>. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor <NUM>. In some cases, the code can be retrieved from the storage unit <NUM> and stored on the memory <NUM> for ready access by the processor <NUM>. In some situations, the electronic storage unit <NUM> can be precluded, and machine-executable instructions are stored on memory <NUM>.

The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

Aspects of the systems and methods provided herein, such as the computer system <NUM>, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium, or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wires, and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a compact disc-read only memory (CD-ROM), digital video disc (DVD) or digital video disc-read only memory DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a random-access memory (RAM), a read-only memory (ROM), a programmable read-only memory (PROM) and erasable programmable read-only memory (EPROM), a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system <NUM> can include or be in communication with an electronic display <NUM> that comprises a user interface (UI) <NUM> for providing, for example, an output or readout of the system coupled to the computer processor. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit <NUM>. The algorithm can, for example, be used to analyze results, such as the presence of one or more target analytes in a biological sample. A final data set can be constructed based on portions of data gathered from each assay of each biological sample.

The removable electrophoresis cell <NUM> is particularly described above with reference to <FIG>. For example, <FIG> illustrate a removable electrophoresis cell <NUM> according to an embodiment. In some embodiments, the removable electrophoresis cell <NUM> has been designed and/or otherwise optimized for manufacturing by injection molding. Portions of the removable electrophoresis cell <NUM> can be similar in form and/or function to corresponding portions and/or features of the electrophoresis cell <NUM> described above with reference to <FIG> and thus, such portions are not described in further detail herein.

As shown, the removable electrophoresis cell <NUM> includes wicking breaks <NUM> to prevent fluid from wicking up the electrodes to one or more contact pad areas <NUM>. The electrophoresis cell <NUM> also includes an integrated chip lifter <NUM> that facilitates removal of a chip (e.g., the chip <NUM>) from the cell <NUM>. In the embodiment shown in <FIG>, when the chip lifter <NUM> is in the down position, it fits flush with the electrophoresis body so as to prevent trapping of air bubbles as well as electric field perturbations that can be caused by changes in fluid height near the edge of the chip (as described in detail above with reference to the electrophoresis cell <NUM>). The electrophoresis cell <NUM> includes weirs <NUM> that are inserted separately, allowing for the electrophoresis cell <NUM> to be molded. To attach the electrode wire (not shown), heat staking features <NUM> are included. The wire can be stretched between the heat staking features <NUM> which are then melted to permanently hold the wire in place. A small (<<NUM>) circular recess <NUM> allows an alternate way of removing the chip from the electrophoresis cell <NUM> by using a pointed prying tool. The small size of the recess <NUM> minimizes bubble trapping and electric field perturbations. As shown in <FIG>, the underside of the electrophoresis cell <NUM> includes stabilization posts <NUM> which force the electrophoresis cell <NUM> to contact the receptacle in an instrument (e.g., the instrument <NUM>) at fixed points. As shown, the electrophoresis cell <NUM> includes spout features <NUM> that can assist in pouring used buffer from the electrophoresis cell after use. In this manner, the electrophoresis cell <NUM> can be used in any suitable scWB process and/or procedure such as any of those described herein.

An integrated opto-electronic instrument has been built that accurately controls all timed steps and provides uniform, repeatable, safe application of electric field and UV illumination for execution of the scWB assay. The integrated instrument minimizes the time between separation and UV photocapture, thus limiting diffusive dispersion and loss of separation resolution. For safe deployment to external users, mechanical interlocking of the high voltage and UV light has been implemented. Below, details on the optimized system components and demonstrate safe operation and acceptable assay performance of the integrated prototype are presented.

Removable electrophoresis cell. Iterative prototyping and design processes were employed to build, test, and optimize a removable electrophoresis cell for use in the final integrated scWB device. The design of the removable electrophoresis cell was configured to, for example, reduce electrolysis bubble formation near the electrodes that spread to cover the liquid surface within the cell, perturbing the electric field and blocking a portion of the UV light during the subsequent UV exposure step. Further, the design was configured to, for example, increase reliable chip adhesion and repeatable buffer addition.

In some embodiments, a chip recess can include finger cut-outs for facile chip removal. Such a chip recess can be sufficiently deep to protect the chip from being dislodged during buffer addition obviating the need for a petroleum jelly fixative. Further, bubble trapping weir structures can be used to separate the platinum electrodes from the main cell compartment and prevent bubbles from floating over the chip surface. In some instances, however, such finger cut-outs can lead to, for example, electric field distortion observed at the edges of the chip (<NUM>° near edge vs. <<NUM>° near center). Numerical simulations confirm that, in some instances, this nonuniformity can be caused by liquid (e.g., buffer solution) height discontinuities due to finger cutouts and the chip not being flush with the bottom surface of the cell. Changes in the liquid height at the edge of the chip can cause the electric field lines to diverge, leading to electric field distortion near the edges of the chip.

To increase field uniformity, changes in the liquid height were avoided. For example, in some embodiments, a removable electrophoresis cell <NUM> includes a chip recess <NUM> configured to fully recess the chip such that the gel layer protruded beyond the chip recess <NUM> and finger cut-outs were removed (see <FIG>). Such a removable electrophoresis cell <NUM> can incorporate the following designs: <NUM>) A relatively large buffer reservoir allowing for the addition of lysis/electrophoresis buffer without spilling, <NUM>) bubble traps (e.g., weirs <NUM>) to prevent electrolysis bubbles from migrating over the surface of the chip and blocking UV light during photocapture while further damping fluid motion above the chip, <NUM>) pogo-pin high-voltage contacts for integration with the instrument lid, and <NUM>) the chip recess <NUM> without finger cutouts to minimize field distortion near the edges of the chip. The removable electrophoresis cell <NUM> can also include electrical contact pads <NUM> and a platinum electrode <NUM>, as described above.

Light source selection. Several candidate light sources were tested. Benzophenone capture chemistries are known to be activated in the range of <NUM>-<NUM> nanometers (nm) and the Lightning cure lamp has a broad spectrum output with significant power at <NUM> and <NUM>. Several compact light sources in the <NUM> to <NUM> wavelength range were evaluated. No capture was observed under high power (<NUM> microWatts (mW)/cm<NUM>) <NUM> LEDs (Hamamatsu). Compact UV fluorescent tube lamps were obtained from UV Systems (Renton, WA) and the performance of bulbs was compared with broad outputs centered at <NUM> and <NUM>, respectively. Both bulbs have significant power output in the <NUM>-<NUM> range. The <NUM> wavelength bulb yielded a significant increase in capture efficiency compared to a <NUM> bulb (<NUM>% vs. <NUM>% for a <NUM> minute exposure). Reducing the exposure time on the <NUM> bulb to <NUM> yielded a measured mean capture efficiency of <NUM>% (compared to <NUM>% for a <NUM> exposure time) suggesting that the exposure time can be reduced from <NUM> minutes with a moderate reduction in the capture efficiency.

Integrated Prototype. An integrated high-voltage electrophoresis and UV exposure unit is used to accurately control all timed steps and provide uniform, repeatable, safe application of electric fields and UV illumination. An integrated opto-electronic instrument <NUM> was built that incorporates the custom-designed removable electrophoresis cell <NUM>, a chip <NUM> (such as those described above), and a <NUM> UV bulb from UV Systems to enable safe and automated sequential operation of electrophoresis and UV exposure steps (see e.g., <FIG>). A touchscreen <NUM> with a graphical user interface was developed to allow users to have control over key assay steps: lysis time, electrophoresis run time, voltage, and UV exposure time (<FIG> and <FIG>).

Safety interlocking features were successfully incorporated and the UV light was visually observed to cease upon opening of the lid. The electrical current was measured with a multimeter and found to cease upon opening the lid. The instrument was additionally fitted with a drain circuit to drain any residual charge from the high voltage pin when the lid is opened. Light-blocking features were incorporated to limit any leakage of UV light to negligible levels.

Methods and Analysis: The ability to lyse cells settled in a scWB chip by covering the chip with ~<NUM> of lysis buffer for <NUM>-<NUM> seconds and then quickly replacing the lysis buffer with electrophoresis buffer was demonstrated. Loss of lysed proteins inside the microwells can be minimized by rapidly replacing the buffer above the chip without excessive fluid perturbation using the removable electrophoresis cell and instrumentation described with reference to <FIG>, <FIG>, and <FIG>.

Preferential partitioning of proteins to open wells versus gel allows for facile loading of protein markers that separate with the expected linear log MW vs. mobility relationship (see <FIG>).

Once incubated with the chip, the marker-containing buffer was removed to avoid continuous injection and reduced resolution of the protein markers during separation. Using the two-buffer workflow (see, e.g., <FIG>), the ability to introduce molecular weight sizing protein markers in the lysis buffer has been shown. Surface tension holds the marker-containing lysis buffer on the surface of the gel, filling the wells, until the electrophoresis buffer is introduced and flows over the chip. Separate from the need to avoid continuous injection, introduction of the protein markers in the smaller volume of lysis buffer (versus the electrophoresis buffer) reduces the required mass (and cost) of ladder proteins by an order of magnitude. Preliminary results for sizing of endogenous beta-tubulin in a Chinese hamster ovary (CHO) cell line using ovalbumin and immunoglobulin G (IgG) markers reduced variability from field perturbations across the chip (raw migration distance had a <NUM>% CV while a sizing correction using ovalbumin and IgG reduced the CV to <NUM>%) (see e.g., <FIG>). <FIG> illustrate molecular weight sizing on <NUM>%T gel, <NUM>%T gel, and <NUM>%T gel, respectively.

As described above, some embodiments herein relate to a computer storage product with a non-transitory computer-readable medium (also can be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transitory propagating signals per se (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as space or a cable). The media and computer code (also can be referred to as code) may be those designed and constructed for the specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as CDs, DVDs, CD-ROMs, and holographic devices; magneto-optical storage media such as optical disks; carrier wave signal processing modules; and hardware devices that are specially configured to store and execute program code, such as ASICs, Programmable Logic Devices (PLDs), Read-Only Memory (ROM), and Random-Access Memory (RAM) devices. Other embodiments described herein relate to a computer program product, which can include, for example, the instructions and/or computer code discussed herein.

Some embodiments and/or methods described herein can be performed by software (executed on hardware), hardware, or a combination thereof. Hardware modules may include, for example, a general-purpose processor, a field programmable gate array (FPGA), and/or an ASIC. Software modules (executed on hardware) can be expressed in a variety of software languages (e.g., computer code), including C, C++, Java™, Ruby, Visual Basic™, and/or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages (e.g., C, FORTRAN, etc.), functional programming languages (Haskell, Erlang, etc.), logical programming languages (e.g., Prolog), object-oriented programming languages (e.g., Java, C++, etc.) or other suitable programming languages and/or development tools. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that structures within the scope of these claims be covered thereby.

Claim 1:
An electrophoresis cell (<NUM>) configured to be disposed within a receptacle (<NUM>) of an assay device for assaying a biological sample for a presence of a target analyte, the electrophoresis cell comprising:
a bottom surface defining a recess area (<NUM>) containing a chip (<NUM>) such that a surface of a polymeric medium of the chip is flush with a non-recessed area of the bottom surface of the electrophoresis cell, the polymeric medium configured for separation of analytes within the biological sample and having activatable functional groups that covalently bond to one or more target analytes when activated; and
an electrode (<NUM>) disposed in the electrophoresis cell and configured to produce an electric field across the recess area in response to a flow of electric current from a power supply;
the electrophoresis cell being characterised by a weir structure (<NUM>) disposed above and on at least one side of the recess area such that the weir structure is spaced apart from the electrode and is between the electrode and the at least one side of the recess area, the weir structure configured to control a flow of a solution below the weir structure and across the chip.