System and method for protein quantification with parallelized capillary-electrophoresis immunoassays

Abstract

An apparatus includes a capillary cartridge including a plurality of capillaries. The apparatus further includes an ultraviolet light source configured to irradiate the plurality of capillaries during the immobilization process. The apparatus further includes a heat shield. At least a portion of the heat shield is disposed parallel to the plurality of capillaries and configured to control transfer of heat away from the plurality of capillaries to reduce a temperature variation between capillaries from the plurality of capillaries during the immobilization process. The apparatus further includes at least one trough holding a solution. An end of each capillary from the plurality of capillaries is configured to be submerged into the solution during the immobilization process.

Description

SYSTEM AND METHOD FOR PROTEIN QUANTIFICATION WITH PARALLELIZEDCAPILLARY-ELECTROPHORESIS IMMUNOASSAYSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 654,775, filed May 31, 2024, which is incorporated herein by reference in its entirety for all purposes.FIELD

[0002] One or more embodiments are related to a system and method for protein quantification with parallelized capillary-electrophoresis immunoassays.BACKGROUND

[0003] Protein characterization and quantification are important in cell and molecular biology research. Precise identification and accurate determination of proteins in complex samples are desirable in many areas such as biochemistry research, medical diagnostics, and pharmaceutical manufacturing process control. A known technique used for protein separation and detection is the western blot, in which a sequence of steps is performed to separate, immobilize, detect, and quantify the proteins of interest. Although western blotting is ubiquitous, its application to quantitative analysis is limited because the multistep and typically manual protocol is prone to variations related to the analyst preparing and conducting the assay, the consumables, and reagents, which translates to poor reproducibility in the determination of the resulting immunoassay signal.

[0004] Capillary electrophoresis (CE) techniques, such as capillary-based western-type separations, isoelectric focusing, etc. enable quantitative and automated sample analysis by eliminating most manual steps used by the traditional gel-based electrophoresis methods. Nonetheless, there are residual sources of variation in CE technology that can cause some differences in the measurement of the same sample. Some implementations described herein improve the performance of CE technology by reducing several sources of residual variation within a capillary cartridge.SUMMARY

[0005] In an embodiment, an apparatus includes a capillary cartridge including a plurality of capillaries. The apparatus further includes an ultraviolet (UV) light source configured to irradiatethe plurality of capillaries during the immobilization process. The apparatus further includes a heat shield. At least a portion of the heat shield is disposed parallel to the plurality of capillaries and configured to control transfer of heat away from the plurality of capillaries to reduce a temperature variation between capillaries from the plurality of capillaries during the immobilization process. The apparatus further includes at least one trough holding a solution. The first end of each capillary from the plurality of capillaries is configured to be submerged into the solution during the immobilization process.

[0006] In an embodiment, a method includes separating, in at least one capillary from an array of capillaries, at least one analyte from a plurality of analytes contained within a sample. The sample is disposed within the at least one capillary. The method further includes activating a UV light source. Each capillary from the array of capillaries includes a photo-activated moiety configured to bind analytes from the array of capillaries to a wall of that capillary when that capillary is irradiated by the UV light source. A heat shield can be configured to control heat transfer away from the array of capillaries. For example, in some such embodiments, the array of capillaries may be disposed between the UV light source and at least a portion of the heat shield. In addition or alternatively, at least a portion of the heat shield can be disposed between the array of capillaries and the UV light source. In this way, the heat shield can reduce a temperature variation between capillaries from the plurality of capillaries when the UV light source is active. The method further includes immunoprobing the array of capillaries after activating the UV light source such that a labeled antibody binds to the at least one analyte.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 is a schematic illustration of a western-style capillary electrophoresis (CE) immunoassay.

[0008] FIG. 2 illustrates a model of a CE instrument, including the capillary cartridge, reagent plate, UV light source, and camera for detection, according to an embodiment.

[0009] FIG. 3A is a perspective view of a capillary cartridge, according to an embodiment.

[0010] FIG. 3B is a schematic showing the various thermal effects and heat transfer mechanisms around the capillary cartridge of FIG. 3 A.

[0011] FIG. 4A is a perspective view of a capillary cartridge and a four-sided shield design, according to an embodiment.

[0012] FIG. 4B is a schematic showing the various thermal effects around the capillary cartridge and the four-sided shield of FIG. 4A.

[0013] FIG. 4C is a cross-sectional view of the four-sided shield of FIG. 4A.

[0014] FIG. 5A is a perspective view of a capillary cartridge and a three-sided shield design, according to an embodiment.

[0015] FIG. 5B is a schematic showing the various thermal effects around the capillary cartridge and the three-sided shield of FIG. 5 A.

[0016] FIG. 5C is a cross-sectional view of the three-sided shield of FIG. 5A.

[0017] FIG. 6A is a perspective view of a capillary cartridge, a three-sided shield, and a comb keeper for stabilizing capillary positions, according to an embodiment.

[0018] FIG. 6B is an enlarged partial rear perspective view of the three-sided shield and the comb keeper of FIG. 6 A.

[0019] FIG. 6C is a cross-sectional view of the three-sided shield and the comb keeper of FIG. 6A.

[0020] FIG. 6D is a perspective view of a capillary cartridge, a three-sided shield, and a comb keeper for stabilizing capillary positions, according to an embodiment.

[0021] FIG. 6E is a cross-sectional view of the three-sided shield and the comb keeper of FIG. 6D.

[0022] FIG. 6F is an enlarged partial perspective view of the three-sided shield and the comb keeper of FIG. 6D.

[0023] FIG. 6G is an exploded view of the capillary cartridge, the three-sided shield, and the comb keeper of FIG. 6D.

[0024] FIG. 6H is a schematic of comb keeper geometry, according to an embodiment.

[0025] FIG. 7A is a perspective view of a capillary cartridge and a baffle-style shield design, according to an embodiment.

[0026] FIG. 7B is a schematic showing the various thermal effects around the capillary cartridge and the baffle-style shield of FIG. 7A.

[0027] FIG. 7C is a cross-sectional view of the baffle-style shield of FIG. 7A.

[0028] FIG. 8A is a perspective view of a capillary cartridge and a box-style shield design, according to an embodiment.

[0029] FIG. 8B is a schematic showing the various thermal effects around the capillary cartridge and the box-style shield of FIG. 8 A.

[0030] FIG. 8C is a cross-sectional view of the box-style shield of FIG. 8 A.

[0031] FIG. 9 illustrates a comparison of temperature profde and uniformity across the capillaries in the cartridge in the un-shi elded, 4-side shielded, and 3 -side-shielded cartridge, according to an embodiment.

[0032] FIG. 10A and 10B illustrate the relationship between capillary temperature variation and immunoassay signal profde with chemiluminescence detection, according to an embodiment.

[0033] FIG. 11 A illustrates a comparison of chemiluminescence signal profdes of a multiplexed lysate-based immunoassay across capillaries in four different runs, according to an embodiment. Variability of assay signal is evidenced by relatively high coefficient of variation.

[0034] FIG. 1 IB illustrates a comparison of chemiluminescence signal profiles of a multiplexed lysate-based immunoassay across capillaries in four different runs in 3 -side shielded cartridges, according to an embodiment. Desirable data quality is demonstrated by lowered coefficient of variation.

[0035] FIG. 12 illustrates shield heater control strategies, according to an embodiment. Illustration of spatial and temporal temperature evolution with proportional, integral, and derivative (PID) temperature and power controls.

[0036] FIG. 13 illustrates the effect of capillary dipping depth on joule heating in the capillary, according to an embodiment. For a given voltage applied across the capillary, the wetted section, or dipped depth, affects the joule heating and fluid temperature inside the capillary.

[0037] FIGS. 14A and 14B illustrate a comparison of buffer trough designs and effect of liquid meniscus on capillary dipping depths, according to an embodiment.

[0038] FIG. 15A and 15B illustrate statistical analysis of immunoassay signal intensity and capillary location in buffer trough grouped by dipping depth bias, according to an embodiment.

[0039] FIGS. 16A, 16B, 16C, and 16D illustrate designs of UV-LED panel arrays to illustrate intensity and uniformity variation with different array patterns, according to an embodiment.

[0040] FIG. 17 illustrates a system block diagram of an instrument to perform an immobilization process, according to an embodiment.

[0041] FIG. 18 illustrates a flowchart of a method to immobilize at least one analyte, according to an embodiment.

[0042] FIG. 19 illustrates a four-sided heat shield, a three-sided heat shield, and a two-side heat shield, according to an embodiment.DETAILED DESCRIPTION

[0043] It is to be understood that both the foregoing general description and the following description are exemplary and explanatory only and are not restrictive of the methods and devices described herein. In this application, the use of the singular includes the plural unless specifically state otherwise. Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising,” “include,” “includes” and “including” are not intended to be limiting.

[0044] Some implementations are related to using an instrument that includes capillaries to perform an immobilization process. The immobilization process includes resolving analytes in capillaries, immobilizing analytes, (e.g., by binding analytes to inner walls of the capillaries upon activation of a triggerable agent) and detecting the analytes that are immobilized in the subset of the plurality of capillaries. During the immobilization process, heat / temperature uniformity at each capillary and across the set of capillaries can be desirable (e.g., improve precision and accuracy of protein quantitation). Accordingly, in some implementations, a heat shield can be used to provide improved (e.g., compared to known art and / or similar devices without a heat shield) heat / temperature uniformity at each capillary and across the set of capillaries.Additionally or alternatively, in some implementations, the capillaries are dipped into a solution held by a partitioned trough to provide improved (e.g., compared to known art and / or similar devices without a partitioned trough) heat / temperature uniformity at each capillary and across the set of capillaries.

[0045] As used throughout the instant application, the following terms shall have the following meanings:

[0046] “Antibody” has its standard meaning and is intended to refer to full-length as well antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), monoclonal, polyclonal, chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.

[0047] ‘Detect” and “detection” have their standard meaning and are intended to encompass detection including the presence or absence, measurement, and / or characterization of an analyte.

[0048] “Label” as used herein refers to a detectable moiety. As will be appreciated by those in the art, suitable labels encompass a wide variety of possible moieties. In general, labels include, but are not limited to, a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies, antigens, aptamers, etc.; c) optical dyes, including colored or fluorescent dyes; d) enzymes such as alkaline phosphatase and horseradish peroxidase, e) particles such as colloids, magnetic particles, etc., and combinations thereof such as fluorescent labeled antibodies, chemiluminescent labeled antibodies, antibodies functionalized with nano materials, small molecules that can specifically bind to targets, etc.

[0049] “Protein” has its standard meaning and is intended to refer to proteins, oligopeptides and peptides, derivatives and analogs, including proteins containing non-naturally occurring amino acids and amino acid analogs, and peptidomimetic structures, and includes proteins made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

[0050] 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.

[0051] As used herein, the terms “about” and “approximately” generally mean plus or minus 10% of the value stated. For example, about 0.5 would include 0.45 and 0.55, about 10 would include 9 to 11, about 1000 would include 900 to 1100.

[0052] As used herein, the term “set” can refer to multiple features or a singular feature with multiple parts. For example, when referring to a set of walls, the set of walls can be considered as one wall with multiple portions, or the set of walls can be considered as multiple, distinct walls. Thus, a monolithically constructed item can include a set of walls. Such a set of walls may include multiple portions that are either continuous or discontinuous from each other. A set of walls can also be fabricated from multiple items that are produced separately and are later joined together (e.g., via a weld, an adhesive, or any suitable method).

[0053] As used herein, the terms “perpendicular,” “normal,” and “orthogonal” generally described a relationship between two geometric constructions (e.g., two lines, two planes, a line and a plane, or the like) in which the two geometric constructions are disposed at substantially90°. For example, a line is said to be perpendicular to another line when the lines intersect at an angle substantially equal to 90°. Similarly, when a planar surface (e.g., a two-dimensional surface) is said to be orthogonal to another planar surface, the planar surfaces are disposed at substantially 90° as the planar surfaces extend to infinity.

[0054] As used herein, the terms “analyte” and / or “target analyte” refer to any molecule or compound to be separated and / or detected with the methods, apparatuses and systems provided herein. Suitable analytes 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, antibiotics, organic materials, hormones, antibodies, antibody fragments, antibody-molecule conjugates (e.g., antibody-drug conjugates), antigens, cellular membrane antigen, 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 (comprising ribonucleic acid and / or deoxyribonucleic acid), transition state analogs, inhibitors, receptors, receptor 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), receptor-ligand complexes, nutrients, electrolytes, growth factors and other biomolecules and / or non-biomolecules, as well as fragments and combinations thereof. In one embodiment, the analyte is a protein or a protein complex, and the sample is a cellular lysate or a purified protein.

[0055] As used herein, the term “sample” refers to a composition that contains an analyte or analytes to be detected. A sample, in one embodiment, is heterogeneous, containing a variety of components (e.g., different proteins) or homogenous, containing one component (e.g., a population of one protein). 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 (e.g., a cell suspension) or denatured form (e.g., a lysate). In some instances, a sample can be a single cell (or contents of a single cell, e.g., as a cellular lysate from the single cell, or a purified protein) or multiple cells (or contents of multiple cells, e.g., as a cellular lysate from the multiple cells, or a purified protein from the multiple cells), a blood sample, a tissue sample, a skin sample, a urinesample, 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.

[0056] In some implementations, “uniform” refers to remaining the same and / or unchanging in form or character. In some implementations, “uniform” refers to a set of values (e.g., temperature values) being substantially constant / similar at given point in time. Some embodiments discussed herein relate to techniques for improving uniformity and / or reducing variation, specifically temperature variations. For example, some embodiments described herein relate to heat shields operable to reduce temperature variations between capillaries. Temperature variations of a heat shield containing embodiment are reduced if temperatures are more uniform with the heat shield than in a similar embodiment in which the heat shield is omitted. As discussed in detail below (e.g., with respect to Figures 10A-1 IB) temperature variations can induce measurement errors, therefore reducing temperature variations is surprisingly effective at reducing measurement errors. In particular, in some implementations and / or for some experiments, surprisingly small temperature non-uniformities, such as temperature variations of 5 degrees C, temperature variations of 4 degrees C, or temperature variations of 3 degrees C, can have surprisingly large impacts on measurement accuracy. Accordingly, in some instances it can be desirable to reduce temperature variation to less than 3 degrees C, less than 2 degrees C, or less than 1 degree C.

[0057] Some embodiments described herein relate to capillary electrophoresis (CE) technologies including, but not limited to the Simple Western™ platform commercially available from ProteinSimple®, a Bio-Techne® brand. Simple Western™ is a capillary-based technology which combines size-based separation or charge-based separation, immunoassay, detection, and data analysis in a fully automated workflow. Embodiments described herein are particularly well suited to western blot-style CE methods, such as that schematically illustrated in FIG. 1. It should be understood, however, that embodiments described herein are not limited to western blot-style analyses, but can be applied to any suitable CE method, particularly methods in which separation is carried out in parallel using an array of capillaries.

[0058] The Simple Western™ platform employs specialized reagents in a controlled- environment instrument to reduce variations. Quantification is achieved by accurate detection of chemiluminescence or fluorescence immunoassay signal. Simultaneous analysis of manysamples is achieved by an array of capillaries in a cartridge format, into which samples from a multi-well plate are loaded during an assay run. Simple Western™ enables automated multiplexed and high-throughput quantitative protein analysis that is less prone to process variations and therefore more accurate and reproducible compared to traditional western blotting.

[0059] FIG. 2 is a model of a CE instrument 20, including a capillary cartridge 21, a reagent plate 22, a UV light source 23, and a camera 24 for detection, according to an embodiment. Samples, antibodies, reagents, and buffer solutions are pipetted into a micro-well plate and loaded into the instrument along with the capillary cartridge. Separation matrix and stacking gel are loaded into the capillaries by lowering the cartridge into the well and trough rows of the reagent plate. The analyte samples are also loaded into the capillary cartridge, which is then lowered and contacts a running buffer. A direct current (DC) voltage is applied across the capillary cartridge to induce electrophoresis, whereby proteins in the samples are separated according to their molecular weights in each capillary. Upon completion of the electrophoretic separation, a combination of thermal energy and ultraviolet (UV) radiation is used to immobilize the proteins onto the capillary inner surfaces. The capillaries and / or the immobilized proteins can be immunoprobed, for example, with a sequence of interleaved antibody -binding and wash steps, in which a primary antibody is used to bind the protein of interest; and a labeled secondary antibody binds to the primary antibody to facilitate detection of the protein of interest. Chemiluminescent detection is accomplished by using a horseradish peroxidase (HRP)- conjugated secondary antibody, whereas fluorescence detection is achieved with fluorescent labeled secondary antibody. The chemiluminescent or fluorescent immunoassay signal is recorded by a charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) camera inside a light-tight chamber in a series of images. Additionally or alternatively, in some implementations, primary antibodies have a fluorescent and / or HRP label attached, reducing / eliminating the need for a secondary antibody.

[0060] Additional details related to detecting an analyte from a sample are discussed in U.S. Patent No. 9,304, 133, titled “METHODS AND DEVICES FOR ANALYTE DETECTION” and filed April 22, 2011, the contents of which are incorporated by reference herein in its entirety. Additional details related to an instrument that can be used to detect an analyte from a sample (e.g., a Simple Western™ instrument) are discussed in U.S. Patent No. 11,933,759, titled “APPARATUS, SYSTEMS, AND METHODS FOR CAPILLARY ELECTROPHORESIS” andfiled September 18, 2017, the contents of which are incorporated by reference herein in its entirety.

[0061] In some applications, particularly those involving pharmacokinetics assessment studies, which need to comply with regulatory requirements, high standards of precision and accuracy are necessary. The inventors named in the present application have recognized that one potential source of lack of uniformity in measurements of analyte concentration and / or quantity is differences in temperature across the array of capillaries during the immobilization process. In particular, as immobilization involves the application of UV radiation and thermal energy, UV intensity and temperature uniformity are important to quantitation precision and accuracy since each sample and corresponding reference or calibrator material are run in unique (e.g., different) and parallel capillaries within a single assay and cartridge. Embodiments described herein, therefore, generally relate to improving temperature and UV intensity uniformity across the capillary cartridge during immobilization. Experimental data have shown that assay signal is sensitive and correlated to temperature variations across the cartridge, and techniques described herein are effective in improving immunoassay data quality and reproducibility.

[0062] Several features of the capillary cartridge (sometimes referred to herein as “cartridge”) are designed to improve temperature and radiation uniformity across all capillaries in the cartridge to improve precision and accuracy of protein quantitation. For example, some implementations include a heat shield in proximity to (e.g., adjacent to, within millimeters of, surround at least a portion of, etc.) the capillary cartridge, a special buffer trough row in the microplate, and a UV-LED panel to provide UV radiation during immobilization.

[0063] First, in some embodiments, a heat shield (sometimes referred to herein as “shield” or “thermal shield" herein) in proximity to (e.g., adjacent to, within millimeters of, surround at least a portion of, etc.) the capillary cartridge is used to reduce / minimize uncontrolled heat transfer between the capillaries across the cartridge to maintain a uniform temperature profile during immobilization. One source of thermal nonuniformity is natural convection heat transfer between the surrounding air and the heated capillary array. These air currents can vary across the array and enhance differences in temperature across the array and along the length of the capillaries. The heat shield can be operable to control transfer of heat away from the array of capillaries to reduce temperature variation between capillaries. For example, the heat shield can mitigate / inhibit natural convection from transferring heat from the capillary array to the surroundingenvironment by confining the air space immediately around each capillary within the cartridge. In some such embodiments, the heat shield can promote conductive and / or convective heat transfer between capillaries, increasing capillary temperature uniformity (i.e., the heat shield may act as a heat spreader). In some embodiments, heaters coupled to the heat shield and disposed on the left and / or right sides of the cartridge can counteract edge heat transfer effects, for example, by controlling heat transfer from the end capillaries. In some implementations, activating heaters on the sides of the cartridge can result in a net heat transfer to end capillaries which minimizes / reduces uncontrolled heat transfer at the edge capillaries.

[0064] Second, in some embodiments, a special buffer trough is designed to reduce / minimize variations in heat loss through capillaries dipped into the buffer solution in the microplate (sometimes referred to herein as “trough”).

[0065] Third, in some embodiments, a UV-LED array (sometimes referred to herein as “UV source,” “UV-LED panel,” or “array of UV-LEDs) is used (e.g., instead of a mercury-vapor (Hg) lamp), to provide a uniform intensity of UV radiation to immobilize proteins.

[0066] It should be understood that the heat shield, buffer trough, and UV-LED array can work alone or in any combination to reduce variations of the immobilization process. In particular, in some implementations, the heat shield is made of UV-transparent material (e.g., fused silica or quartz) to permit UV light and radiative heat transfer from the UV-LED array to the capillaries, while controlling convective heat transfer from the capillaries. Additionally, in some implementations, the heat shield is made from a material with high thermal conductivity to reduce / minimize thermal non-uniformity effects from the UV-LED panel source and other thermal sources. In some embodiments, the heat shield can be coated with a thin (e.g., <0.1 mm) layer of a high thermal conductivity material such as a chemical vapor deposition (CVD) diamond layer to improve the heat spreading performance of the heat shield.

[0067] Several designs of the thermal shield have been considered. In one embodiment, each of the capillaries in the cartridge are isolated in four-sided enclosures such that all four sides are shielded; said differently, along a predetermined portion of each cartridge (e.g., along the entire length of the cartridge, along the entire length of the cartridge that is not submerged in a solution, along a length of the cartridge adjacent / normal to the heat shield, etc.), 100% of the surface area at the predetermined portion of that cartridge is covered / adjacent to the heat shield. In another embodiment, only three sides of each capillary are shielded; said differently, along apredetermined portion of each cartridge (e.g., along the entire length of the cartridge, along the entire length of the cartridge that is not submerged in a solution, along a length of the cartridge adjacent / normal to the heat shield, etc.), approximately 75% of the surface area at the predetermined portion of that cartridge is covered / adjacent to the heat shield. In yet another embodiment, only two sides of each capillary are shielded; said differently, along a predetermined portion of each cartridge (e.g., along the entire length of the cartridge, along the entire length of the cartridge that is not submerged in a solution, along a length of the cartridge adjacent / normal to the heat shield, etc.), approximately 50% of the surface area at the predetermined portion of that cartridge is covered / adjacent to the heat shield.

[0068] FIG. 19 illustrates a four-sided, three-sided, and two-side heat shield, according to an embodiment. Pattern 2002 shows a partial top view of a four-sided heat shield 2008; a set of capillaries can include capillaries Cl, C2, and C3, and the four-sided heat shield 2008 fully surrounds capillaries Cl, C2, and C3. Pattern 2004 shows a partial top view of a three-sided heat shield 2010; a set of capillaries can include capillaries C4, C5, and C6, and the three-sided heat shield 2010 surrounds only the left, back, and right portion of capillaries C4, C5, and C6. Pattern 2006 shows a partial top view of a two-sided heat shield 2012; a set of capillaries can include capillaries C7, C8, can C9, and the two-sided heat shield 2012 surrounds only the left and right portions of capillaries C7, C8, and C9.

[0069] In some embodiments, comb keeper structures (sometimes referred to herein as “comb keeper”) are used to secure and align the capillaries inside each slot or compartment of the heat shield.

[0070] As discussed above, both the four-sided and three-sided heat shield designs can be used in combination with heaters that occupy the positions adjacent to the left- and right-most capillary to compensate for the thermal boundary conditions of those side capillaries. In other embodiments, a heat shield design with multiple heater zones may also achieve desirable temperature uniformity during immobilization. In yet another embodiment, placement of a thermally insulating wall at an optimal distance from the left- and right-most capillaries can improve temperature uniformity (e.g., a perfectly insulating wall positioned at a distance of half the capillary spacing from each of the left and right edge capillaries will eliminate any edge effect, so in practice a real insulating wall with a high thermal resistance can substantially reduce edge effects).

[0071] Another source of potential capillary temperature non-uniformity is uneven heat transfer from the capillary to the buffer solution. In particular, heat transfer can be a function of wetted length of the capillary. Because of meniscus effects, capillaries closest to the walls of the trough (typically end capillaries) have a greater wetted length, and therefore greater heat transfer between capillary and buffer solution. Some embodiments described herein, therefore, include or relate to troughs configured to reduce heat transfer variability between buffer solution and capillary. The influence of the wetted length on heating is also described with respect to FIGS. 14A and 14B, according to an embodiment. At a fixed voltage, the joule heating power in the unwetted section of the capillary (e.g., the majority of the capillary where the UV-initiated binding reaction is occurring) is a function of the wetted length.

[0072] The UV-LED panel array is designed to improve the uniformity of the immobilization process and / or to compensate for other sources of non-uniformity. The UV-LED array pattern is configured for UV radiation uniformity. In some embodiments, edge LED dies are supplied with higher power (e.g., relative to LEDs that are not edge LEDs) to compensate edge non-uniformity thermal and / or UV intensity effects such that the overall rate of immobilization is uniform.

[0073] A factor for precision and accuracy of protein quantitation using CE techniques is uniformity among the capillaries in the cartridge. In some implementations, capillaries have an outer diameter of about 360 pm and are on the order of 50mm long. In some implementations, the cartridge has 25 capillaries, with a spacing of 2 to 5 mm between capillaries. Even though the same immunoassay sequence, including loading, separation, incubation, and detection can occur simultaneously in all capillaries in the cartridge, variation of micro-environment inside the instrument can lead to non-uniformity of immunoassay signal from the same target. For example, uncontrolled free-convection inside the instrument can lead to temperature non-uniformity across the capillaries in the cartridge during separation, immobilization, and antibody incubation, which results in variation of immunoassay signal and a lack of repeatability and precision of protein quantitation.

[0074] With reference to FIG. 3 A, a capillary cartridge 31 includes a plurality of capillaries 32 extending from a reservoir 33 formed by a body 34. With reference to FIG. 3B, various thermal effects around the capillary cartridge during immobilization are illustrated. In the immobilization step, a combination of thermal energy and UV radiation is used to induce binding of proteins in the sample onto the inner surface of the capillaries. UV radiation is supplied by anarray of UV-LED, the arrangement of which is configured (e.g., optimized) to irradiate all capillaries of the cartridge uniformly. Thermal energy can be introduced using any suitable technique, such as using an inferred (IR) radiation source, strip heaters coupled to capillaries, or passing an electrical current through the wall or the lumen / contents of the capillaries in the cartridge to induce Joule heating. Heating of the capillaries also heats up the air around the cartridge, inducing heat transfer between the capillaries and to the surroundings by free convection (see e.g., Arrow 2 at Figure 3B). In particular, the edge capillaries (see e.g., capillaries 32A and 32B at Figure 3B), which have no neighbor to one side, experience different heat transfer rates than the capillaries at, for example, the center of the cartridge. As a result, in known CE systems the edge capillaries can have lower temperature and lower immunoassay signal (see e.g., Arrows 3 at Figure 3B).

[0075] With continued reference to FIG. 3B, a prominent natural convection effect can occur along the length of each capillary. As the cartridge is heated, hot air at the bottom of the cartridge rises to transfer heat to the top of the cartridge, which results in a temperature gradient along the capillaries (see e.g., Arrows 4 at Figure 3B). At the top of the cartridge, the buffer reservoir acts as a heat sink such that the top of the capillaries is slightly cooled (see e.g., Arrows 5 at Figure 3B). The capillary tips (e.g., near the bottom of the cartridge) are dipped into a trough of buffer solution, which also acts as a heat sink. Heat transfer rate, and therefore temperature, of the capillary is also affected by the immersed depth of the capillaries such that a spatial bias of dipped depth within a cartridge can result in temperature non-uniformity across the capillaries (see e g., Arrows 6 at Figure 3B). Convective heat transfer between the cartridge and the instrument internal environment (see e.g., Arrow 7 at Figure 3B) can also cause temperature nonuniformity among the capillaries (e.g., improper or non-optimal temperature regulation within the larger instrument enclosure may create unwanted temperature gradients, natural convection, and / or forced convection at the location of the capillary array). Such unwanted temperature variations across the capillary array can lead to variations in immunoassay signal. Natural convection can be difficult to control, thus impacting the reproducibility of CE immunoassays.

[0076] Some implementations improve thermal uniformity during the immobilization process with a combination of several innovations. First, the capillaries are heated by a stable power source (e.g., IR source, voltage source, etc.) to reduce variation in heating power. A thermal shield is used to limit non-uniform heat transfer mechanisms.

[0077] With reference to FIGS. 4A and 4C, a capillary cartridge 41 is shown with a heat shield 41, referred to herein as a “front-and-back-shield” embodiment. In this embodiment, the heat shield 45 is added to encase the cartridge 41 such that each capillary 42 is compartmentalized (e.g., around all four sides of each capillary) and convection-isolated from its neighbors. Similarly stated, a front portion 45 A of the heat shield 45 is disposed adjacent to a front portion of the capillary array 42, a back portion 45B of the heat shield 45 is disposed adjacent to a back portion of the capillary array 43. In the illustrated embodiment, a combination of fins 46 (sometimes referred to herein as “baffles” or “ribs”) isolate each capillary from its neighboring capillaries. In the illustrated embodiments, the ribs 46 are formed on the front portion 45 A of the heat shield 45. When assembled, the heat shield 45 forms a compartment 47 for each of the capillaries 42. In some embodiments, each compartment 47 is at least twice as wide as the diameter of the capillary compartmentalized in that compartment but not wider than 10 times the capillary diameter (e.g., compartment width is 2mm). In some implementations, the thermal shield encases at least 50% of the capillary length (e.g., covers 37.5mm). In the illustrated embodiment, the heat shield 45 includes a first heater 44A at a first end and a second heater 44B at second end. The first heater 44A is at least partially received within a notch 48 formed in the front portion 45 A of the heat shield 45.

[0078] With reference to FIG. 4B, heat transfer by free convention across the capillaries in the cartridge is reduced, blocked, or minimized by the fins 46 on the heat shield 45 (see e.g., cancelling of Arrow 2 at Figure 4B). Heaters 44A and 44B on the left- and right-most locations of the heat shield act as thermal neighbors to compensate for the extra heat loss of the edge capillaries 42A and 42B, respectively (see e.g., Arrows 3 at Figure 4B). Free convection heat transfer across the cartridge is limited by the heat shield (see e.g., Arrows 4 at Figure 4B) and the confined air space around each capillary reduces natural convection along the length of the capillary and reduces variability in natural convection from capillary to capillary. In some embodiments, a special buffer solution trough design to reduce variation in dipping depth of each capillary is employed (see e.g., Arrows 6 at Figure 4B). The four-sided heat shield design also blocks any air flow (e.g., bulk air flow across / through the capillaries) to and from the larger environment inside the instrument to reduce or minimize thermal disturbances by free or forced convection (see e.g., cancelling of Arrow 7 at FIG. 4B).

[0079] With reference to FIGS. 5 A and 5C, a capillary cartridge 51 is shown with a heat shield 55, referred to herein as “front-shield only” embodiment. In the illustrated embodiment, the heat shield 55 is a three-sided heat shield that includes a combination of fins 56 to isolate each capillary from its neighboring capillaries. In other words, the heat shield 56 surrounds three sides of each capillary 52.

[0080] In some implementations, an advantage of the heat shield 55 over the four-sided shield 45 of FIGS. 4A-4C, is that the heat shield 55 can be rotated or translated to and from the array of capillaries 52 in several directions. In some embodiments, the heat shield 55 is rotated about a generally horizontal axis between a first position in which the heat shield 55 is positioned away from the capillaries (e.g., a stored position), and a second position (FIG. 5A) in which the heat shield 55 is positioned at the capillaries 52 (e.g., an in-use position). In contrast, a four-sided heat shield would typically be constructed to split into two halves or the capillaries are inserted into the heat shield from above, which can complicate assembly and / or removal of the array of capillaries from the heat shield.

[0081] In some implementations, the front shield is disposed adjacent to a front portion of the capillary array (e.g., between the capillaries and the UV-LED), while a back portion of the capillaries are left exposed. Similarly stated, unlike the embodiment shown and described with reference to Figures 4A and 4B there is no back disposed adjacent to the back portion of the capillary array. Nonetheless, the front portion of the shield is operable to block bulk air flow, and therefore reduce convective flows through the capillary array. In addition, longer fins (e.g., compared to the fins of the four-side design including front and back shields) on the thermal shield can be operable to reduce free convection across the capillaries (see e.g., cancelling of Arrow 2 at Figure 5B). In some implementations, the length of the fin is designed to achieve an aspect ratio from 1 to 10 of each shield compartment (e.g., compartment dimension is 6mm long and 2mm wide). Like the four-sided embodiment, heaters on the left- and right-most locations compensate for the “no-neighbor” boundary condition of the edge capillaries (see e.g., Arrows 3 at Figure 5B). When the capillaries are heated, hot air from the bottom of the cartridge is mostly confined to each capillary length by the longer fins. Because only three sides of the capillaries are enveloped, a limited amount of free convection is possible, although conduction (see e.g., Arrows 4 at Figure 5B) may still be present. This design can also benefit from a special buffer solution trough design to reduce variation in dipping depths (see e.g., Arrows 6 at Figure 5B).The three-sided shield only blocks air flow to and from the instrument environment from the front side. Disturbances at the back side are reduced / minimized, by the longer fins (see e.g., Arrow 7 at Figure 5B) and flow across the array (see e.g. (7) at Figure 5B) is also blocked by the three-sided shield.

[0082] One inherent difficulty in designing a heat shield for the capillary cartridge can be the tolerance of the position of the capillaries. The distance between capillaries (pitch) in the cartridge can be small and it can be difficult to control the pitch exactly in manufacturing. In addition, the capillaries can be flexible such that they may not be straight and perfectly normal to the plane of the top surface of the cartridge. The effectiveness of the heat shield can be affected by the distance of the capillary to either side of the isolation fin. If the distances between the capillary to the left-fin and the right-fin are not equal, or if the capillary touches a fin of the heat shield, heat transfer will not be uniform.

[0083] With reference to FIGS. 6A-6C, a heat shield 61 is illustrated with a comb keeper 62 positioned at a bottom 63 of the heat shield 61. The comb keeper 62 includes a main body portion 64 that is secured to the bottom 63 of the heat shield 61. The comb keeper 62 includes a plurality of slots 65 formed in an edge 66 of the comb keeper 62. The comb keeper 62 and slots 65 are positioned such that one slot 65 is aligned with each of the compartments 67 formed by the heat shield 65. The comb keeper 62 is used to stabilize, or “pin” the location of capillary inside the heat shield such that the capillary is centered in its compartment. As a result, heat transfer from each capillary is more consistent and assay signal variation is reduced. The comb keeper 62 advantageously stabilizes capillary positions and accounts for unwanted or uncontrolled capillary bending, for example.

[0084] With reference to FIGS. 6D-6G, a heat shield 71 is illustrated with a comb keeper 72 positioned at a bottom 73 of the heat shield 71. In the illustrated embodiment, the comb keeper 72 has a plurality of slots 75 with a more aggressive “champagne flute” shape such that capillaries that were bent, slanted, or curved can also be pinned inside the center of the compartment.

[0085] With reference to FIG. 6H, in some embodiments, a nominal position 81 of the capillary 82 is at 3 mm in the y-direction and 1 mm in the x-direction. In one embodiment, the comb keeper 83 includes a slot 84 with a base 85 located at the nominal position 81 of the capillary 82. In other words, the slot 84 of the comb keeper 83 positions the capillary 82 at thenominal position 81 . If the capillary is tilted towards the heat shield, the capillary will contact the comb keeper and be deflected back to the nominal position. However, if the capillary is tilted away from the shield (e.g., in the y-direction), with the comb keeper 83 there is a chance that the comb keeper 83 won’t contact the capillary 82 at all. Advantageously, a comb keeper 86 includes a slot 87 with a base 88 located away from the nominal position 81 of the capillary 82. In other words, the slot 87 of the comb keeper 86 positions the capillary 82 away from the nominal position 81. As such, the final location after the capillary contacts and is guided by the comb keeper 86 is different than the nominal position of the capillary. By biasing the location of the base 88 of the slot 87 in comb keeper 86 further away from the nominal position 81 of the capillary 82, there is advantageously an increased probably of contact and therefore more reproducible positioning of the capillary.

[0086] With reference to FIGS. 7A-7C, a baffle-style heat shield 91 design in which free convection around the capillaries are isolated with baffles, according to an embodiment. In some implementations, dimensions of the baffle style shield are similar to the four-sided (e.g., as shown in FIGS. 4A and 4B) or three-sided (e.g., as shown in FIGS. 5A and 5B) heat shield designs. Comb keepers (e.g., as shown in FIGS. 6A-6G) can also be included in the baffle-style heat shield. The baffle-style heat shield is particularly well suited for heat shield materials that are not UV- or optically transparent (e.g., plastic). Heat loss to the baffle-style heat shield during capillary heating is reduced / minimal because the heat shield has less thermal mass relative to the embodiments of FIGS. 4A-5B and has poor conductivity (e.g., made of plastic).

[0087] With reference to FIGS. 8A-8C, a box-style heat shield 101 design is illustrated. The heat shield 101 can be simpler to manufacture and may be less sensitive to manufacturing tolerances, as there are no baffles or ribs between capillaries. The box-style heat shield may be constructed from, for example, quartz or other suitable material that provides UV-transparency. In some implementations, the width of the box-style shield is at least as wide as the capillary array, but not wider than 2x the cartridge width. The thickness of the box-style heat shield can approximately 2 to 10 times the diameter of the capillaries. In some embodiments, a flat heater can be affixed onto the front or back shield surface of the box-style heat shield to provide uniform heating to the cartridge. The dimension of the flat heater could be, for example, similar to height and width of the box-style shield, but those skilled in the art can improve / optimize the heater design to reduce / minimize temperature variation among the capillaries in the cartridge.

[0088] FIG. 9 shows the temperature profile of the capillaries in the cartridge with different heat shield designs with edge heaters, according to an embodiment. Each plot shows the temperature deviation of each capillary from the mean temperature of all capillaries in the cartridge; in this example (and other examples discussed herein), the cartridge included 25 linearly arranged capillaries, where position 1 is the left-most capillary and the capillary is located further right as the position number increases (e.g., capillary 25 is the right-most capillary). With no heat shield, lower temperatures were observed for edge capillaries (at positions 1 and 25) due to the “no-neighbor” effect discussed herein. With a four-sided heat shield encasing the cartridge supplemented with side heaters (e.g., as shown in Figures 4A and 4B), a uniform temperature profile with deviation < 1 deg Celsius can be achieved. A three-sided heat shield with side heaters (e.g., as shown in FIGS. 5A-5C) resulted in a temperature profile with deviation approximately < 1.5 deg Celsius. As shown, there is no bias towards lower temperature at the edge (capillary positions 1 and 25) for the four-sided and three-sided heat shields.

[0089] Correlation between immobilization temperature and immunoassay signal across the capillaries in a cartridge is illustrated in FIGS. 10A and 10B, according to an embodiment. FIGS. 10A and 10B shows capillary temperature during immobilization along with chemiluminescence signal of multiple targets detected at different cartridge heating powers in the immobilization step. The data was obtained with the three-sided shield design, but with side heaters turned off to illustrate the correlation of temperature to assay signal. As discussed herein, higher rate of heat loss at the edge results in the characteristic rainbow-shaped temperature profile where edge capillaries have lower temperatures. Subsequently, lower immunoassay signals were observed at the edge capillaries of the cartridge. The result is consistent across different protein targets and at different heating powers, indicating that temperature uniformity during immobilization is a major driver of immunoassay signal uniformity.

[0090] The immobilization step in CE immunoassays is a transient process. During immobilization, UV exposure and / or heating is used to increase temperature of the reagents inside the capillaries from room temperature to, for example, approximately 50°C to 90°C. The side heaters in the heat shield are designed to compensate for extra heat loss from the edge capillaries. However, different strategies can be employed to achieve uniform temperature in the capillaries during the immobilization process at different times. FIG. 12 depicts severaltemperature control schemes to illustrate spatial and temporal temperature control, according to an embodiment. When the cartridge is heated without side heaters compensation (three left-most plots in FIG. 12), temperatures of all capillaries increase uniformly and quickly at the beginning of the immobilization step. As immobilization proceeds, the rate of temperature increase of the edge capillaries is lower than the center capillaries, leading to diverging temperatures across the capillaries in the cartridge. Towards the end of immobilization, temperatures of center capillaries continue to increase at a faster rate than the edge capillaries.

[0091] One can control the side heaters such that the temperature of the heater is constant throughout the immobilization. However, constant temperature control can lead to an “unstable” temperature profile of the capillaries (see, e.g., the middle three vertical plots in FIG. 12). Due to the transient nature of the process, cartridge temperature increases when thermal energy is introduced to the capillaries, using any suitable technique, such as using an inferred (IR) radiation source, strip heaters coupled to capillaries, passing an electrical current through the wall or the lumen / contents of the capillaries in the cartridge to induce Joule heating. If the side heater temperature is controlled to match the capillary temperature at early immobilization, a diverging temperature profile similar to when no side heater is employed is seen. On the other hand, if the side heater temperature is set higher to match the capillary temperature at the end of immobilization, the edge capillaries will be hotter than the middle capillaries at the beginning of the immobilization. The profile will become more uniform towards the end of immobilization. This type of spatial and temporal dependance is described as “unstable” because the temperature profile is uniform only for a short period during immobilization.

[0092] In some implementations, the control scheme of the side heaters is a constant power control, in which a constant power proportional to thermal energy introduced to the capillaries of the cartridge is applied to the side heaters during immobilization. With a constant power control scheme, the side heaters temperature increases steadily throughout the immobilization process, compensating the transient heat loss of the edge capillaries throughout the immobilization process. This results in a “stable” temperature profile in which capillary temperature is uniform throughout the entire immobilization step, as shown in three right-most plots in FIG. 12. Surprisingly, a constant power control scheme has been shown to produce more stable temperature profiles than more sophisticated control systems, such as the use of a PID controller to target a specific set point.

[0093] Some embodiments involve a buffer solution trough designed such that the “dippingdepths” of the capillaries compensate for other thermal effects during immobilization. During immobilization, the capillaries are dipped into a buffer solution in a trough on the reagent plate. When a partially wetted capillary is heated by Joule heating, the heating power is approximately inversely proportional to the square of the wetted length at sufficiently high temperature such that solution conductivity of the wetted region is lower compared to that of the un- wetted region. In other words, the temperature of a capillary depends on its dipping depth, as illustrated in FIG. 13. Dipping depths of all capillaries are generally the same when the cartridge is dipped into a microwell plate in which each capillary is dipped into a single well. However, when microplates for immunoassays are designed to minimize plate preparation time, buffer solution common to all capillaries are pre-fdled into “trough rows.” Trough rows sometimes have grouped partitions to prevent “sloshing” of reagents during plate preparation. In each partition, the buffer solution forms a meniscus with a contact angle to the walls of the partition. This effect is illustrated in FIGS. 14A and 14B, according to an embodiment. The meniscus produces a dipping depth bias among the few capillaries in the grouped partition (as shown in FIG. 14A), in which the capillary at the center of the partition (e.g., capillary 13) has the shortest wetted length, whereas the capillaries near the edge in the grouped partition (e.g., capillaries 12 and 14) has the longest dipping depth. For a given Joule heating power into the cartridge, the grouped partition results in a systematic temperature non-uniformity, whereby capillaries occupying the center positions in the group partitions achieve higher temperatures and higher immunoassay signal, while capillaries at the edge positions in the group partitions would have lower signal.

[0094] FIG. 14B depicts a buffer trough 110 to eliminate the “meniscus-bias” of signal nonuniformity, according to an embodiment. In this embodiment, the buffer trough 110 is individual -partitioned for capillaries 3-23, which means that each capillary occupies its own partition of the trough. The edge capillaries (1 & 2 and 24 & 25) share a trough partition. In other words, the buffer trough 110 includes partitions 111 to form individual compartments 112 (configured to receive a single capillary) and two group compartments 113 (configured to receive more than one capillary). In the illustrated embodiment, the two group compartments 113 are positioned on the ends of the trough 110. The partitions 111 are designed to create the same meniscus shape for all capillaries in the cartridge and are connected such that all compartments share the same hydrostatic pressure to reduce / avoid any nonuniformity inhydrodynamic flow between capillaries. The individual-partition trough can be further engineered to compensate edge effects. For example, extra heat loss from edge capillaries in the cartridge can be compensated by a buffer trough where the edge partitions are shared such that the dipping depths of edge capillaries are lower than all other capillaries in the cartridge. This design can compensate for inadequate edge heater power, or other transient thermal effects not compensated by the side heaters on the thermal shield (as shown in FIG. 14B).

[0095] Stated another way, the trough can be designed to provide a uniform dipping depth for all capillaries, or the trough may be designed to create specific dipping depths at each capillary position so as to work in concert with a particular thermal shield design or to compensate for edge effects that cannot be eliminated by a specific shield design. Accordingly, as illustrated at FIG. 14B, in some implementations, the buffer trough can be partitioned such that some capillaries each occupy their own partition of the trough while some capillaries share partitions with other capillaries. For example, a buffer trough can be designed such that the two left-most capillaries share a single partition and / or the two right-most capillaries share a different single partition, and each capillary between the two left-most capillaries and the two right-most capillaries has or occupies its own different partition.

[0096] Any other suitable combination of individually partitioned and multiple-capillary partitions is also possible. For example, three, four, or five capillaries can share an edge partition, while pairs or triplets of capillaries between the edgemost capillaries share a partition. Alternatively, for example in implementations with excess edge heat loss, the capillaries disposed in an end portion of a capillary cartridge can be multiply partitioned, while capillaries in a central portion of the capillary cartridge can be individually partitioned. In other embodiments, the buffer trough is not partitioned. Said differently, the buffer trough is not configured to separate one or more capillaries from one or more other capillaries. For example, if 25 capillaries are used during an immunoassay process, all 25 capillaries can be submerged at least partially using the buffer trough without being separated from each other via a partition(s).

[0097] In yet other embodiments, the buffer trough is partitioned such that each capillary occupies its own partition (e.g., fully individual -partitioned). For example, the trough 110 of FIG. 14B is modified such that (1) capillaries 1 and 2 each have their own partition instead of sharing a partition and (2) capillaries 24 and 25 each have their own partition instead of sharing a partition.

[0098] FIGS. 16A-16D shows the intensity profiles of different UV-LED panel designs, according to an embodiment. The UV-LED panel consists of an array of UV-LED devices (i.e., individual UV-LED dies) arranged in a rectangular pattern. FIG. 16A shows intensity profiles for a panel having 54 dies, FIG. 16B shows intensity profiles for a panel having 30 dies, FIG. 16C shows intensity profiles for a panel having 24 dies, and FIG. 16D shows intensity profiles for a panel having 23 dies. The size of the UV-LED panel can be similar to or slightly larger than the dimensions of the cartridge (height ~ 35mm to 60mm and width ~ 80mm to 100mm). An undersized panel design results in edge roll-off in UV intensity, whereas an oversized panel may generate excessive heat. The intensity and uniformity of UV radiation across capillaries can be adjusted and tuned by the array pattern. In one embodiment, a dense array pattern results in higher average UV intensity (see e.g., FIG. 16A). In another embodiment, the array pattern is more uniform (see e g., FIGS. 16B and 16C). It is inevitable to have edge effects where the intensity rolls off around the edges of the array. One solution is to design the array to be larger than the cartridge areas. However, when space is limited, an alternate embodiment employs an array pattern that is denser around the edge to increase intensity around the array edge and improve the uniformity of UV intensity. In another embodiment, higher power is supplied to the edge dies on the panel to increase UV radiation intensity and improve uniformity.

[0099] FIGS. 11 A and 1 IB compare assay signal profiles obtained with and without the heat shield, according to an embodiment. FIGS. 11A and 1 IB each show four different assay runs detecting multiple target proteins. In the assay runs on the instrument without a heat shield, the signal shows a profile bias characteristic of temperature non-uniformity, with high coefficient of variation (% CV). On the other hand, assay runs on the instrument equipped with the three-sided heat shield and side heaters showed relatively flat signal profiles with low coefficient of variation.

[0100] FIGS. 15A and 15B show a comparison of assay signal in a grouped partition trough with 3 capillaries sharing a partition and an individual partition trough, respectively, according to an embodiment. In both FIGS. 15A and 15B, the capillaries are grouped by “edge of cartridge” (referring to FIGS. 14A and 14B, capillaries 1 and 25), “side of trough” (referring to FIGS. 14A and 14B, capillaries 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, and 24), and “center of trough” (referring to FIGS. 14A and 14B, capillaries 4, 7, 10, 13, 16, 19, and 22).

[0101] In FIG. 15 A, the diamond for each group indicates the mean of each group with the height of each diamond indicating the 95% confidence interval of the mean. The statistical analysis confirms the observation of assay signal profile that capillaries dipped in the center of the grouped-partitioned trough have higher signal, as shown in FIG. 15 A. The statistically significant bias is eliminated with the individual-partition trough, as shown in FIG. 15B. In addition, the assay data shown in FIG. 15A was generated without side heaters compensation (corresponding to the three left-most plots in FIG. 12) which results in edge capillary signal that is significantly lower than the mean signal, as seen in FIG. 15 A. In contrast, the assay data shown in FIG. 15B was generated with a constant power control scheme (corresponding to the three right-most plots in FIG. 12) which results in edge capillary signal that is indistinguishable from the mean signal as seen in FIG. 15B.

[0102] The data in this disclosure comprise mostly measurements using chemiluminescence detection. Similar results should be obtainable using any detection modality since techniques described herein controls variations that can occur prior to detection steps.

[0103] FIG. 17 shows a system block diagram of an instrument 1800 operable to perform capillary electrophoresis, including an immobilization process, according to an embodiment. Instrument 1800 includes processor 1802, memory 1804, capillary cartridge 1806, UV source 1808, heat shield 1810, and trough 1812, two or more of which can be operatively coupled to one another (e.g., via a system bus).

[0104] Processor 1802 can be, for example, a hardware-based integrated circuit (IC) or any other suitable processing device configured to run and / or execute a set of instructions or code. For example, processor 1802 can be a general-purpose processor, a central processing unit (CPU), an accelerated processing unit (APU), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a programmable logic array (PLA), a complex programmable logic device (CPLD), a programmable logic controller (PLC) and / or the like. In some implementations, processor 1802 can be configured to run any of the methods and / or portions of methods discussed herein.

[0105] Memory 1804 can be, for example, a random-access memory (RAM), a memory buffer, a hard drive, a read-only memory (ROM), an erasable programmable read-only memory (EPROM), and / or the like. Memory 1804 can be configured to store any data used by the processors to perform the techniques (methods, processes, etc.) discussed herein. In someinstances, memory 1804 can store, for example, one or more software programs and / or code that can include instructions to cause processor 1802 to perform one or more processes, functions, and / or the like. In some implementations, memory 1804 can include extendible storage units that can be added and used incrementally. In some implementations, memory 1804 can be a portable memory (for example, a flash drive, a portable hard disk, and / or the like) that can be electrically and / or communicatively coupled to the processors. In some instances, memory 1804 can be remotely operatively coupled with a compute device (not shown in FIG. 1).

[0106] Capillary cartridge 1806 can refer to a cartridge that include capillaries. The capillaries in capillary cartridge 1806 can have any orientation, such as vertical, horizontal, or anything in between. For example, in some implementations, each capillary can be oriented such that a second end of that capillary is disposed above a first end of that capillary during an immobilization process. Similarly stated, each capillary can be vertically oriented such that a second end of that capillary is disposed directly above a first end of that capillary during an immobilization process. Capillary cartridge 1806 can also include a buffer solution / heat sink. In some implementations, the second end of each capillary is coupled to a buffer solution / heat sink.

[0107] Trough 1812 can include partitions and a solution. At least a portion of the capillaries from capillary cartridge 1806 can be submerged into the solution (e.g., near the first end of the capillaries). The capillaries can be submerged so that subsets of one or more capillaries are divided / separated by the partitions in trough 1812.

[0108] Heat shield 1810 can include heaters 1814 and, together, provide heat to the capillaries included in capillary cartridge 1806. Further, UV source 1808 can provide UV light to the capillaries included in capillary cartridge 1806. In some implementations, at least a portion of heat shield 1810 and / or heaters 1814 is located between capillary cartridge 1806 and UV sources 1808 during the immobilization process. In some implementations, heat shield 1810 and / or heaters 1814 is UV transparent such that all and / or most of UV light from UV source 1808 can reach the capillaries.

[0109] UV source 1808, heat shield 1810, heater 1814, and / or trough 1812 can be used to provide a more uniform heat / temperature distribution to the capillaries at capillary cartridge 1806 compared to known methods. In some implementations, instrument 1800 is the same as aninstrument described in U.S. Patent No. 11,933,759, but adds UV source 1808, heat shield 1810, heaters 1814, and trough 1812 to improve heat or temperature uniformity at the capillaries.

[0110] FIG. 18 shows a flowchart of a method 1900 to immobilize at least one analyte, according to an embodiment. In some implementations, method 1900 is performed by instrument 1800 from FIG. 17.

[0111] At STEP 1902, at least one analyte from a plurality of analytes contained within a sample is separated in at least one capillary form an array of capillaries (e.g., capillary cartridge 1806 in FIG. 17). The sample is disposed within the at least one capillary. At STEP 1904, a UV light source (e.g., UV source 1808) is activated. Each capillary from the array of capillaries includes a photo-activated moiety configured to bind analytes from the array of capillaries to a wall of that capillary when that capillary is irradiated by the UV light source. At least a portion of a heat shield (e.g., heat shield 1810, heaters 1814) is disposed between the array of capillaries and the UV light source. The heat shield is configured to control transfer of heat away from the array of capillaries to reduce a temperature variation between capillaries from the array of capillaries when the UV light source is active. At STEP 1906, each capillary in the array of capillaries is immunoprobed after activating the UV light source such that a labeled antibody binds to the at least one analyte.

[0112] In an embodiment, an apparatus (e.g., instrument 1800) includes a capillary cartridge (e.g., capillary cartridge 1806). The capillary cartridge includes a plurality of capillaries. Each capillary from the plurality of capillaries has a first end configured to be submerged into a trough during an immobilization process and a second end opposite the first end. The apparatus further includes an ultraviolet (UV) light source (e.g., UV source 1808 from FIG. 17) configured to irradiate the plurality of capillaries during the immobilization process. The apparatus further includes a heat shield (e.g., heat shield 1810, heaters 1814). At least a portion of the heat shield is disposed parallel to the plurality of capillaries and configured to control transfer of heat away from the plurality of capillaries to reduce a temperature variation between capillaries from the plurality of capillaries during the immobilization process. The apparatus further includes at least one trough (e.g., trough 1812) holding a solution. The first end of each capillary from the plurality of capillaries is configured to be submerged into the solution during the immobilization process.

[0113] In some implementations, the heat shield is UV-transparent.

[0114] In some implementations, the heat shield is configured to impede convective heat transfer away from the plurality of capillaries while permitting radiative heat transfer to and / or from the plurality of capillaries.

[0115] In some implementations, the heat shield is configured to reduce the temperature variation between capillaries from the plurality of capillaries, at least in part, through convective and / or conductive heat transfer between capillaries from the plurality of capillaries while impeding convective heat transfer away from the plurality of capillaries.

[0116] In some implementations, the plurality of capillaries is arranged in a linear array with a first capillary from the plurality of capillaries defining a first end of the linear array and a last capillary from the plurality of capillaries defining a second end of the linear array. The heat shield includes (1) a first side heater disposed adjacent to the first capillary and configured to heat the first capillary during the immobilization process and (2) a second side heater disposed adjacent to the last capillary and configured to heat the last capillary from the plurality of capillaries during the immobilization process.

[0117] In some implementations, the heat shield includes a plurality of ribs isolating each capillary from the plurality of capillaries into an individual channel that impedes convective heat transfer between each capillary from the plurality of capillaries. In some implementations, the plurality of capillaries is arranged in a linear array and at least one rib from the plurality of ribs is located between each adjacent pair of capillaries from the plurality of capillaries.

[0118] In some implementations, the portion of the heat shield is configured to impede convective heat transfer from the plurality of capillaries, for example, by blocking bulk air flow across and / or through the plurality of capillaries.

[0119] In some implementations, the portion of the heat shield is a first portion of the heat shield disposed on a first side of the plurality of capillaries. A second portion of the heat shield is disposed on a second side of the plurality of capillaries opposite the first side. The first portion of the heat shield and / or the second portion of the heat shield can be configured to impede convective heat transfer from the plurality of capillaries. For example, the first portion of the heat shield and / or the second portion of the heat shield can be operable to block bulk air flows through / across the plurality of capillaries. Similarly stated, the first portion of the heat shield and / or the second portion of the heat shield can be operable to block and / or impede air currents that flow normal to the plurality of capillaries.

[0120] In some implementations, the portion of the heat shield is a first portion of the heat shield disposed on a first side of the plurality of capillaries (e.g., between the capillaries and the UV light source) and configured to impede convective heat transfer from the plurality of capillaries. The first portion of the heat shield is UV-transparent and configured to allow UV light from the UV light source to reach the plurality of capillaries through the first portion of the heat shield. A second portion of the heat shield is disposed on a second side of the plurality of capillaries opposite the first side. The first portion of the heat shield and the second portion of the heat shield can be (individually and / or collectively) configured to block bulk air flow through / across the plurality of capillaries.

[0121] In some implementations, the apparatus further includes a comb keeper coupled to the heat shield. The comb keeper is configured to contact each capillary from the plurality of capillaries to align each capillary within the heat shield and / or prevent the plurality of capillaries from directly contacting the heat shield.

[0122] In some implementations, the comb keeper is configured to contact each capillary from the plurality of capillaries at a location that is closer to the first end of that capillary than the second end of that capillary.

[0123] In some implementations, a first portion of each capillary from the plurality of capillaries is coupled to the capillary cartridge, the apparatus further comprising a comb keeper coupled to the heat shield. The comb keeper configured to contact a second portion of each capillary from the plurality of capillaries to align each capillary from the plurality of capillaries within the heat shield.

[0124] In some implementations, the location of the second portion of each capillary is different than a nominal position after contacting the comb keeper.

[0125] In some implementations, the heat shield has a geometric form of a hollow prism that surrounds the plurality of capillaries.

[0126] In some implementations, the at least one trough includes a plurality of partitions and the first end of each capillary from the plurality of capillaries is configured to be inserted into a different partition from the plurality of partitions during the immobilization process.

[0127] In some implementations, the at least one trough includes at least two partitions.

[0128] In some implementations, the immobilization process includes resolving one or more analytes in at least a subset of the plurality of capillaries. The immobilization process furtherincludes binding at least one of the one or more analytes to an inner wall of each capillary from the subset of the plurality of capillaries upon activation of a triggerable agent within that capillary such that the at least one of the one or more analytes is immobilized to a wall of that capillary, the triggerable agent including a reactive group. The immobilization process further includes detecting the at least one of the one or more analytes that are immobilized in the subset of the plurality of capillaries.

[0129] In some implementations, each capillary from the plurality of capillaries has a substantially similar temperature during the immobilization process.

[0130] In some implementations, a method includes separating, in at least one capillary from an array of capillaries, at least one analyte from a plurality of analytes contained within a sample. The sample is disposed within the at least one capillary. The method further includes activating a UV light source. Each capillary from the array of capillaries includes a photo-activated moiety configured to bind analytes from the array of capillaries to a wall of that capillary when that capillary is irradiated by the UV light source. A heat shield can be configured to control heat transfer away from the array of capillaries. For example, in some such embodiments, the array of capillaries may be disposed between the UV light source and at least a portion of the heat shield. In addition or alternatively, at least a portion of the heat shield can be disposed between the array of capillaries and the UV light source. In this way, the heat shield can reduce a temperature variation between capillaries from the plurality of capillaries when the UV light source is active. The method further includes immunoprobing the array of capillaries after activating the UV light source such that a labeled antibody binds to the at least one analyte.

[0131] In some implementations, the method includes, after separating the at least one analyte, rotating the heat shield to a position adjacent to the at least one capillary. In some implementations, the method includes, after separating the at least one analyte, rotating the heat shield to a position parallel to the at least one capillary.

[0132] Combinations of the foregoing concepts and additional concepts discussed here (provided such concepts are not mutually inconsistent) are contemplated as being part of the subject matter disclosed herein. The terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0133] The skilled artisan will understand that the drawings primarily are for illustrative purposes and are not intended to limit the scope of the subject matter described herein. The drawings are not necessarily to scale; in some instances, various aspects of the subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference characters generally refer to like features (e.g., functionally similar and / or structurally similar elements).

[0134] To address various issues and advance the art, the entirety of this application (including the Cover Page, Title, Headings, Background, Summary, Brief Description of the Drawings, Detailed Description, Embodiments, Abstract, Figures, Appendices, and otherwise) shows, by way of illustration, various embodiments in which the embodiments may be practiced. As such, all examples and / or embodiments are deemed to be non-limiting throughout this disclosure.

[0135] It is to be understood that the logical and / or topological structure of any combination of any program components (a component collection), other components and / or any present feature sets as described in the Figures and / or throughout are not limited to a fixed operating order and / or arrangement, but rather, any disclosed order is an example and all equivalents, regardless of order, are contemplated by the disclosure.

[0136] Various concepts may be embodied as one or more methods, of which at least one example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. Put differently, it is to be understood that such features may not necessarily be limited to a particular order of execution, but rather, any number of threads, processes, services, servers, and / or the like that may execute serially, asynchronously, concurrently, in parallel, simultaneously, synchronously, and / or the like in a manner consistent with the disclosure. As such, some of these features may be mutually contradictory, in that they cannot be simultaneously present in a single embodiment. Similarly, some features are applicable to one aspect of the innovations, and inapplicable to others.

[0137] Embodiments, unless clearly indicated to the contrary, should be understood to mean“at least one.”

[0138] The phrase “and / or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0139] As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.

[0140] As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, inone embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0141] Some embodiments described 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 Compact Disc / Digital Video Discs (CD / DVDs), Compact Disc-Read Only Memories (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 Application-Specific Integrated Circuits (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.

[0142] 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 processor, a field programmable gate array (FPGA), and / or an application specific integrated circuit (ASIC). Software modules (executed on hardware) can include instructions stored in a memory that is operably coupled to a processor and can be expressed in a variety of software languages (e.g., computer code), including C, C++, Java™, Ruby, Visual Basic™, Python™, 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 aweb 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.

[0143] The terms “instructions” and “code” should be interpreted broadly to include any type of computer-readable statement(s). For example, the terms “instructions” and “code” may refer to one or more programs, routines, sub-routines, functions, procedures, etc. “Instructions” and “code” may include a single computer-readable statement or many computer-readable statements.

[0144] While specific embodiments of the present disclosure have been outlined above, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the embodiments set forth herein are intended to be illustrative, not limiting.

Claims

CLAIMS1. An apparatus, comprising: a capillary cartridge including a plurality of capillaries; an ultraviolet (UV) light source configured to irradiate the plurality of capillaries during an immobilization process; a heat shield, at least a portion of the heat shield disposed parallel to the plurality of capillaries and configured to control transfer of heat away from the plurality of capillaries to reduce a temperature variation between capillaries from the plurality of capillaries during the immobilization process; and at least one trough holding a solution, an end of each capillary from the plurality of capillaries configured to be submerged into the solution during the immobilization process.

2. The apparatus of claim 1, wherein the heat shield is UV-transparent.

3. The apparatus of claim 1, wherein the heat shield is configured to impede convective heat transfer away from the plurality of capillaries while permitting radiative heat transfer to and / or from the plurality of capillaries.

4. The apparatus of claim 1, wherein the heat shield is configured to reduce the temperature variation between capillaries from the plurality of capillaries, at least in part, through convective and / or conductive heat transfer between capillaries from the plurality of capillaries while impeding convective heat transfer away from the plurality of capillaries.

5. The apparatus of claim 1, wherein: the plurality of capillaries is arranged in a linear array with a first capillary from the plurality of capillaries defining a first end of the linear array and a last capillary from the plurality of capillaries defining a second end of the linear array; and the heat shield includes (1) a first side heater disposed adjacent to the first capillary and configured to heat the first capillary during the immobilization process and (2) a second sideheater disposed adjacent to the last capillary and configured to heat the last capillary from the plurality of capillaries during the immobilization process.

6. The apparatus of claim 1, wherein the heat shield includes a plurality of ribs isolating each capillary from the plurality of capillaries into an individual channel that impedes convective heat transfer between each capillary from the plurality of capillaries.

7. The apparatus of claim 6, wherein the plurality of capillaries is arranged in a linear array and at least one rib from the plurality of ribs is located between each adjacent pair of capillaries from the plurality of capillaries.

8. The apparatus of claim 1, wherein the portion of the heat shield is configured to impede bulk air flow across the plurality of capillaries.

9. The apparatus of claim 1, wherein: the portion of the heat shield is a first portion of the heat shield disposed on a first side of the plurality of capillaries; and a second portion of the heat shield is disposed on a second side of the plurality of capillaries opposite the first side, the first portion of the heat shield and the second portion of the heat shield configured to impede bulk air flow across the plurality of capillaries.

10. The apparatus of claim 1, wherein: the portion of the heat shield is a first portion of the heat shield disposed on a first side of the plurality of capillaries between the plurality of capillaries and the UV light source; the first portion of the heat shield is UV-transparent and configured to allow UV light from the UV light source to reach the plurality of capillaries through the first portion of the heat shield; and a second portion of the heat shield is disposed on a second side of the plurality of capillaries opposite the first side, the first portion of the heat shield and the second portion of the heat shield configured to impede bulk air flow across the plurality of capillaries.

11. The apparatus of claim 1, wherein the heat shield has a geometric form of a hollow prism that surrounds the plurality of capillaries.

12. The apparatus of claim 1, wherein the at least one trough includes a plurality of partitions and the end of each capillary from the plurality of capillaries is configured to be inserted into a different partition from the plurality of partitions during the immobilization process.

13. The apparatus of claim 1, wherein the at least one trough includes at least two partitions.

14. The apparatus of claim 1, wherein: the plurality of capillaries includes a first capillary, a second capillary disposed adjacent to the first capillary, a third capillary, a fourth capillary disposed adjacent to the third capillary, and a subset of capillaries disposed between the second capillary and the third capillary; the at least one trough is partitioned such that the first capillary and the second capillary are configured to be inserted into a first partition, the third capillary and the fourth capillary are configured to be inserted into a second partition, and the subset of capillaries are configured to be inserted into a third partition.

15. The apparatus of claim 1, wherein the immobilization process includes: resolving one or more analytes in at least a subset of the plurality of capillaries; binding at least one of the one or more analytes to an inner wall of each capillary from the subset of the plurality of capillaries upon activation of a triggerable agent within that capillary such that the at least one of the one or more analytes is immobilized to a wall of that capillary, the triggerable agent including a reactive group; and detecting the at least one of the one or more analytes that are immobilized in the subset of the plurality of capillaries.

16. The apparatus of claim 1, wherein each capillary from the plurality of capillaries has a substantially similar temperature during the immobilization process.

17. The apparatus of claim 1, wherein the end of each capillary from the plurality of capillaries is a first end and each capillary from the plurality of capillaries is vertically oriented such that a second end of that capillary is disposed directly above the first end of that capillary.

18. The apparatus of claim 17, further comprising: a comb keeper coupled to the heat shield, the comb keeper configured to contact each capillary from the plurality of capillaries to (1) vertically orient the plurality of capillaries and (2) prevent the plurality of capillaries from directly contacting the heat shield.

19. The apparatus of claim 1, wherein a first portion of each capillary from the plurality of capillaries is coupled to the capillary cartridge, the apparatus further comprising; a comb keeper coupled to the heat shield, the comb keeper configured to contact a second portion of each capillary from the plurality of capillaries to align each capillary from the plurality of capillaries within the heat shield.

20. The apparatus of claim 19, wherein a location of the second portion of each capillary is different than a nominal position after contacting the comb keeper.

21. A method, comprising: separating, in at least one capillary from an array of capillaries, at least one analyte from a plurality of analytes contained within a sample, the sample disposed within the at least one capillary; activating a UV light source, each capillary from the array of capillaries including a photo-activated moiety configured to bind analytes from the array of capillaries to a wall of that capillary when that capillary is irradiated by the UV light source, at least a portion of a heat shield disposed between the array of capillaries and the UV light source, the heat shield configured to control transfer of heat away from the array of capillaries to reduce a temperature variation between capillaries from the array of capillaries when the UV light source is active; andimmunoprobing the array of capillaries after activating the UV light source such that a detection label binds to the at least one analyte.

22. The method of claim 21, further comprising, after separating the at least one analyte, rotating the heat shield to the at least one capillary.

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