Capillary preparation for protein electrophoresis

Abstract

The capillary preparation solution is formulated to coat the inner surface of capillaries used in capillary electrophoresis (CE). [Solution] The solution may be used to prepare the capillary before the CE run, and may also be applied to the capillary between one or more different CE runs to maintain the effectiveness of the preparation.

Description

Technical Field

[0001] The present invention generally relates to capillary electrophoresis (CE) of samples containing biomolecules such as proteins. In particular, the present invention relates to dynamic conditioning of capillaries for use in CE.

Background Art

[0002] Capillary electrophoresis (CE) refers to a group of analytical techniques useful for separating compounds ranging from small molecules to large biomolecules such as proteins, peptides and nucleic acids. Depending on the type of CE analysis performed, CE can separate compounds based on their charge and / or size. CE is often characterized by fast analysis speed, minimal sample consumption, high resolution, high sensitivity, high throughput, and compatibility and orthogonality with other analytical techniques such as liquid chromatography (LC) and mass spectrometry (MS).

[0003] Sample analysis by CE may be enhanced by operating a plurality of capillaries (or their capillary windows) in parallel, with each capillary containing an individual sample. In this case, the detector may be configured to measure analytes in the plurality of capillaries simultaneously. Moreover, many of the components of CE systems can be implemented on a microfluidic scale. That is, one or more dimensions of such components are on the order of micrometers, or additionally other dimensions are on the order of millimeters. Hence, many of these components can be implemented in / on one or more microfluidic chips, or coupled to one or more microfluidic chips. In this case, the tubular capillaries may be formed (or replaced thereby) as closed channels formed between layers of the microfluidic chip.

[0004] In electrophoretic electrophoresis (CE), the capillary (or channel) is filled with a buffer solution (often called the "running buffer," i.e., background electrolyte (BGE)) formulated to supply ions for electrolyte activation and maintain a substantially constant pH within a range considered optimal for a particular CE run. The capillary may also contain an electrophoretic separation medium, which is often a polymer gel that functions as a molecular sieve within the capillary. The polymer gel may be introduced into the capillary together with the buffer solution or separately. The polymer "gel" may or may not be crosslinked. The sample solution is then introduced into one end of the capillary as a plug (by hydrodynamic or electrokinetic means). The end of the capillary is in fluid communication with the respective tank containing the buffer solution. Each tank has electrodes that are electrically connected to a circuit containing a high-voltage (HV) power supply (with one of the electrodes often grounded). Next, the HV power supply is operated to apply a voltage between the electrodes and therefore along the length of the capillary. The applied voltage moves the positively charged sample through the polymer gel to the negative electrode (cathode). Samples with different charges and / or sizes move through the polymer gel at different speeds, thereby separating them from one another. Generally, high pressure is desirable for electrophoretic separation, but is ultimately limited by Joule heating (i.e., ohmic heating, i.e., heating by electrical resistance). Joule heating induces a temperature gradient in the buffer solution, which leads to a loss of resolution.

[0005] The applied voltage also induces electroosmotic flow (EOF) of the buffer solution in the capillary, which is usually in the direction of the cathode. EOF is often strong enough to move negatively charged samples toward the cathode rather than the anode, but at a slower rate than positively charged samples due to the opposite charge of the negatively charged sample. In other words, the anode-oriented attraction of a negatively charged sample counteracts the EOF-oriented direction toward the cathode. A suitable detector (e.g., configured to measure UV or UV-Vis absorbance or fluorescence) is typically positioned near the cathode and configured to perform on-capillary measurements of the sample flowing toward the cathode. Therefore, assuming that the EOF (End of Focus) is in the direction of the cathode, the smallest sample with the maximum positive charge arrives at the detector first, followed by larger samples with lower positive charges, then neutral samples (which move independently due to the EOF), then the largest sample with the minimum positive charge, and finally smaller samples with higher negative charges. An electrophoretic map is constructed using the detector's output signal, where the separated samples appear as peaks (signal intensity as a function of time).

[0006] In particular, EOF occurs in molten silica capillaries (or channels). A silanol (S-OH) group is attached to the inner wall surface of the molten silica capillary. In the presence of a normal buffer solution (e.g., pH > 2 or 3), the silanol group is converted into a negatively charged silanoate (SO₄). - The cations are ionized to the silanoate group. As a result, the inner surface of the capillary becomes negatively charged, attracting the positively charged cations of the buffer solution, thereby forming a positively charged electrical double layer on the inner surface. The electrical double layer consists of a "fixed" layer firmly bonded to the silanoate group and a "movable" layer that is attracted towards the negatively charged cathode when a voltage is applied. The bulky buffer solution moves along with the movable layer, thereby becoming the end of flow (EOF).

[0007] While natural proteins can be subjected to cerebrospinal chromatography (CE), it is often desirable to denature the proteins before performing CE experiments. A common denaturing agent is the anionic surfactant sodium dodecyl sulfate (SDS). When added to a protein-containing sample solution, SDS forms a complex with the protein, with a typical weight ratio of 1.4 g SDS per gram of protein. The resulting complex is a denatured protein (linear polypeptide) covered with a uniform negative charge. In this way, different proteins can be separated by CE based solely on their different molecular sizes (and not on charge or shape / conformation). SDS can also be added to the running buffer solution. The denaturing effect may be enhanced by adding other reagents effective in reducing covalent disulfide bonds (e.g., thiols such as beta-mercaptoethanol (β-ME), dithiothreitol (DTT), or dithioerythritol (DTE)).

[0008] The ability of SDS protein CE to effectively separate proteins depends on many different factors. Failure to properly consider these factors can severely impair CE performance and limit the capillary's lifespan. Factors to control include undesirably high EOF, adsorption or electrostatic attraction of positively charged proteins or peptides to the negatively charged inner surface of the capillary, efficient separation time, reproducibility of specific CE runs / experiments, dropout (missing peaks in the electrophoresis), material costs (including chemicals and reagents), and run-to-run stability. Adsorption of samples to the inner capillary surface can cause peak tailing, band broadening, and therefore loss of resolution. Some of these factors have been addressed by applying static or dynamic coatings to the inner surface of the capillary. Such coatings may consist of polymers, surfactants, or small molecules. Such coatings may be effective in suppressing, removing, or even altering the direction of EOF, and / or in suppressing or preventing interactions (e.g., adsorption or binding) between the sample and the inner surface of the capillary. However, the main focus regarding enhancing capillary performance has been solely on capillary preparation, with little to no consideration given to maintenance. While high performance can be achieved through capillary preparation alone, it will begin to decline if the capillary wall is damaged or becomes damaged. On the other hand, maintenance in the form of cleaning and / or removing adsorbed sample will help maintain the capillary wall in good working condition. Furthermore, both static and dynamic coatings tend to degrade over time. In this regard, dynamic coatings may be considered preferable to static coatings because they can be replenished when they begin to degrade and performance starts to decline.

[0009] One known method for reducing the EOF in a capillary requires increasing the salt concentration in the electrophoresis buffer. While reducing the EOF can enhance performance, the increased salt concentration also increases Joule heating, which degrades performance. The increase in Joule heating may be mitigated by reducing the applied voltage used for CE, but lower voltages also degrade performance and increase the travel time of the sample through the capillary. Alternatively, the increase in Joule heating may be mitigated by providing means for cooling the capillary, but this increases the cost and complexity of the CE system. Another known method involves using dynamic coating and sieving polymers in a single conjugated matrix. However, such methods can add significant production or manufacturing costs, which increase further when high-throughput CE analysis is required or when parallel (multi-capillary) CE is being performed. Furthermore, the formulation of the conjugated matrix may be limited to only specific types of sieve polymers suitable for this method, thereby potentially restricting the number of possible CE applications that can be used. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent Application Publication 2017 / 0335178, Specification A1 [Non-patent literature]

[0011] [Non-Patent Document 1] The state of the art of dynamic coatings, Electrophoresis, Vol. 22, Issue 4, p.603-611(2001)(Righetti et al.) [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] There is a continuous need to provide solutions for preparing capillaries used in CE, such as SDS-protein CE. [Means for solving the problem]

[0013] To address all or part of the above-mentioned problems, and / or other problems that may have been observed by those skilled in the art, the Disclosure provides methods, processes, systems, equipment, tools and / or apparatus, as illustrated by the examples in the embodiments described below.

[0014] According to one embodiment, a water-soluble capillary preparation solution for coating the inner surface of capillaries used in capillary electrophoresis (CE) comprises water, cationic starch, a compound effective in reducing nonspecific binding of proteins and inhibiting protein aggregation, a polyglycol-based surfactant, and a biocide.

[0015] According to one embodiment, a water-soluble capillary preparation solution for coating the inner surface of capillaries used in capillary electrophoresis (CE) essentially consists of water, cationic starch, a compound effective in reducing nonspecific binding of proteins and inhibiting protein aggregation, a polyglycol-based surfactant, and a biocide.

[0016] In one embodiment, the concentration of cationic starch in the capillary preparation solution is in one of the following ranges: 0.01-20% (w / w), 0.01-10% (w / w), 0.01-1% (w / w), 0.01-1% (w / w), and 0.1-0.5% (w / w).

[0017] In one embodiment, the concentration of the compound effective in reducing nonspecific binding of proteins in the capillary preparation solution and in suppressing protein aggregation is in one of the ranges of 1-30 mM, 1-20 mM, and 1-10 mM.

[0018] In one embodiment, the concentration of the polyglycol-based surfactant in the capillary conditioning solution is in one of the ranges of 0.05 to 1% (w / w) and 0.1 to 0.5% (w / w).

[0019] In one embodiment, the concentration of the biocide in the capillary conditioning solution is in one of the ranges of 1 to 10 M and 1 to 5 M.

[0020] In one embodiment, when the capillary conditioning solution is formed as the coating on the inner surface of the capillary, the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and effective for inhibiting protein aggregation, the polyglycol-based surfactant, and the biocide make the capillary conditioning solution effective for reducing the electroosmotic flow of fluid through the capillary during a CE run performed on a sample analyte in the capillary.

[0021] In one embodiment, when the capillary conditioning solution is formed as the coating on the inner surface of the capillary, the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and effective for inhibiting protein aggregation, the polyglycol-based surfactant, and the biocide make the capillary conditioning solution effective for preventing adsorption of sample analytes to the inner surface of the capillary during a CE run performed on the sample analytes in the capillary.

[0022] In one embodiment, the cationic starch comprises a quaternary ammonium cation.

[0023] In one embodiment, the compound effective for reducing non-specific binding of proteins and effective for inhibiting protein aggregation comprises arginine.

[0024] In one embodiment, the biocide comprises urea.

[0025] In one embodiment, the capillary preparation solution is applied as a dynamic coating to the inner surface of the capillary.

[0026] According to another embodiment, the capillary used for capillary electrophoresis (CE) includes an inner surface that partitions the lumen of the capillary and a coating that lines the inner surface, the coating being formed of a capillary preparation solution according to any of the embodiments disclosed herein.

[0027] In one embodiment, the inner surface of the capillary is negatively charged.

[0028] In one embodiment, the inner surface of the capillary has a composition selected from the group consisting of fused silica, polymer, polyetheretherketone (PEEK), linear polyacrylamide (LPA), polyvinyl acetate (PVA), polyimide (PI), and polytetrafluoroethylene (PTFE).

[0029] According to another embodiment, a method for preparing a capillary preparation solution formulated according to any of the embodiments disclosed herein includes the step of mixing the cationic starch, the compound effective in reducing nonspecific binding of proteins and effective in inhibiting protein aggregation, the polyglycol-based surfactant, and the biocide in water.

[0030] In one embodiment, the present invention includes the step of boiling cationic starch in water before mixing cationic starch, a compound effective in reducing nonspecific binding of proteins and inhibiting protein aggregation, a polyglycol-based surfactant, and a biocide in water.

[0031] In one embodiment, the present invention includes the step of mixing cationic starch, a compound effective in reducing nonspecific binding of proteins and inhibiting protein aggregation, a polyglycol-based surfactant, and a biocide in water, and then filtering the capillary preparation solution to remove fine particles therefrom.

[0032] According to another embodiment, a method for preparing a capillary for use in capillary electrophoresis (CE) includes the step of coating the inner surface of the capillary by contacting it with a capillary preparation solution according to any of the embodiments disclosed herein.

[0033] According to another embodiment, a method for separating a sample of a specimen by capillary electrophoresis (CE) includes the steps of: coating the inner surface of a capillary with a capillary preparation solution according to any embodiment disclosed herein; filling the capillary with a buffer solution; and performing a CE run on the specimen by injecting the specimen into the capillary and applying a voltage across the capillary according to operating parameters effective for separating the specimen by CE.

[0034] In one embodiment, the method includes a step of flushing the capillary with a flushing agent either before coating the inner surface of the capillary with a capillary preparation solution, and / or after performing a CE run. The duration of the flushing step may be selected or preset as desired or as needed. The flushing step may be repeated after a preset number of CE runs (i.e., capillary use or sample injection) have been completed (for example, after every nth CE run).

[0035] In one embodiment, the flushing agent is one of the following: sodium hydroxide (NaOH), potassium hydroxide (KOH), hydrogen chloride (HCl), or a combination of two or more of these.

[0036] In one embodiment, the method includes a step of repeating the coating step with an additional amount of capillary preparation solution after performing a CE run, or after repeating the CE run one or more times for one or more other samples.

[0037] In one embodiment, the sample being separated in the capillary by CE is a protein.

[0038] In one embodiment, the sample separated in the capillary by CE is a denatured protein. Therefore, the method may include a step of denaturing the protein by contacting it with a denaturant, such as sodium dodecyl sulfate (SDS), before injecting the sample into the capillary. An additional amount of denaturant may also be included with the buffer solution.

[0039] In one embodiment, the buffer solution has a pH in the range of 3 to 9 or 3 to 7. Depending on the type of starch used in the capillary preparation solution, it may be necessary to avoid an alkaline environment to prevent starch precipitation.

[0040] In one embodiment, the buffer may be based on or contain borates, EDTA (ethylenediaminetetraacetic acid), zwitterionic compounds such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris buffer, MOPS buffer, Bis-Tris buffer, ACES buffer, Bisine buffer, CAPS buffer, CAPSO buffer, CHES buffer, or Trisine buffer, or it may contain one, two or more of these.

[0041] In one embodiment, the present invention includes the step of introducing an electrophoretic separation medium into a capillary before separating the sample by CE. The electrophoretic separation medium has a composition effective as a molecular sieve for the sample of the sample subsequently introduced into the capillary. The electrophoretic separation medium may be introduced separately from the buffer solution or together with the buffer solution.

[0042] In one embodiment, the electrophoretic separation medium may contain one or more of the following: polyacrylamide (PAM), linear polyacrylamide (LPA), agarose, polydimethylacrylamide, polyvinylpyrrolidone (PVP), dextran, pullulan, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyethylene oxide (PEO), and one or more derivatives thereof.

[0043] In one embodiment, the step of performing a CE run includes the step of performing optical measurements on one or more specimens separated in a capillary.

[0044] Other apparatuses, devices, systems, methods, features, and advantages of the present invention will be apparent to those skilled in the art in consideration of the following drawings and detailed description. All such additional systems, methods, features, and advantages are included in this description, are within the scope of the present invention, and are intended to be protected by the appended claims.

[0045] The present invention can be better understood by referring to the following drawings. The components in the drawings are not necessarily isometric, but rather are emphasized in order to illustrate the principle of the invention. In the drawings, similar reference numerals indicate the corresponding parts in different figures. [Brief explanation of the drawing]

[0046] [Figure 1] This is a schematic diagram of an example of a capillary electrophoresis (CE) system capable of performing the subject matter disclosed herein. [Figure 2] This is an electrophoretic image obtained as a result of performing a CE run on a sample placed in a capillary whose inner wall has not been prepared with the type of coating disclosed herein (i.e., an uncoated capillary). [Figure 3]This is an electrophoretic image obtained as a result of performing a CE run on a sample placed in a capillary whose inner wall is coated with a test solution different from the capillary preparation solution disclosed herein. [Figure 4A] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4B] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4C] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4D] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4E] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4F] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4G] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4H] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4I] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4J] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4K] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 4L]These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5A] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5B] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5C] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5D] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5E] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5F] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5G] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5H] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5I] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5J] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5K] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 5L] These are electrophoretic images obtained as a result of performing CE runs on samples containing proteins as the target specimens. [Figure 6] This is an electrophoretic map obtained as a result of performing a CE run on a sample placed in a capillary tube. [Figure 7] This is an electrophoretic map obtained as a result of performing a CE run on a sample placed in a capillary tube.

[0047] Unless otherwise specified, all illustrations in the drawings are considered schematic. [Modes for carrying out the invention]

[0048] All “Aspects,” “Examples,” “Embodiments,” and “Embodiments” described in this disclosure are deemed non-limiting and non-exclusive. Therefore, the fact that certain “Aspects,” “Examples,” “Embodiments,” or “Embodiments” are expressly described herein does not exclude other “Aspects,” “Examples,” “Embodiments,” and “Embodiments,” whether expressly described or not, from the scope of this disclosure. In this disclosure, the terms “Aspects,” “Examples,” “Embodiments,” and “Embodiments” are deemed to be interchangeable, i.e., to have interchangeable meanings.

[0049] In this disclosure, the terms “substantially,” “approximately,” or “about” may be interpreted as encompassing a range of values ​​that include + / - 10% of a particular number when modifying such number.

[0050] In this disclosure, the term “protein” refers to a biomolecule comprising at least one long polypeptide (for example, longer than 20 amino acid residues).

[0051] In this disclosure, the term “starch” refers to a polymerized carbohydrate (polysaccharide) consisting of several glucose units linked by glycosidic bonds.

[0052] In this disclosure, the terms “cationic starch” or “cationic starch derivative” refer to naturally occurring starches and modified starches (e.g., cationic, anionic, or amphoteric starches). Depending on the type of starch, it may be necessary to derivatize the starch in order to exhibit positively charged functional groups. Such functional groups may include, for example, quaternary ammonium cations. The starch may be derivatized according to any currently known or hereafter developed techniques. The initially obtained starch may be neutral, anionic, or amphoteric and may subsequently be made cationic by any appropriate treatment of any currently known or hereafter developed techniques. The starch may initially be provided in powder form, or alternatively as an aqueous phase (e.g., solution, slurry, suspension, paste, etc.). An example of additional starches is described in Patent Document 1 above, the entire contents of which are incorporated herein by reference.

[0053] In this disclosure, the term "arginine" refers to the amino acid having the chemical formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H.

[0054] In this disclosure, the term “polyglycol-based surfactant” generally refers to polyglycol-based compounds (or mixtures thereof) having surfactant properties, which may be nonionic and / or unmodified. Examples of polyglycol-based surfactants include, but are not limited to, TERGITOL surfactant, which is commercially available from The Dow Chemical Company (Midland, Michigan, USA). Examples of TERGITOL surfactants include, but are not limited to, TERGITOL TMN-10 surfactant (secondary alcohol ethoxylate), TERGITOL TMN-6 surfactant (secondary alcohol ethoxylate), TERGITOL TMN-100X surfactant (secondary alcohol ethoxylate), TERGITOL 15-S-9 surfactant (secondary alcohol ethoxylate), TERGITOL 15-S-7 surfactant (secondary alcohol ethoxylate), TERGITOL NP-40 (nonylphenol ethoxylate), TERGITOL NP-10 (nonylphenol ethoxylate), and TERGITOL NP-9 (nonylphenol ethoxylate).

[0055] In this disclosure, the term "urea" refers to an amide having the chemical formula CO(NH2)2.

[0056] In this disclosure, the term “static coating” refers to a coating covalently bonded to the inner surface of the capillary.

[0057] In this disclosure, the term “dynamic coating” refers to a coating that is adsorbed to the inner surface of a capillary without covalent bonding.

[0058] In this disclosure, the term “semi-permanent coating” refers to a coating that is intermediate between a static coating and / or a dynamic coating, or that possesses one or more properties or attributes shared by a static coating and / or a dynamic coating. Depending on the CE application, a semi-permanent coating may be replaced with a static coating or a dynamic coating.

[0059] Another type of coating is sometimes called a "skorpio" coating. This type of coating initially acts as a dynamic coating by bonding to the inner surface of the capillary through transient hydrogen and ionic bonds, and subsequently becomes covalently bonded to the inner surface in a manner similar to a permanent coating. See Non-Patent Document 1 above (its entire contents are incorporated herein by reference).

[0060] Figure 1 is a schematic diagram of a capillary electrophoresis (CE) system 100 as one general example of a CE system (or equipment, apparatus, etc.) capable of carrying out the subject matter currently disclosed. A capillary 104, or a plurality of parallel-arranged capillaries 104 (e.g., 4, 16, 96, 384, etc.), is mounted on the CE system 100.

[0061] Each capillary 104 has a hollow, cylindrical (tubular) capillary body. Typically, the cross-section of the capillary body is circular and annular, and as a result, the capillary body has a radial thickness between the inner surface and the outer surface of the capillary. The inner surface of the capillary defines (coaxially surrounds as the boundary of the inner pore of the capillary) the inner pore (or cavity) (also referred to herein as the capillary pore), and therefore also defines the inner diameter (ID) (capillary ID). If the capillary 104 is bare (e.g., uncoated) or in a bare area of ​​the capillary 104, the outer surface of the capillary defines the outer diameter (OD) (capillary OD). For example, the capillary ID may be in the range of 0.5 μm to 200 μm, and the capillary OD may be in the range of 50 μm to 400 μm. Each capillary 104 may have a significant capillary length relative to its outer diameter (OD). For example, the capillary length may be on the order of centimeters (cm), such as in the range of 20 cm to 80 cm.

[0062] Examples of materials for the capillary body include, but are not limited to, silica, fused silica, fused quartz, doped (synthetic) fused silica, and polymers such as polytetrafluoroethylene (PTFE). The capillary 104 may also include an external protective layer (or coating, film, etc.) that contacts and coaxially surrounds the outer surface of the capillary body. The protective layer is configured to protect the capillary 104 from damage or breakage and to impart strength or robustness to the capillary 104, allowing it to be bent to a certain extent without damage or breakage. The protective layer may consist of a variety of materials suitable for this purpose, such as various polymers (examples include, but are not limited to, polyimide (PI), acrylates, silicon, and fluoropolymers). In addition, or alternatively, the capillary 104 may include an inner layer that lines the inner surface of the capillary body. Examples of inner layers include, but are not limited to, various polymers such as polyetheretherketone (PEEK), linear polyacrylamide (LPA), polyvinyl acetate (PVA), polyimide (PI), and polytetrafluoroethylene (PTFE).

[0063] In the context of this disclosure, the “inner surface” of the capillary 104 refers to the surface that is directly exposed to the capillary pore (or that directly surrounds the capillary pore and defines the boundary of the capillary pore). Therefore, depending on whether the capillary 104 has an inner layer as described above or not, the “inner surface” of the capillary 104 may be the inner surface of the capillary body itself (e.g., fused silica) or the inner surface of the inner layer lining the inner surface of the capillary body.

[0064] In one aspect of this disclosure, the inner surface of each capillary 104 is coated with a capillary preparation solution prepared and formulated as described herein. Depending on whether the capillary 104 has an inner layer as described above, the inner surface on which the coating formed with the capillary preparation solution is located may be the inner surface of the capillary body itself, or it may be the inner surface of an inner layer lining the inner surface of the capillary body.

[0065] Each capillary 104 may also contain an electrophoretic separation medium, such as a appropriately formulated polymer gel, which acts as a molecular sieve in the CE, as will be understood by those skilled in the art.

[0066] Before introducing the sample into the capillary 104 (for electrophoretic separation), the inlet end of the capillary 104 is fluidly connected to the sample source 108 (for example, by immersion in the sample source 108), which may be one or more containers holding one or more samples in solution, or contain such containers. For example, as described, in the case of multiple capillaries 104, the sample source 108 may be a multi-well plate having different wells holding each sample (sample-containing solution), or contain such a plate. The outlet end of the capillary 104 is fluidly connected to the buffer tank 112, which is filled with buffer solution (electrophoretic buffer used in CE) (for example, by immersion in the buffer tank 112). The capillary 104 is also filled with electrophoretic buffer by capillary action (capillary action) or by applying a pressure difference between the ends of each capillary 104. The buffer tank 112, or both the sample source 108 and the buffer tank 112, contain electrodes. Typically, the electrodes of the sample source 108 (or multiple electrodes in each well of a multiwell plate) act as anodes 116, and the electrodes of the buffer tank 112 act as cathodes 120 and may be electrically grounded. The anodes 116 and cathodes 120 are coupled to a high-voltage (HV) source 124. The electrolytic running buffer solution completes the electrical circuit between the HV source 124 and the capillary 104 (via the anodes 116 and cathodes 120).

[0067] The CE system 100 also includes a detection device configured to detect and measure the sample after the sample has been separated by electrophoresis. Typically, the detection device is optically based and configured for on-capillary detection (detection of the sample directly present in the capillary 104). Thus, in the example described, the detection device includes a light source 128 and a photodetector (or camera) 132. Multiple capillaries 104 may be mounted on a cartridge 136, or on other types of supports configured to optically align the capillaries 108 with the light source 128 and the photodetector 132. The capillaries 104 are typically positioned relative to the detector such that the light source 128 illuminates the capillaries 104 in a detection area along their length (for example, located near a buffer tank 112 containing a cathode 120). Accordingly, the light emitted from the detection area is collected by the photodetector 132. Examples of light sources 128 include, but are not limited to, broadband light sources (e.g., flash lamps), light-emitting diodes (LEDs), laser diodes (LDs), and lasers. Multiple light sources 128 may be provided so that the user can select a desired excitation wavelength. Examples of photodetectors 132 include, but are not limited to, active pixel sensors (APS) such as cameras, photomultiplier tubes (PMTs), photodiodes (PDs), charge-coupled devices (CCDs), and complementary metal-oxide-semiconductor (CMOS) devices, which are sensitive to the emission wavelength to be detected. The capillary 104, the detection device, and any other photosensitive components are usually enclosed by an instrument console (not shown) to prevent interaction with stray light or ambient light. In general, any type of detection / measurement technique that complies with CE standards can be used, one example being fluorescence-based techniques, as will be understood by those skilled in the art.

[0068] As a general example of a method for performing a CE run, the sample to be analyzed and capillary 104 are prepared as needed. Preparation of capillary 104 may include flushing or washing the inner surface of capillary 104 with a suitable flushing agent, such as sodium hydroxide (NaOH), potassium hydroxide (KOH), or hydrogen chloride (HCl). For a given capillary 104, the flushing step may be performed for a certain (e.g., predetermined) amount of time as needed, and the flushing step may be repeated as needed after a certain (e.g., predetermined) number of CE runs. As an example, the flushing step may involve flushing capillary 104 with 1 M NaOH for 10 minutes, and such a flushing step may be repeated after every 20th CE run (i.e., before every 20th sample injection). Then, capillary 104 is attached to the CE system 100. As part of preparing capillary 104, the electrophoretic separation medium (e.g., polymer gel) may be added to capillary 104. Depending on the sample being analyzed, the electrophoretic separation medium may be added separately from the electrophoresis buffer or as part of the electrophoresis buffer. Depending on the configuration of the CE system 100, the electrophoretic separation medium may be added to capillary 104 before or after attaching capillary 104 to the CE system 100. Then, the sample solution plugs are injected from the sample source 108 into each of the provided capillaries 108. Sample injection may be performed electrokinetically, as in the example described. In this case, the HV source 124 is operated to apply a voltage pulse along the length of capillary 104, i.e., between the inlet and outlet ends of capillary 104, thereby drawing the sample plugs through each inlet end. Alternatively, sample injection may be performed by applying a pressure difference over the length of the capillary 104 in any suitable manner, thereby drawing the sample plug into the capillary 104. In the latter case, the sample source 108 does not need to have an anode 116, because the HV source 124 is not used in the sample injection step.

[0069] After the sample plug is placed into the capillary 104, the sample source 108 is replaced with a buffer tank (not shown) similar to the buffer tank 112 located at the outlet end of the capillary 104. Thus, the inlet end of the capillary 104 is fluidly connected to (e.g., immersed in) this inlet-side buffer tank. The inlet-side buffer tank contains an anode 116, similar to the one shown in Figure 1, which is associated with the sample source 108 and is electrically connected to the HV source 124. The anode 116 may have multiple electrodes, one for each capillary 104, which are arranged parallel or coaxially with the inlet end of the capillary 104. Next, to initiate electrophoretic separation, the HV source 124 is operated to apply a voltage across the capillary 104. The parameters of the applied voltage (magnitude and duration, and optionally, as pulses such as desired width, frequency, and pulse waveform) are selected so that the applied voltage is effective in performing CE on the sample. In other words, the applied voltage causes different components (samples) of the sample to move through the capillary 104 at different speeds (including through any electrophoretic separation medium provided in the capillary 104), thereby separating the different components into specific bands. These different bands are then detected and measured by a detection device (light source 128 and photodetector 132). The CE system then uses the electrical output signals from the detection device to generate electrophoretic diagrams corresponding to the measurements performed in each capillary 104 queried by the detection device, as will be understood by those skilled in the art.

[0070] In some cases, the method for performing a CE run is denaturing protein electrophoresis, such as SDS-protein electrophoresis. In this case, the target sample is protein, and the protein is denatured before performing the CE run. Therefore, the sample is prepared by contacting it with a denaturing agent such as SDS, which is usually done before injecting the sample into capillary 104. Some amount of SDS may be added to the electrophoresis buffer.

[0071] One aspect of this disclosure relates to a method for preparing each capillary 104 used in CE, particularly in SDS-protein electrophoresis. This method includes the step of coating the inner surface of the capillary 104 with the capillary preparation solution disclosed herein. The coating may involve flowing the capillary preparation solution through the lumen (pore) of each capillary 104 to wash the inner surface of the capillary 104 with the capillary preparation solution. The flow of the capillary preparation solution through the capillary 104 may be established by an active technique (e.g., creating a pressure difference between the ends of the capillary 104 to push or pull the capillary preparation solution towards the inlet end, or electrokinetically by using an HV source 124 if the capillary preparation solution is electrokinetically reactive to an electric field), a passive technique (e.g., capillary force or core material), or any other suitable technique. If necessary, the flowing step may be repeated one or more times to enhance the coating. The coating on the inner surface may be maintained (or regenerated) by repeating the coating step between consecutive CE runs. The adjustment (coating) step may be repeated after each CE run, or after every two or three runs, for example. In other words, the frequency of the coating step may vary and may depend on one or more factors such as the type of sample being analyzed, the method / test being performed, the chemicals being used, the type of capillary being used, and environmental parameters. Typically, the adjustment step is performed before the flushing step described above and before the step of adding the gel or other electrophoretic separation medium.

[0072] In one embodiment, the inner surface of the capillary 104 may be prepared to facilitate or optimize the formation of the coating on the inner surface (for example, to promote or improve the mechanism involved in creating an interface between the inner surface and a suitable coating, at least as a dynamic coating) before forming the coating (by applying the capillary preparation solution as described above). As an example, the inner surface may be prepared by exposing it to corona discharge or plasma discharge.

[0073] Another aspect of this disclosure relates to a capillary 104 including a coating that lines the inner surface of the capillary 104, wherein the coating is formed of a capillary preparation solution disclosed herein.

[0074] Another aspect of this disclosure relates to a method for separating a sample by electrophoresis using the capillary preparation solution disclosed herein. In this method, the inner surface of a capillary 104 (or a number of capillaries 104) is coated with the capillary preparation solution as described above. The capillary 104 is then filled with a buffer solution (electrophoresis buffer). The sample is then injected into the capillary 104, and an electrophoresis run is performed on the sample by applying a voltage across the capillary 104 according to operating parameters effective for separating the sample by electrophoresis.

[0075] In general, there are no restrictions on the type of electrophoresis buffer or the pH of the electrophoresis buffer used in the methods disclosed herein. The electrophoresis buffer may be selected based on various factors, for example, the capillary preparation solution and sample being used. Examples of electrophoresis buffers include, but are not limited to, those based on or containing borates, EDTA (ethylenediaminetetraacetic acid), and zwitterionic compounds such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The pH of the electrophoresis buffer may be, for example, in the range of 3 to 9, or in the range of 3 to 7, or less than 7.

[0076] In general, there are no restrictions on the type of electrophoretic separation medium used in the methods disclosed herein. The electrophoretic separation medium may be selected based on various factors, for example, the capillary preparation solution and sample being used. Examples of electrophoretic separation media include, but are not limited to, polyacrylamide (PAM), linear polyacrylamide (LPA), agarose, polydimethylacrylamide, polyvinylpyrrolidone (PVP), dextran, pullulan, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyethylene oxide (PEO), and one or more derivatives thereof.

[0077] Another aspect of this disclosure relates to the composition of the capillary preparation solution used in the CE in the method disclosed herein. In one example, the capillary preparation solution is an aqueous solution containing in water the following components: cationic starch, a compound effective in reducing nonspecific protein binding and inhibiting protein aggregation, a nonionic polyglycol-based surfactant, and a biocide to prevent microbial growth. An example of a compound effective in reducing nonspecific protein binding and inhibiting protein aggregation is arginine. Polyglycol-based surfactants such as NP-40 (Tergitol) assist in cleaning the capillary by increasing protein solubility. Other surfactants such as Tween-20 or Triton-X100 may be suitable for this purpose. An example of a biocide is urea. Urea also has an effect on protein solubility, for example, by helping to remove proteins bound to the inner capillary wall. Other examples of biocides that may be suitable for use in the prepared solutions of this disclosure include N,N'-methylenebismorpholine, biphenyl-2-ol, 3-iodo-2-propynylbutylcarbamate, chlorocresol, 2-bromo-2-(bromomethyl)pentanezinitrile, N-(trichloromethylthio)phthalimide, or ProClin, provided by Merck & Co., Inc. (Rahway, New Jersey, USA). TM It is one of the preservatives.

[0078] For example, the relative concentrations of these components in water may be in the range of 0.01–20% (w / w) or 0.01–10% (w / w) or 0.01–1% (w / w) or 0.1–0.5% (w / w) for cationic starch, in the range of 1–30 mM or 1–20 mM or 1–10 mM for arginine (or other compound effective in reducing nonspecific binding of proteins and effective in inhibiting protein aggregation), in the range of 0.05–1% (w / w) or 0.1–0.5% (w / w) for polyglycol-based surfactant (or other suitable surfactant), and in the range of 1–10 M or 1–5 M for urea (or other suitable biocide). More generally, the relative proportions of cationic starch, arginine, polyglycol-based surfactant, and urea in aqueous solution can be selected to make the capillary preparation solution effective in reducing the electroosmotic flow of the fluid through capillary 104 during the CE run performed on the sample in capillary 104. Alternatively, the relative proportions of cationic starch, arginine, polyglycol-based surfactant, and urea can be selected to make the capillary preparation solution effective in preventing the adsorption of the sample onto the inner surface of capillary 104 during the CE run performed on the sample in capillary.

[0079] The capillary preparation solution may be prepared by mixing starch, arginine, a polyglycol-based surfactant, and urea in water. Before mixing, the starch may be boiled and / or stirred in water. After mixing, the capillary preparation solution may be passed through a suitable filter to remove particulate matter.

[0080] In one specific, but not limited, example, a starch slurry was prepared by adding starch (25 g) to deionized water (150 mL) and then mixing with a stirrer for 10 seconds. The starch used was CHARGEMASTER R33F cationic starch (Grain Processing Corporation, Muscatene, Iowa, USA). The starch slurry was then mixed with 450 ml of water. oThe mixture was then added to an additional volume (2000 mL) of deionized water boiling at C. The resulting mixture was then stirred using a stirring bar (250-350 RPM) to reduce its temperature (375). o The mixture was stirred in (C) for 30 minutes, and then allowed to cool. A urea solution was prepared by adding urea (1501.5 g) to deionized water (6000 mL) and mixing with a paddle (585 ± 10 RPM) for 30 minutes. Next, a mixture of boiled starch (3 L), L-arginine (17.4 g), and TERGITOL TMN-10 surfactant (50 mL) was added to the urea solution, and the components were mixed with a paddle (585 ± 10 RPM) for 180 minutes. The resulting solution was then passed through a 1.2 μm ALPHA polypropylene microfiber filter (Meissner Filtration Products, Inc., Camarillo, California, USA).

[0081] Figures 2–7 are electrophoretic diagrams illustrating the advantages obtained from using coated capillaries as disclosed herein.

[0082] Figure 2 is an electrophoresis plot of the signal intensity (in relative fluorescence units, i.e., RFU) as a function of size (in kilodaltons, i.e., kDa) of detected / measured peaks resulting from a CE run on a sample containing protein as the target sample. The sample was placed in an uncoated capillary containing only gel separation medium (i.e., an uncoated capillary with the capillary preparation solution disclosed herein, or with any other type of coating). Figure 2 shows the assimilation of the lower marker, the dye peak, and the slowness of the migration time. The lower marker is a sample compound of known size and concentration added to the sample. This is used for the quantification of the sample compound. In contrast, the ladder is used to determine the size of the sample compound. The dye peak results from a portion of the fluorescent dye added to the sample (to enable detection via fluorescence measurement / visualization) that is not actually bound to the protein. Figure 2 also shows that the last (eighth) peak is not with the runtime of the method set during the progress of the test. All peaks must be detected exactly within this runtime. Figure 2 shows that this was not the case for the last peak. The red trace in Figure 2 is the baseline. Due to poor capillary adjustment, the baseline increased as the sample moved through the capillary.

[0083] Figure 3 is an electrophoresis plot (RFU vs. kDa) obtained as a result of performing a CE run on a sample containing the same protein analyzed in the CE run in Figure 2, using the same capillary except that the capillary was coated with a different test solution than the capillary preparation solution disclosed herein. The test solution was a polysiloxane-based hydrophobic solution. Figure 3 shows peak tailing, the absence of the final peak (expected at approximately 240 kDa), and an elevated baseline.

[0084] Figures 4A–4L show 12 electrophoretic maps (signal intensity in RFU as a function of time in MM:SS) obtained as a result of performing 12 separate CE runs in parallel capillaries with the same sample (containing protein as the target sample) in each capillary. The 12 CE runs were performed in each capillary under the same conditions. Each capillary was uncoated, i.e., not coated with the capillary preparation solution as disclosed herein. Figures 4A–4L show peak dropout and / or loss of resolution, and the loss of effectiveness of the capillaries after a few CE runs. These problems were attributed to the accumulation / adsorption of protein to the inner surface of the capillary wall.

[0085] For comparison, Figures 5A to 5L show 12 electrophoretic maps (signal intensity in RFU as a function of time in HH:MM:SS) obtained by performing 12 separate CE runs in parallel capillaries with the same sample (containing protein as the target sample) in each capillary. The 12 CE runs were performed in each capillary under the same conditions. The protein-containing sample was the same as that in the CE runs related to Figures 4A to 4L. The difference is that the electrophoretic maps in Figures 5A to 5L were generated by coating the capillaries with the capillary preparation solution disclosed herein before performing electrophoresis. Figures 5A to 5L show improved performance compared to Figures 4A to 4L due to the use of the capillary preparation solution disclosed herein.

[0086] Figure 6 shows the electrophoresis (RFU vs. kDa) obtained as a result of performing a CE run on a protein-containing sample in a capillary. Although a prepared solution was used, it was not suitable for adequately cleaning and coating the inner capillary wall. As indicated by the leftmost arrow, the dye front merged with the lower marker, resulting in erroneous quantification. As indicated by the next arrow, some of the broader peaks have secondary peaks. The contour of the peak characterized by the rightmost arrow is inaccurate. This peak has the same peak height (same RFU) as the other peaks, but this is the lowest concentrated protein in the ladder, and therefore its RFU should be lower than the other peaks.

[0087] Figure 7 shows the electrophoresis (RFU vs. kDa) obtained as a result of performing a CE run on a protein-containing sample in a capillary coated with the capillary preparation solution disclosed herein. The results show that the CE run performs well (e.g., good ladder contour).

[0088] Terms such as “communicate” and “communicate with” (for example, “the first component communicates with” and “communicate with” the second component) will be understood in this specification to be used to suggest structural, functional, mechanical, electrical, signaling, optical, magnetic, electromagnetic, ionic, or fluidic relationships between two or more components or elements. Thus, the fact that one component is said to communicate with a second component is not intended to preclude the possibility that an additional component exists between the first and second components and / or is operably associated or engaged with the first and second components.

[0089] It will be understood that various aspects and details of the present invention may be modified without departing from the scope of the invention. Furthermore, the above description is for illustrative purposes only and not for limitation, and the present invention is defined by the claims.

Claims

1. A capillary preparation solution for coating the inner surface of a capillary used in capillary electrophoresis (CE), wherein the capillary preparation solution is: water, Cationic starch, Compounds that are effective in reducing nonspecific protein binding and inhibiting protein aggregation. Polyglycol-based surfactants, and biocides A capillary preparation solution that is an aqueous solution containing the following features.

2. The concentration of the cation starch is within a range selected from the group consisting of 0.01–20% (w / w), 0.01–10% (w / w), 0.01–1% (w / w), 0.01–1% (w / w), and 0.1–0.5% (w / w). The concentration of the compound, which is effective in reducing nonspecific binding of proteins and in suppressing protein aggregation, is within a range selected from the group consisting of 1 to 30 mM, 1 to 20 mM, and 1 to 10 mM. The concentration of the polyglycol-based surfactant is within a range selected from the group consisting of 0.05 to 1% (w / w) and 0.1 to 0.5% (w / w). The capillary preparation solution according to claim 1, wherein the concentration of the biocide is in the range of 1 to 10 M and 1 to 5 M.

3. The capillary preparation solution according to claim 1, wherein the cationic starch comprises a quaternary ammonium cation.

4. The capillary preparation solution according to claim 1, wherein the compound effective in reducing nonspecific binding of proteins and effective in suppressing protein aggregation comprises arginine.

5. The capillary preparation solution according to claim 1, wherein the biocide comprises urea.

6. The capillary preparation solution according to claim 1, wherein, when the capillary preparation solution is formed as the coating on the inner surface of the capillary, the relative ratio of the cationic starch, the compound effective in reducing nonspecific binding of proteins and effective in suppressing protein aggregation, the polyglycol-based surfactant, and the biocide, the capillary preparation solution is effective in reducing the electroosmotic flow of the fluid passing through the capillary during a CE run performed on a sample in the capillary.

7. The capillary preparation solution according to claim 1, wherein, when the capillary preparation solution is formed as the coating on the inner surface of the capillary, the relative ratio of the cationic starch, the compound effective in reducing nonspecific binding of proteins and effective in suppressing protein aggregation, the polyglycol-based surfactant, and the biocide makes the capillary preparation solution effective in preventing the adsorption of the sample to the inner surface during a CE run performed on the sample in the capillary.

8. A capillary used for electrophoresis (CE), wherein the capillary is The inner surface that partitions the lumen of the capillary, A capillary comprising a coating that lines the inner surface, the coating being formed of the capillary preparation solution described in claim 1.

9. The capillary according to claim 8, wherein the inner surface is negatively charged.

10. The capillary according to claim 8, wherein the inner surface has a composition selected from the group consisting of fused silica, polymer, polyether ether ketone (PEEK), linear polyacrylamide (LPA), polyvinyl acetate (PVA), polyimide (PI), and polytetrafluoroethylene (PTFE).

11. A method for preparing the capillary preparation solution according to claim 1, the method comprising the step of mixing the cationic starch, the compound effective in reducing nonspecific binding of proteins and effective in inhibiting protein aggregation, the polyglycol-based surfactant, and the biocide in water.

12. The method according to claim 11, further comprising the step of boiling the cation starch in water before the mixing step.

13. The method according to claim 11, further comprising the step of filtering the capillary preparation solution after the mixing step to remove particulate matter therefrom.

14. A method for preparing a capillary for use in capillary electrophoresis (CE), the method comprising the step of coating the inner surface of the capillary by bringing it into contact with the capillary preparation solution described in claim 1.

15. A method for separating a sample of specimens by capillary electrophoresis (CE), wherein the method is: The steps include coating the inner surface of the capillary with the capillary preparation solution described in claim 1, The steps include filling the aforementioned capillary with a buffer solution, The CE run on the aforementioned sample is performed as follows: Injecting the sample into the capillary, A method comprising the step of applying a voltage across the capillary according to operating parameters effective for separating the sample by CE.

16. The method according to claim 15, further comprising the step of coating the inner surface of the capillary with an additional amount of the capillary preparation solution, after the step of performing the CE run, or after repeating the step of performing the CE run one or more times for one or more other samples.

17. The method according to claim 15, wherein the sample is a protein.

18. The method according to claim 17, wherein the protein is denatured by contacting it with a denaturing agent before the step of introducing it.

19. The method according to claim 15, further comprising the step of introducing an electrophoretic separation medium into the capillary before performing the CE run.

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