Capillary conditioning for protein electrophoresis

Abstract

A capillary conditioning solution is formulated for coating an inside surface of a capillary utilized for capillary electrophoresis (CE). The solution may be utilized to condition the capillary before a CE run, and may be applied to the capillary between one or more different CE runs to maintain the effectiveness of the conditioning.

Description

CAPILLARY CONDITIONING FOR PROTEIN ELECTROPHORESISTECHNICAL FIELD

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

[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 undertaken, CE can separate compounds on the basis of their electric charge and / or size. CE is often characterized by fast analysis times, 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 multiple capillaries (or capillary windows thereof) in parallel, with each capillary containing an individual sample. In this case, the detector may be configured to measure the analytes in the multiple capillaries simultaneously. Moreover, many of the components of a CE system can be realized on the scale of microfluidics, meaning that 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 embodied in / on, or be coupled to, one or more microfluidic chips. In this case, tubular capillaries may be formed as (or replaced by) closed channels formed between layers of the microfluidic chips.

[0004] In CE, a capillary (or channel) is filled with a buffer solution (often termed a “running buffer,” or background electrolyte (BGE)) formulated to provide ions for electrolytic activity and to maintain a substantially constant pH within a range deemed optimal for the particular CE run. The capillary may also contain an electrophoretic separation medium, which is often a polymer gel that functions as a molecular sieve in the capillary. The polymer gel may be loaded into the capillary with the buffer solution or separately from the buffer solution. The polymer “gel” may or may not be cross-linked. The sample solution is then loaded (by hydrodynamic or electrokinetic means) as a pluginto one end of the capillary. The ends of the capillary are placed in fluidic communication with respective reservoirs containing the buffer solution. The reservoirs have respective electrodes electrically communicating with a circuit that includes a high-voltage (HV) power supply (with one of the electrodes often grounded). The HV power supply is then operated to apply a voltage between the electrodes and thus across the length of the capillary. The applied voltage causes positively charged analytes of the sample to migrate through the polymer gel toward the negative electrode (cathode). Analytes of different charge and / or size migrate at different speeds through the polymer gel, and thereby become separated from each other. Generally, a high voltage is desirable for electrophoretic separation, but ultimately is limited by Joule heating (or ohmic heating, i.e. heating due to electrical resistance). Joule heating induces temperature gradients in the buffer solution, which leads to a loss in resolution.

[0005] The applied voltage also induces electroosmotic flow (EOF) of the buffer solution in the capillary, typically also in the direction of the cathode. The EOF is often strong enough to also cause negatively charged analytes of the sample to migrate toward the negative electrode (cathode), instead of toward the positive electrode (anode), but at slower speeds than the positively charged analytes due to the opposite charge of the negatively charged analytes. In other words, the attraction of the negatively charged analytes toward the anode runs counter to the direction of the EOF toward the cathode. An appropriate detector (e.g., configured to measure UV or UV-Vis absorbance, or fluorescence, etc.) is typically located near the cathode and configured to make on-capillary measurements of the analytes flowing toward the cathode. Thus, assuming the EOF is in the direction of the cathode, the smallest analytes of highest positive charge reach the detector first, followed by larger analytes of lower positive charge, followed by neutral analytes (moving solely due to the EOF), followed by the largest analytes of lowest negative charge, and followed by smaller analytes of higher negative charge. The output signals of the detector are utilized to construct an electropherogram, in which the separated analytes appear as peaks (signal intensity as a function of time).

[0006] In particular, EOF occurs in fused silica capillaries (or channels). Silanol (S-OH) groups are attached to the inner wall surface of a fused silica capillary. In the presence of a typical buffer solution (e.g., pH > 2 or 3), the silanol groups are ionized to negatively charged silanoate (S-O-) groups. Consequently, the inner surface of the capillary is negatively charged and attracts positively charged cations of the buffer solution, thereby forming an electrical double layer of positive chargesat the inner surface. The electrical double layer consists of a “fixed” layer bound tightly to the silanoate groups, and a “mobile” layer that is pulled in the direction of the negatively charged cathode when the voltage is applied. The bulk buffer solution migrates with the mobile layer, thus resulting in EOF.

[0007] While native proteins can be subjected to CE, it is often desirable to first denature proteins before running the CE experiment. The typical denaturing agent is the anionic surfactant sodium dodecyl sulfate (SDS). When added to the protein-containing sample solution, SDS forms a complex with the protein, the typical weight ratio being 1.4 g SDS per gram of protein. The resulting complex is an unfolded protein (linear chain polypeptide) coated with a uniform negative charge. In this way, different proteins can be separated by CE solely on the basis of their different molecular sizes (and not 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 for reducing covalent disulfide bonds, for example thiols such as beta-mercaptoethanol ( -ME), dithiothreitol (DTT), or dithioerithritol (DTE).

[0008] The ability of SDS-Protein CE to separate proteins effectively depends on many different factors. Without adequate consideration of these factors, CE performance can be severely inhibited and capillary lifetime can be limited. Factors that should be controlled include undesirably high EOF, adsorption or electrostatic attraction of positively charged sample proteins or peptides to the negatively charged inside surface of the capillary, efficient separation time, reproducibility of a particular CE run / experiment, dropouts (missing peaks in the electropherogram), cost of materials (including chemicals and reagents), stability from run to ran, etc. Adsorption of analytes to the inside capillary surface causes peak tailing, band broadening, and thus loss of resolution. Some of these factors have been addressed by applying a static coating or a dynamic coating to the inside surface of the capillary. Such coatings may consist of polymers, surfactants, or small molecules. Such coatings may be effective to suppress, eliminate or even change the direction of EOF, and / or suppress or prevent interactions between the analytes and the inside surface of the capillary (c.g., adsorption or binding). However, the primary focus for enhancing capillary performance has been on capillary conditioning alone, with little or no regard for maintenance. Capillary conditioning alone can allow for high performance, but will begin to decline if the wall of the capillary is or becomes compromised. On the other hand, maintenance in the form of cleaning and / or removing adsorbed analytes wouldassist in keeping the capillary wall in good working order. Moreover, both static and dynamic coatings tend to deteriorate over time. In this regard, a dynamic coating may be considered advantageous over a static coating in that the dynamic coating can be replenished when it starts to degrade and performance starts to decline.

[0009] One known approach to lowering EOF in the capillary calls for providing a high salt concentration in the running buffer. Lowering EOF can boost performance, but increasing salt concentration also increases Joule heating, which lowers performance. The increased Joule heating may be mitigated by lowering the applied voltage utilized for CE, but a lower voltage also lowers performance and increases the migration time of the analytes through the capillary. Alternatively, the increased Joule heating may be mitigated by providing means for cooling the capillary, which increases the cost and complexity of the CE system. Another known approach is to utilize a dynamic coating and sieving polymer in a single conjugated matrix. However, such approach can add significant production or manufacturing costs, which are increased further when a high throughput CE analysis is needed or when parallel (multi-capillary) CE is being implemented. Moreover, the formulation of the conjugated matrix may be limited to only certain types of sieving polymers compatible with this approach, thereby limiting the number of possible CE applications available.

[0010] There is an ongoing need to provide solutions for conditioning capillaries utilized in CE such as SDS-Protein CE.SUMM RY

[0011] To address the foregoing problems, in whole or in part, and / or other problems that may have been observed by persons skilled in the art, the present disclosure provides methods, processes, systems, apparatus, instruments, and / or devices, as described by way of example in implementations set forth below.

[0012] According to an implementation, an aqueous capillary conditioning solution for coating an inside surface of a capillary utilized for capillary electrophoresis (CE) includes: water; cationic starch; a compound effective for reducing non-specific binding of proteins and suppressing protein aggregation; a polyglycol-based surfactant; and a biocide.

[0013] According to an implementation, an aqueous capillary conditioning solution for coating an inside surface of a capillary utilized for capillary electrophoresis (CE) consists essentially of:water; cationic starch; a compound effective for reducing non-specific binding of proteins and suppressing protein aggregation; a polyglycol-based surfactant; and a biocide.

[0014] In an implementation, the concentration of the cationic starch in the capillary conditioning solution is in one of the following ranges: 0.01 to 20 % (w / w); 0.01 to 10 % (w / w); 0.01 to 1 % (w / w); 0.01 to 1 % (w / w); and 0.1 to 0.5 % (w / w).

[0015] In an implementation, the concentration of the compound effective for reducing nonspecific binding of proteins and suppressing protein aggregation in the capillary conditioning solution is in one of the following ranges: 1 to 30 mM; 1 to 20 mM; and 1 to 10 mM.

[0016] In an implementation, the concentration of the poly glycol-based surfactant in the capillary conditioning solution is in one of the following ranges: 0.05 to 1 % (w / w); and 0.1 to 0.5 % (w / w).

[0017] In an implementation, the concentration of the biocide in the capillary conditioning solution is in one of the following ranges: 1 to 10 M; and 1 to 5 M.

[0018] In an implementation, when the capillary conditioning solution is formed as the coating on the inside surface of the capillary', the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide render the capillary conditioning solution effective to reduce electroosmotic flow of liquid through the capillary during a CE run performed on sample analytes in the capillary.

[0019] In an implementation, when the capillary conditioning solution is formed as the coating on the inside surface of the capillary', the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide render the capillary conditioning solution effective to prevent adsorption of sample analytes to the inside surface of the capillary during a CE run performed on the sample analytes in the capillary.

[0020] In an implementation, the cationic starch comprises quaternary ammonium cations.

[0021] In an implementation, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation comprises arginine.

[0022] In an implementation, the biocide comprises urea.

[0023] In an implementation, the capillary conditioning solution is applied as a dynamic coating on the inside surface of the capillary.

[0024] According to another implementation, a capillary utilized for capillary electrophoresis (CE) includes: an inside surface defining an inner lumen of the capillary; and a coating lining the inside surface, wherein the coating is formed from a capillary conditioning solution according to any of the implementations disclosed herein.

[0025] In an implementation, the inside surface of the capillary is negatively charged.

[0026] In an implementation, the inside surface of the capillary has a composition selected from the group consisting of: fused silica; a polymer; polyether ether ketone (PEEK); linear polyacrylamide (LPA); polyvinyl acetate (PVA); poly imide (PI); and polytetrafluoroethylene (PTFE).

[0027] According to another implementation, a method for preparing a capillary conditioning solution formulated according to any of the implementations disclosed herein includes mixing the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide in water.

[0028] In an implementation, the method includes, before mixing the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide in water, boiling the cationic starch in water.

[0029] In an implementation, the method includes, after mixing the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide in water, filtering the capillary conditioning solution to remove particulates therefrom.

[0030] According to another implementation, a method for conditioning a capillary utilized for capillary electrophoresis (CE) includes contacting an inside surface of the capillary with a capillary conditioning solution according to any of the implementations disclosed herein to coat the inside surface.

[0031] According to another implementation, a method for separating analytes of a sample by capillary electrophoresis (CE) includes: coating an inside surface of a capillary with a capillary conditioning solution according to any of the implementations disclosed herein; filling the capillary with a buffer solution; and performing a CE run on the sample by: injecting the sample into thecapillary; and applying a voltage across the capillary according to operating parameters effective to separate the analytes by CE.

[0032] In an implementation, the method includes flushing the capillary with a flushing agent at one or both of the following times: before coating the inside surface of the capillary with the capillary conditioning solution; after performing of the CE run. The duration of the flushing step may be selected or predetermined as desired or needed. The flushing step may be repeated after a predetermined number of CE runs (i.e., uses of the capillary, or sample injections) have been completed (e.g., after every nth CE run).

[0033] In an implementation, the flushing agent is one of: sodium hydroxide (NaOH); potassium hydroxide (KOH); hydrogen chloride (HC1); or a combination of two or more of the foregoing.

[0034] In an implementation, the method includes, after performing the CE run, or after repeating the performing of the CE run one or more times on one or more other samples, repeating the coating step with an additional amount of the capillary conditioning solution.

[0035] In an implementation, the analytes being separated in the capillary by CE are proteins.

[0036] In an implementation, the analytes being separated in the capillary by CE are denatured proteins. Thus, the method may include, before injecting the sample into the capillary, denaturing the proteins by contacting the proteins with a denaturing agent such as, for example, sodium dodecyl sulfate (SDS). An additional amount of the denaturing agent may also be included with the buffer solution.

[0037] In an implementation, the buffer solution has a pH in a range from 3 to 9 or from 3 to 7. Depending on the type of starch utilized in the capillary conditioning solution, an alkaline environment may need to be avoided to prevent precipitation of the starch.

[0038] In an implementation, the buffer may be or include one of, or a combination of two or more of, the following: those based on or containing borate, EDTA (ethylenediaminetetraacetic acid), etc.; zwitterionic compounds such as, for example, HEPES (4-(2-hydroxyethyl)-l- pipcrazinccthancsulfonic acid); A Tris Buffer; MOPS Buffer; Bis-Tris Buffer; ACES Buffer; Bicinc Buffer; CAPS Buffer; CAPSO Buffer; CHES Buffer; Tricine Buffer.

[0039] In an implementation, the method includes, before separating the analytes by CE, loading an electrophoretic separation medium into the capillary. The electrophoretic separation medium has a composition effective as a molecular sieve for the analytes of the sample subsequently loaded intothe capillary. The electrophoretic separation medium may be loaded separately from, or along with, the buffer solution.

[0040] In an implementation, the electrophoretic separation medium is or includes one or more of the following: polyacrylamide (PAM); linear polyacrylamide (LPA); agarose; poly dimethylacrylamide; polyvinylpyrrolidone (PVP); dextran; pullulan; methylcellulose; hydroxymethylcellulose; hydroxyethylcellulose; hydroxypropylcellulose; polyethylene oxide (PEO); and derivatives of one or more of the foregoing.

[0041] In an implementation, the performing of the CE run includes making an optical measurement of one or more of the analytes separated in the capillary.

[0042] Other devices, apparatus, systems, methods, features and advantages of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The invention can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In the figures, like reference numerals designate corresponding parts throughout the different views.

[0044] Figure 1 is a schematic view of an example of a capillary electrophoresis (CE) system in which the presently disclosed subject matter may be implemented.

[0045] Figure 2 is an electropherogram resulting from performing a CE run on a sample loaded in a capillary whose inside wall was not conditioned with a coating of the type disclosed herein (i.e., an uncoated capillary).

[0046] Figure 3 is an electropherogram resulting from performing a CE run on a sample loaded in a capillary whose inside wall was coated with a test solution different from the capillary conditioning solution disclosed herein.

[0047] Figures 4A-4L are twelve electropherograms resulting from performing twelve respective CE runs on sample containing proteins as target analytes.

[0048] Figures 5A-5L are twelve electropherograms resulting from performing twelve respective CE runs on sample containing proteins as target analytes.

[0049] Figure 6 is an electropherogram resulting from performing a CE run on a sample loaded in a capillary.

[0050] Figure 7 is an electropherogram resulting from performing a CE run on a sample loaded in a capillary.

[0051] The illustrations in all of the drawing figures are considered to be schematic, unless specifically indicated otherwise.DETAILED DESCRIPTION

[0052] In this disclosure, all “aspects,” “examples,” “embodiments,” and “implementations” described are considered to be non-limiting and non-exclusive. Accordingly, the fact that a specific “aspect,” “example,” “embodiment,” or “implementation” is explicitly described herein does not exclude other “aspects,” “examples,” “embodiments,” and “implementations” from the scope of the present disclosure even if not explicitly described. In this disclosure, the terms “aspect,” “example,” “embodiment,” and “implementation” are used interchangeably, i.e., are considered to have interchangeable meanings.

[0053] In this disclosure, the term “substantially,” “approximately,” or “about,” when modifying a specified numerical value, may be taken to encompass a range of values that include + / — 10% of such numerical value.

[0054] In this disclosure, the term “protein” refers to a biomolecule containing at least one long (e.g., greater than 20 amino acid residues) polypeptide.

[0055] In this disclosure, the term “starch” refers to a polymeric carbohydrate (polysaccharide) consisting of several glucose units joined by glycosidic bonds.

[0056] In this disclosure, the term “cationic starch” or “cationic starch derivative” refers to naturally-occurring starches and modified starches (e.g., cationic, anionic, or amphortcric starches). Depending on the type of starch, the starch may need to be derivatized to present positively charged functional groups. Such functional groups may include, for example, quaternary ammonium cations. The starch may be derivatized according to any technique now known or later developed. The starch initially acquired may be neutral, anionic or amphorteric and thereafter rendered cationic by anysuitable process now known or later developed. The starch may be initially provided in the form of a powder, or alternatively as an aqueous phase (e.g., solution, slurry, suspension, paste, etc.). Additional examples of starches are described in U.S. Patent Application Publication No. 2017 / 0335178 Al, the entire contents of which are incorporated by reference herein.

[0057] In this disclosure, the term “arginine” refers to the amino acid having the chemical formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H.

[0058] In this disclosure, the term “polyglycol-based surfactant” generally refers to any polyglycol-based compound (or blend of compounds) having surfactant properties, and which may also be nonionic and / or non-denaturing. Examples of polyglycol-based surfactants include, but are not limited to, TERGITOL surfactants 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).

[0059] In this disclosure, the term “urea” refers to the amide having the chemical formula CO(NH2)2.

[0060] In this disclosure, the term “static coating” refers to a coating that is covalently bonded to the inside surface of a capillary.

[0061] In this disclosure, the term “dynamic coating” refers to a coating that is adsorbed to the inside surface of a capillary without covalent bonding.

[0062] In this disclosure, the term “semi-permanent coating” refers to a coating that has one or more properties or attributes intermediate to or shared by a static coating and / or a dynamic coating. Depending on the CE application, a semi-permanent coating may be substituted for a static coating or dynamic coating.

[0063] Another type of coating may be referred to as a “skorpio” coating. This type of coating behaves initially as a dynamic coating by binding to the inside surface of the capillary by transient hydrogen and ionic bonds, and subsequently becoming covalently bonded to the inside surface in the manner of a permanent coating. See Righetti et al., “The state of the art of dynamic coatings”,Electrophoresis, Vol. 22, Issue 4, p. 603-611 (2001), the entire contents of which are incorporated by reference herein.

[0064] Figure 1 is a schematic view of a capillary electrophoresis (CE) system 100 as one general example of a CE system (or apparatus, instrument, etc.) in which the presently disclosed subject matter may be implemented. A capillary 104, or multiple (e.g., 4, 16, 96, 384, etc.) capillaries 104 arranged in parallel, are mounted in the CE system 100.

[0065] Each capillary 104 has a hollow cylindrical (tube-shaped) capillary body. Typically, the cross-section of the capillary body is circular and annular, such that the capillary body has a radial thickness between a capillary inside surface and a capillary outside surface. The capillary inside surface defines (coaxially surrounds, as a boundary of) the inner bore (or lumen) of the capillary (also referred to herein as the capillary bore) and thus also defines the inside diameter (l.D.) of the capillary (capillary I.D.). If the capillary 104 is bare (e.g., uncoated), or at a bare section of the capillary 104, the capillary outside surface defines the outside diameter (O.D.) of the capillary (capillary O.D.). As one example, the capillary l.D. may be in a range from 0.5 pm to 200 pm, and the capillary O.D. may be in a range from 50 pm to 400 pm. Each capillary 104 may have a significant capillary length relative to its outer diameter (O.D.). As one example, the capillary length may be on the order of centimeters (cm), such as in a range from 20 cm to 80 cm.

[0066] Examples of the material of the capillary body include, but are not limited to, silica, fused silica, fused quartz, doped (synthetic) fused silica, and polymers like polytetrafluoroethylene (PTFE). The capillary 104 may also include an outer protective layer (or coating, film, etc.) in contact with and coaxially surrounding the outer surface of the capillary body. The protective layer is configured to protect the capillary 104 from damage or breakage, and to add strength or robustness to the capillary 104 to allow the capillary 104 to be bent to a certain degree without being damaged or broken. The protective layer may be composed of various materials suitable for this purpose, such as various polymers, examples of which include, but are not limited to, polyimide (PI), acrylate, silicone, and fluoropolymers. Additionally or alternatively, the capillary 104 may include an inner layer that lines the inside surface of the capillary body. Examples of the inner layer include, but are not limited to, various polymers such as polyether ether ketone (PEEK); linear polyacrylamide (LPA); polyvinyl acetate (PVA); polyimide (PI); and polytetrafluoroethylene (PTFE).

[0067] In the context of the present disclosure, the “inside surface” of the capillary 104 is the surface directly exposed to (or directly surrounding and defining the boundary of) the capillary bore. Thus, depending on whether the capillary 104 has or does not have an inner layer as just described, the “inside surface” of the capillary 104 may be the inside surface of the capillary body itself (e.g., fused silica) or it may be the inside surface of an inner layer lining the inside surface of the capillary body.

[0068] In an aspect of the present disclosure, the inside surface of each capillary 104 is coated with a capillary conditioning solution that is prepared and formulated as described herein. Depending on whether the capillary 104 has or does not have an inner layer as just described, the inside surface on which the coating formed from the capillary conditioning solution is disposed may be the inside surface of the capillary body itself or it may be the inside surface of an inner layer lining the inside surface of the capillary body.

[0069] Each capillary 104 may also include an electrophoretic separation medium such as an appropriately formulated polymer gel that serves as a molecular sieve during CE, as appreciated by persons skilled in the art.

[0070] Prior to loading samples (to be subjected to electrophoretic separation) into the capillaries 104, the inlet ends of the capillaries 104 are placed in fluidic communication with (e.g., immersed in) a sample source 108, which may be or include one or more containers holding one or more samples in solution. For example, as illustrated, in the case of multiple capillaries 104 the sample source 108 may be or include a multi-well plate having different wells that hold respective samples (samplecontaining solutions). The outlet ends of the capillaries 104 are placed in fluidic communication with (e.g., immersed in) a buffer reservoir 112 that is filled with a buffer solution (the running buffer utilized for CE). The capillaries 104 are also filled with the running buffer, by capillary action or by applying a pressure differential between the ends of each capillary 104. The buffer reservoir 112, or both the sample source 108 and the buffer reservoir 112, include electrodes. Typically, the electrode of the sample source 108 (or multiple electrodes in each well of a multi-well plate) serves as an anode 116, and the electrode of the buffer reservoir 112 serves as a cathode 120 and may be electrically grounded. The anode(s) 116 and the cathode 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 capillaries 104 (via the anode(s) 116 and cathode 120).

[0071] The CE system 100 also includes a detection device configured for detecting and measuring analytes of the sample after the analytes have been separated by electrophoresis. Typically, the detection device is optics-based and configured for on-capillary detection (detection of analytes residing directly in the capillaries 104). Thus, in the illustrated example, the detection device includes a light source 128 and a light detector (or camera) 132. Multiple capillaries 104 may be mounted in a cartridge 136 or other type of support configured to properly optically align the capillaries 108 with the light source 128 and the light detector 132. The capillary or capillaries 104 are typically positioned relative to the detection device such that the light source 128 illuminates the capillary or capillaries 104 at a certain detection area along their length (which, for example, may be located near the buffer reservoir 112 containing the cathode 120). In response, the light emitted from the detection area is collected by the light detector 132. Examples of a light source 128 include, but are not limited to, a broadband light source (e.g., flash lamp), a light emitting diode (LED), a laser diode (LD), a laser, etc. Multiple light sources 128 may be provided to enable a user to select a desired excitation wavelength. Examples of a light detector 132 include, but are not limited to, a camera, a photomultiplier tube (PMT), a photodiode (PD), a charge-coupled device (CCD), an activepixel sensor (APS) such as a complementary metal-oxide-semiconductor (CMOS) device, etc., which are sensitive to the emission wavelengths to be detected. The capillaries 104, detection device, and any other light-sensitive components typically are enclosed by an instrument console (not shown) to prevent interaction with stray or ambient light. Generally, any type of detection / measurement technique compatible with CE may be utilized, one example being fluorescence-based techniques appreciated by persons skilled in the art.

[0072] As a general example of a method for performing a CE run, the samples to be analyzed and the capillaries 104 are prepared as needed. The preparation of the capillaries 104 may include flushing or rinsing the inside surfaces of the capillaries 104 with an appropriate flushing agent such as, for example, sodium hydroxide (NaOH), potassium hydroxide (KOH), or hydrogen chloride (HC1). 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 after a certain (e.g., predetermined) number of CE runs as needed. As one example, the flushing step may entail flushing the capillary 104 with 1 M NaOH for ten minutes, and such flushing step may be repeated every twentieth CE run (i.e., prior to every twentieth sample injection). The capillaries 104 are thenmounted to the CE system 100. Also as part of preparing the capillaries 104, an electrophoretic separation medium (e.g., polymer gel) may be loaded into the capillaries 104. Depending on the samples being analyzed, the electrophoretic separation medium may be loaded separately from, or as part of, the running buffer. Depending on the configuration of the CE system 100, the electrophoretic separation medium may be loaded into the capillaries 104 before or after mounting the capillaries 104 into the CE system 100. Plugs of the sample solution are then injected from the sample source 108 into each capillary 108 provided. Sample injection may be done electrokinetically as in the illustrated example. In this case, the HV source 124 is operated to apply a voltage pulse across the lengths of the capillaries 104, i.e., between the inlet ends and the outlet ends of the capillaries 104, thereby drawing the sample plugs through the respective inlet ends. Alternatively, sample injection may be done by applying a pressure differential across the lengths of the capillaries 104 in any suitable manner, thereby aspirating the sample plugs into the capillaries 104. In this latter case, the sample source 108 does not need to have anodes 116, as the HV source 124 is not utilized for the sample injection step.

[0073] After loading the sample plugs into the capillaries 104, the sample source 108 is replaced with a buffer reservoir (not shown) similar to the buffer reservoir 112 positioned at the outlet ends of the capillaries 104. Thus, the inlet ends of the capillaries 104 are placed in fluidic communication with (e.g., immersed in) this inlet-side buffer reservoir. The inlet-side buffer reservoir includes an anode 116 electrically communicating with the HV source 124, similar to the one shown in Figure 1 associated with the sample source 108. The anode 116 may comprise a plurality of electrodes, one for each capillary 104, which are positioned in parallel with or coaxial with the inlet ends of the capillaries 104. To initiate electrophoretic separation, the HV source 124 is then operated to apply a voltage across the capillaries 104. The parameters of the applied voltage (magnitude and duration, and optionally as pulses of desired width, frequency, pulse shape, etc.) are selected such that the applied voltage is effective for implementing CE on the samples. That is, the applied voltage causes different components (analytes) of the samples to migrate through the capillaries 104 (including through any electrophoretic separation medium provided in the capillaries 104) at different speeds, thereby separating the different components into distinctive bands. The different bands are then detected and measured by the detection device (light source 128 and the light detector 132). The CE system then utilizes the electrical output signals from the detection device to generateelectropherograms corresponding to the measurements made in each capillary 104 interrogated by the detection device, as appreciated by persons skilled in the art.

[0074] In some examples, the method for performing a CE run is denatured-protein electrophoresis such as SDS-protein electrophoresis. In this case, the analytes of interest in the samples are proteins, and the proteins are denatured before carrying out the CE runs. Thus, the samples are prepared by contacting the samples with a denaturing agent such as SDS, which is typically done before injecting the samples into the capillaries 104. An amount of SDS may additionally be included in the running buffer.

[0075] One aspect of the present disclosure relates to a method for conditioning each capillary 104 utilized for CE and particularly for SDS-protein electrophoresis. This method involves coating the inside surfaces of the capillaries 104 with the capillary conditioning solution disclosed herein. The coating may be accomplished by flowing the capillary conditioning solution through the lumen (inner bore) of each capillary 104 to rinse the inside surface of the capillary 104 with the capillary conditioning solution. The flow of the capillary conditioning solution through the capillary 104 may be established by an active technique (e.g., creating a pressure differential between the ends of the capillary 104 to push or pull the capillary conditioning solution into the inlet ends, or electrokinetically by using the HV source 124 if the capillary conditioning solution is electrokinetically responsive to an electric field), a passive technique (e.g., capillary forces or wicking) or by any other suitable technique. If needed, the flowing step may be repeated one or more times to build up the coating. The coating on the inside surface may be maintained (or regenerated) by repeating the coating step between successive CE runs. The conditioning (coating) step may be repeated after each CE run, or after every two or three runs, etc. That is, the frequency at which the coating step is performed may vary, and may depend on one or more factors such as, for example, the type of sample being analyzed, the method / assay being implemented, the chemicals being utilized, the type of capillary being utilized, environmental parameters, etc. Typically, the conditioning step is performed after the flushing step described earlier and before the step of loading the gel or other electrophoretic separation medium.

[0076] In an implementation, before forming the coating (by applying the capillary conditioning solution as just described), the inside surface of the capillary 104 may be prepared as needed for facilitating or optimizing the formation of the coating on the inside surface (e.g., to promote orenhance the mechanism(s) involved in creating an interface between the inside surface and the coating that is suitable at least as a dynamic coating). As one example, the inside surface may be prepared by exposing the inside surface to a corona discharge or a plasma discharge.

[0077] Another aspect of the present disclosure relates to a capillary 104 that includes a coating lining the inside surface of the capillary 104, where the coating is formed from the capillary conditioning solution disclosed herein.

[0078] Another aspect of the present disclosure relates to a method for separating analytes of a sample by CE, utilizing the capillary conditioning solution disclosed herein. In this method, the inside surface of a capillary 104 (or multiple capillaries 104) is coated with the capillary conditioning solution as just described. The capillary 104 is then filled with a buffer solution (running buffer). A CE run is then performed on the sample by injecting the sample into the capillary 104, and applying a voltage across the capillary 104 according to operating parameters effective to separate the analytes by CE.

[0079] Generally, no limitation is placed on the type of running buffer utilized in the methods disclosed herein or the pH of the running buffer. The running buffer may be selected based on various factors such as, for example, the capillary conditioning solution and the sample being utilized. Examples of the running buffer include, but are not limited to, those based on or containing borate, EDTA (ethylenediaminetetraacetic acid), etc., zwitterionic compounds such as, for example, HEPES (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid). The pH of the running buffer may be, for example, in a range from 3 to 9, or in a range from 3 to 7, or a pH of less than 7.

[0080] Generally, no limitation is placed on the type of electrophoretic separation medium utilized in the methods disclosed herein. The electrophoretic separation medium may be selected based on various factors such as, for example, the capillary conditioning solution and the sample being utilized. Examples of the electrophoretic separation medium include, but are not limited to, polyacrylamide (PAM), linear polyacrylamide (LPA), agarose, polydimethylacrylamide, polyvinylpyrrolidone (PVP), dextran, pullulan, mcthylccllulosc, hydroxymcthylccllulosc, hydroxyethylcellulose, hydroxypropylcellulose, polyethylene oxide (PEO), derivatives of one or more of the foregoing, etc.

[0081] Another aspect of the present disclosure relates to the composition of the capillary conditioning solution utilized for CE in the methods disclosed herein. In one example, the capillaryconditioning solution is an aqueous solution that includes the following components in water: cationic starch; a compound effective for reducing non-specific binding of proteins and suppressing protein aggregation; a nonionic polyglycol-based surfactant; and a biocide to prevent microbial growth. An example of a compound effective for reducing non-specific binding of proteins and suppressing protein aggregation is arginine. A polyglycol-based surfactant such as NP-40 (Tergitol) supports the cleaning of the capillary by increasing protein solubility. Other surfactants may be suitable for this purpose, such as Tween-20 or Triton-XIOO. 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 inside capillary wall. Other examples of biocides which may be suitable to be used for the conditioning solution of the present disclosure are N,N'-methylenebismorpholine, biphenyl-2-ol, 3-iodo-2- propynylbutylcarbamate, Chlorocresol, 2-bromo-2-(bromomethyl)pentanedinitrile, N- (trichloromethylthio)phthalimide or any one of a ProCline™ preservative as offered by for instance by Merck & Co., Inc., Rahway, New Jersey, USA.

[0082] As examples, the relative concentrations of these components in water may be as follows: cationic starch having a concentration in a range from 0.01 to 20 % (w / w), or 0.01 to 10 % (w / w), or 0.01 to 1 % (w / w), or 0.1 to 0. 5% (w / w); arginine (or other compound effective for reducing nonspecific binding of proteins and suppressing protein aggregation) having a concentration in a range from 1 to 30 mM, or 1 to 20 mM, or 1 to 10 mM; polyglycol-based surfactant (or other suitable surfactant) having a concentration in a range from 0.05 to 1 % (w / w), or 0.1 to 0.5 % (w / w); and urea (or other suitable biocide) having a concentration in a range from 1 to 10 M, or 1 to 5 M. More generally, the relative proportions of the cationic starch, the arginine, the polyglycol-based surfactant, and the urea in the aqueous solution may be selected to render the capillary conditioning solution effective to reduce electroosmotic flow of liquid through the capillary 104 during a CE run performed on sample analytes in the capillary 104. Alternatively or additionally, the relative proportions of the cationic starch, the arginine, the polyglycol-based surfactant, and the urea may be selected to render the capillary conditioning solution effective to prevent adsorption of sample analytes to the inside surface of the capillary 104 during a CE run performed on the sample analytes in the capillary.

[0083] The capillary conditioning solution may be prepared by mixing the starch, arginine, polyglycol-based surfactant, and urea in water. Before mixing, the starch may be boiled and / or stirredin the water. After mixing, the capillary conditioning solution may be run through a suitable filter to remove any particulates.

[0084] In one specific but non-limiting example, a starch slurry was prepared by adding starch (25 g) to deionized water (150 mL) followed by mixing with a shaker device for 10 seconds. The starch utilized was CHARGEMASTER R33F cationic starch (Grain Processing Corporation, Muscatine, Iowa, USA). The starch slurry was then added to an additional volume (2000 mL) of deionized water boiling at 450 °C. The resulting mixture was then stirred using a stir bar (250-350 RPM) at a reduced temperature (375 °C) for 30 minutes, and thereafter 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. The mixture of boiled starch (3 L), L-arginine (17.4 g), and TERGITOL TMN-10 surfactant (50 mL) was then added to the urea solution, and the components were mixed with a paddle (585 ±10 RPM) for 180 minutes. The resulting solution was then run through a 1.2 pm ALPHA polypropylene microfiber filter (Meissner Filtration Products, Inc, Camarillo, California, USA).

[0085] Figures 2-7 are electropherograms demonstrating advantages attained from utilizing coated capillaries as disclosed herein.

[0086] Figure 2 is an electropherogram plotted as signal intensity (in relative fluorescence units or RFU) as a function of the size (in kiloDaltons or kDa) of detected / measured peaks resulting from performing a CE run on a sample containing proteins as target analytes. The sample was loaded in an uncoated capillary containing only a gel separation medium, i.e., a capillary that was not coated with the capillary conditioning solution disclosed herein or with any other type of coating. Figure 2 shows merging of lower marker and dye peaks and slow migration time. The lower marker is a sample compound of known size and concentration that is added to the sample. It is used for quantification of sample compounds (in contrast, the ladder is used for determining the size of the sample compounds. The dye peak results from the portion of the fluorescent dye added to the sample (to enable detection via fluorescence mcasurcmcnt / visualization) that docs not actually bind to the protein. Figure 2 also shows that the last (eighth) peak is not with runtime of the method that was set during the assay development. All peaks should be entirely detected 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 tothe poor conditioning of this capillary, the baseline increased as the sample moved through the capillary.

[0087] Figure 3 is an electropherogram (RFU vs. kDa) resulting from performing a CE run on a sample containing the same proteins analyzed in the CE run of Figure 2, and utilizing the same capillary except that the capillary was coated with a test solution different from the capillary conditioning solution disclosed herein. The test solution was a hydrophobic polysiloxane-based solution. Figure 3 shows peak tailing, the last peak (expected at about 240 kDa) being missing, and an elevated baseline.

[0088] Figures 4A-4L are twelve electropherograms (signal intensity in relative fluorescence units or RFU as a function of time in MM:SS) resulting from performing twelve respective CE runs in parallel capillaries with the same sample (containing proteins as target analytes) in each capillary. The twelve CE runs were performed in the respective capillaries under the same conditions. Each capillary was uncoated, i.e., not coated with a capillary conditioning solution as disclosed herein. Figures 4A-4L show peak dropouts and / or loss of resolution and that the capillary loses effectiveness after a handful of CE runs. These problems were attributed to a buildup / adsorption of proteins on the inside surfaces of the capillary walls.

[0089] By comparison, Figures 5A-5L are twelve electropherograms (signal intensity in relative fluorescence units or RFU as a function of time in HH:MM:SS) resulting from performing twelve respective CE runs in parallel capillaries with the same sample (containing proteins as target analytes) in each capillary. The twelve CE runs were performed in the respective capillaries under the same conditions. The protein-containing sample was the same as in the CE runs pertaining to Figures 4A- 4L. The difference was that the electropherograms of Figures 5A-5L were generated by performing electrophoresis after the capillary was coated with a capillary conditioning solution as disclosed herein. Figures 5A-5L show improved performance in comparison to Figures 4A-4L due to the use of the capillary conditioning solution disclosed herein.

[0090] Figure 6 is an electropherogram (RFU vs. kDa) resulting from performing a CE run on a protein-containing sample in a capillary. A conditioning solution was utilized but was inadequate for fully cleaning and coating the inside capillary wall. As shown by the leftmost arrow, the dye front merges with the lower marker, resulting in erroneous quantification. As shown by the next arrow, some of the broader peaks have secondary peaks. The profile of the peak marked by the rightmostarrow is incorrect. This peak has the same peak height (same RFU) as other peaks, but it is the least concentrated protein in the ladder so the RFU should be lower than the other peaks.

[0091] Figure 7 is an electropherogram (RFU vs. kDa) resulting from performing a CE run on a protein-containing sample in a capillary coated with a capillary conditioning solution as disclosed herein. As a result, the CE run shows good performance, for example, a good ladder profile.

[0092] It will be understood that terms such as “communicate” and “in . . . communication with” (for example, a first component “communicates with” or “is in communication with” a second component) are used herein to indicate a structural, functional, mechanical, electrical, signal, optical, magnetic, electromagnetic, ionic or fluidic relationship between two or more components or elements. As such, the fact that one component is said to communicate with a second component is not intended to exclude the possibility that additional components may be present between, and / or operatively associated or engaged with, the first and second components.

[0093] It will be understood that various aspects or details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation — the invention being defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A capillary conditioning solution for coating an inside surface of a capillary utilized for capillary electrophoresis (CE), the capillary conditioning solution being an aqueous solution comprising: water; cationic starch; a compound effective for reducing non-specific binding of proteins and suppressing protein aggregation; a polyglycol-based surfactant; and a biocide.

2. The capillary conditioning solution of claim 1, wherein: the concentration of the cationic starch is in a range selected from the group consisting of: 0.01 to 20 % (w / w); 0.01 to 10 % (w / w); 0.01 to 1 % (w / w); 0.01 to 1 % (w / w); and 0.1 to 0.5 % (w / w); the concentration of the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation is in 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 in a range selected from the group consisting of: 0.05 to 1 % (w / w); and 0.1 to 0.5 % (w / w); and the concentration of the biocide is in a range from 1 to 10 M; and 1 to 5 M.

3. The capillary conditioning solution of claim 1, wherein the cationic starch comprises quaternary ammonium cations.

4. The capillary conditioning solution of claim 1, wherein the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation comprises arginine.

5. The capillary conditioning solution of claim 1, wherein the biocide comprises urea.

6. The capillary conditioning solution of claim 1, wherein, when the capillary conditioning solution is formed as the coating on the inside surface of the capillary, the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the poly glycol-based surfactant, and the biocide render the capillary conditioning solution effective to reduce electroosmotic flow of liquid through the capillary during a CE run performed on sample analytes in the capillary.

7. The capillary conditioning solution of claim 1, wherein, when the capillary conditioning solution is formed as the coating on the inside surface of the capillary, the relative proportions of the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the poly glycol-based surfactant, and the biocide render the capillary conditioning solution effective to prevent adsorption of sample analytes to the inside surface during a CE run performed on the sample analytes in the capillary.

8. A capillary utilized for electrophoresis (CE), the capillary comprising: an inside surface defining an inner lumen of the capillary; and a coating lining the inside surface, wherein the coating is formed from the capillary conditioning solution of claim 1.

9. The capillary of claim 8, wherein the inside surface is negatively charged.

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

11. A method for preparing the capillary conditioning solution of claim 1, the method comprising mixing the cationic starch, the compound effective for reducing non-specific binding of proteins and suppressing protein aggregation, the polyglycol-based surfactant, and the biocide in water.

12. The method of claim 11, comprising, before the mixing, boiling the cationic starch in water.

13. The method of claim 11, comprising, after the mixing, filtering the capillary conditioning solution to remove particulates therefrom.

14. A method for conditioning a capillary utilized for capillary electrophoresis (CE), the method comprising contacting an inside surface of the capillary with the capillary conditioning solution of claim 1 to coat the inside surface.

15. A method for separating analytes of a sample by capillary electrophoresis (CE), the method comprising: coating an inside surface of a capillary with the capillary conditioning solution of claim 1 ; filling the capillary with a buffer solution; and performing a CE run on the sample by: injecting the sample into the capillary; and applying a voltage across the capillary according to operating parameters effective to separate the analytes by CE.

16. The method of claim 15, comprising, after the performing of the CE run, or after repeating the performing of the CE run one or more times on one or more other samples, repeating the coating of the inside surface of the capillary with an additional amount of the capillary conditioning solution.

17. The method of claim 15, wherein the analytes are proteins.

18. The method of claim 17, comprising, before the loading, denaturing the proteins by contacting the proteins with a denaturing agent.

19. The method of claim 15, comprising, before the performing of the CE run, loading an electrophoretic separation medium into the capillary.

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