A method for the preparation of a functionalized gel for capillary electrophoresis analysis

Abstract

The application provides a preparation method of a functionalized gel for capillary electrophoresis analysis, comprising the following steps: configuring a potassium borate buffer solution containing tris-hydroxymethyl aminomethane, ethylenediamine tetraacetic acid and anhydrous potassium tetraborate; adding a solid polymer product into the potassium borate buffer solution, and dissolving at room temperature to obtain the functionalized gel; mixing a sugar aqueous solution, a labeled molecule acetic acid solution and a reducing agent mixture according to a specified proportion, and incubating at a specified temperature to obtain a derivatized monosaccharide sample, and diluting the derivatized monosaccharide sample; mixing the derivatized monosaccharide sample with formamide according to a specified proportion to obtain a sample to be detected, filling a capillary with the functionalized gel, using the potassium borate buffer solution as a buffer solution at both ends of the capillary, and separating on a capillary electrophoresis instrument according to specified electrophoresis conditions.

Description

Technical Field

[0001] This invention relates to the field of information technology, and in particular to a method for preparing a functionalized gel for capillary electrophoresis analysis. Background Technology

[0002] In the field of biochemistry research, carbohydrate analysis has always held a crucial position because carbohydrates are not only fundamental substances for life activities but also closely related to the occurrence of many diseases and the regulation of biological functions. Especially in the separation and detection of monosaccharides, the basic carbohydrate units, high-precision analytical techniques are irreplaceable in revealing the mysteries of biological processes. However, current research and applications still face many challenges in monosaccharide separation technology, and breakthroughs are urgently needed to meet increasingly complex research requirements.

[0003] Existing monosaccharide separation methods often reveal several key shortcomings in practical operation. Many traditional techniques require sophisticated sample handling during separation and are easily affected by external conditions, leading to poor result stability. Furthermore, the combination of separation media and buffer environments is often suboptimal, making it difficult to maintain consistent performance under demands for high precision and sensitivity. This mismatch is particularly pronounced in the analysis of complex samples, limiting the widespread application of these techniques.

[0004] A deeper issue lies in the compatibility between the separation medium and the buffer environment, which has become a core technical challenge. Separation media typically need to form a stable working system with the buffer solution under specific conditions, and the construction of this system directly affects the resolution of monosaccharide samples during the separation process. If the chemical or physical properties of the medium and the buffer solution are incompatible, it will lead to an unstable separation environment. For example, in some cases, the medium may degrade due to the composition of the buffer solution, or the ionic strength of the buffer solution may not be sufficient to support the normal function of the medium. This incompatibility manifests in practical operation as poor separation results. For instance, when analyzing a mixture of multiple monosaccharides, some monosaccharides may not be effectively distinguished due to the inhomogeneity of the medium, or even overlap may occur, severely affecting the accuracy of the analysis.

[0005] Therefore, designing a separation medium that can maintain stability under complex conditions and ensure its high compatibility with buffer environments to achieve efficient resolution of monosaccharide samples has become a key issue in current research and applications. Summary of the Invention

[0006] This invention provides a method for preparing a functionalized gel for capillary electrophoresis analysis, mainly comprising: A gel-forming material and a borate ion source are provided, wherein the borate ions are derived from their potassium or ammonium salts. The gel-forming material includes acrylamide and a crosslinking monomer. The acrylamide and the crosslinking monomer are added to deionized water in a specified ratio to form a reaction solution. An initiator solution and a catalyst are added to the reaction solution to carry out a polymerization reaction. The polymerization product is freeze-dried to obtain a solid polymer product. A potassium borate buffer solution containing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate is prepared. The solid polymer product is added to the potassium borate buffer solution and allowed to dissolve at room temperature to obtain the functionalized gel. A sugar solution, a label molecule acetic acid solution, and a reducing agent mixture are mixed in a specified ratio and incubated at a specified temperature to obtain a derivatized monosaccharide sample. The derivatized monosaccharide sample is then diluted. The derivatized monosaccharide sample is mixed with formamide in a specified ratio to obtain a sample to be tested. The functionalized gel is used to fill a capillary tube, and the potassium borate buffer solution is used as the buffer solution at both ends of the capillary tube. Separation is performed on a capillary electrophoresis apparatus under specified electrophoresis conditions. Further, the provision of the gel-forming material and borate ion source includes: adding the acrylamide and the crosslinking monomer to deionized water in a specified ratio to form the reaction solution, wherein the crosslinking monomer is N,N-dimethylacrylamide; adding the initiator solution and the catalyst to the reaction solution, wherein the initiator solution is ammonium persulfate solution and the catalyst is N,N,N',N'-tetramethylethylenediamine; stirring the mixture in a water bath at a specified temperature for a specified time to carry out the polymerization reaction; and freeze-drying the polymerization product to obtain the solid polymer product. Further, the preparation of a potassium borate buffer solution containing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate includes: weighing the tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate in a specified ratio and placing them in a volumetric flask, dissolving them in deionized water, and then bringing the volume to obtain the potassium borate buffer solution. Further, the step of adding the solid polymer product to the potassium borate buffer and dissolving it at room temperature to obtain the functionalized gel includes: adding a specified amount of the solid polymer product to the potassium borate buffer and dissolving it at room temperature for a specified time to obtain the functionalized gel; and using the functionalized gel to fill the capillary and the pump. Further, the step of mixing the sugar solution, the acetic acid solution of the tag molecule, and the reducing agent mixture in a specified ratio, incubating at a specified temperature to obtain a derivatized monosaccharide sample, and diluting the derivatized monosaccharide sample includes: mixing the sugar solution, the acetic acid solution of the tag molecule, and the tetrahydrofuran reducing agent mixture in a specified ratio, wherein the tag molecule is 8-aminopyrene-1,3,6-trisulfonic acid, and the reducing agent is sodium cyanoborohydride; incubating at a specified temperature for a specified time to obtain a reaction product; and diluting the reaction product to a specified volume to obtain the derivatized monosaccharide sample.Further, the step of mixing the derivatized monosaccharide sample with formamide in a specified ratio to obtain the sample to be tested, filling the capillary with the functionalized gel and using the potassium borate buffer as the buffer at both ends of the capillary, and separating the sample on a capillary electrophoresis apparatus under specified electrophoresis conditions includes: adding formamide to the reaction plate, adding the derivatized monosaccharide sample to the formamide and mixing them in a specified ratio to obtain the sample to be tested; setting the constant temperature chamber temperature, pre-electrophoresis voltage, pre-electrophoresis time, sample loading voltage, sample loading time, electrophoresis voltage, and electrophoresis time as the specified electrophoresis conditions on the capillary electrophoresis apparatus; and performing pre-electrophoresis, sample loading, and electrophoresis separation of the sample to be tested according to the specified electrophoresis conditions. Further, the step of adding the initiator solution and the catalyst to the reaction solution includes: using a glass syringe to draw a specified amount of the ammonium persulfate solution and adding it to the reaction vessel, then drawing a specified amount of the N,N,N',N'-tetramethylethylenediamine and adding it to the reaction vessel, and stirring for a specified time in a water bath at a specified temperature to complete the polymerization reaction. Furthermore, the step of mixing the sugar solution, the acetic acid solution of the tag molecule, and the tetrahydrofuran reducing agent in a specified ratio includes: dissolving the monosaccharide in water to obtain the sugar solution, adding the acetic acid solution of the tag molecule, and then adding the tetrahydrofuran reducing agent to obtain a mixture; and incubating the mixture at a specified temperature to obtain the reaction product.

[0007] The technical solutions provided by the embodiments of the present invention may include the following beneficial effects: This invention discloses a method for separating monosaccharide samples by capillary electrophoresis based on functionalized gels. Addressing the need for high-precision, high-sensitivity separation media in the separation of monosaccharide-derived samples, and the operational challenge of compatibility between buffer systems and gel materials, this invention designs specific gel-forming materials and borate ion sources. Combined with the polymerization reaction of acrylamide and crosslinking monomers, a solid polymer product is prepared and dissolved in a carefully prepared potassium borate buffer to form a functionalized gel. Simultaneously, the preparation of derivatized monosaccharide samples and electrophoresis conditions are optimized to ensure stability and resolution during separation. This invention solves the compatibility problem between gel materials and buffer systems through freeze-drying, room-temperature dissolution, and control of specific electrophoresis parameters, ultimately achieving highly efficient separation of monosaccharide samples. This significantly improves the analytical accuracy and sensitivity of capillary electrophoresis, providing reliable technical support for the analysis of complex carbohydrates in the field of biochemistry. Attached Figure Description

[0008] Figure 1 This is a flowchart of a method for preparing a functionalized gel for capillary electrophoresis analysis according to the present invention.

[0009] Figure 2 Image showing a mixture of potassium borate gum, mannose, and galactose. Figure 3Image showing a mix of regular POP7 gum and galactomannose; Figure 4 A diagram showing a mixture of potassium borate, ribose, xylose, and fructose. Figure 5 This is a mix of regular POP7 gum ribose, xylose, and fructose. Detailed Implementation

[0010] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] like Figure 1 The preparation method of a functionalized gel for capillary electrophoresis analysis in this embodiment may specifically include: S1 provides a gel-forming material and a borate ion source, wherein the borate ions are derived from its potassium or ammonium salt, and the gel-forming material includes acrylamide and a crosslinking monomer.

[0012] A gel-forming material and a borate ion source are provided. The gel-forming material includes acrylamide and a crosslinking monomer, and the borate ion source is selected from its potassium or ammonium salt. The gel-forming material is mixed with deionized water to form an initial solution. An appropriate amount of initiator and catalyst are added to the initial solution to promote the polymerization reaction. The mixture is stirred at a preset temperature to form a polymerized mixture. The polymerized mixture is freeze-dried to obtain a solid intermediate product for subsequent dissolution preparation. The intermediate product is added to a pre-prepared buffer solution containing borate ions and allowed to dissolve at room temperature to prepare the target gel material.

[0013] For example, the gel preparation process first involves providing a gel-forming material and a borate ion source. The gel-forming material consists of acrylamide and crosslinking monomers, and the borate ion source is derived from potassium or ammonium salts. This material selection helps to form a stable network structure because acrylamide, as the main monomer, can polymerize into chain polymers, while crosslinking monomers such as N,N-dimethylacrylamide connect these chains through double bonds to form a three-dimensional network gel, thereby improving the mechanical strength and elasticity of the material. This approach can bring about a uniformly distributed borate ion to enhance the pH responsiveness of the gel, which is beneficial for the selective adsorption of sugar isomers in subsequent separation applications.

[0014] In one possible implementation, for the formation of the initial solution, the gel-forming material is mixed with deionized water, and then an initiator such as ammonium persulfate and a catalyst such as N,N,N',N'-tetramethylethylenediamine are added to promote the free radical polymerization reaction. Stirring at a preset temperature, such as 20 degrees Celsius, can effectively initiate the addition polymerization between monomers to form polymer chains. This polymerization reaction is achieved by the initiator generating free radicals that attack the double bonds of the monomers, gradually elongating the chains, and the catalyst accelerates the reaction rate, thereby obtaining a viscous polymer mixture. This treatment can bring higher polymerization efficiency and product purity, and is beneficial to the stability and reusability of the gel. For example, in a laboratory setting, this step ensures that the mixture is transformed from a clear solution into a homogeneous gel, avoiding the problem of uneven local polymerization.

[0015] It should be noted that when the polymerized mixture is freeze-dried, the solvent can be removed while the porous structure of the polymer is preserved by rapidly freezing and sublimating the water under vacuum, thus obtaining a solid intermediate product. Compared with thermal drying, this drying method can prevent thermal degradation, maintain the biocompatibility of the material, and facilitate the rapid reconstruction of the gel network during subsequent dissolution. For example, in actual operation, the powdered product obtained after freeze-drying is easy to store and transport, reduces the risk of degradation caused by humidity, and thus supports the reliability of long-term experiments.

[0016] Specifically, the intermediate product is added to a pre-prepared buffer solution containing borate ions and allowed to dissolve at room temperature. This allows the borate ions to form reversible bonds with the hydroxyl groups on the polymer chain, thus preparing the target gel material. This dissolution process allows for uniform ion penetration, forming a dynamically cross-linked gel, which is beneficial for improving the swelling capacity and the ability to capture target molecules. For example, in sugar separation applications, this gel can selectively bind to specific sugar structures, achieving effective differentiation of isomers, thereby providing higher accuracy and sensitivity in food analysis. These steps support each other, ensuring continuity and optimized results from material supply to final gel preparation.

[0017] S2, the acrylamide and the crosslinking monomer are added to deionized water in a specified ratio to form a reaction solution.

[0018] Acrylamide and crosslinking monomers are added to deionized water in a predetermined ratio and stirred thoroughly to form a homogeneous initial reaction solution, ensuring uniform distribution of each component. An appropriate amount of initiator is added to the initial reaction solution to initiate the polymerization reaction, and the mixture is continuously stirred at a predetermined temperature to allow the monomers in the solution to gradually form a polymer network structure. When the polymerization reaction reaches a predetermined stage, the temperature and stirring speed in the reaction environment are adjusted to further stabilize the polymer network structure, forming a preliminary gel-like mixture. The preliminary gel-like mixture is placed in a specific container and allowed to stand for a period of time to allow its internal structure to further crosslink and improve, ultimately obtaining a reaction solution that meets the requirements for subsequent gel preparation.

[0019] For example, acrylamide and crosslinking monomers are added to deionized water in a preset ratio and stirred thoroughly to form a homogeneous initial reaction solution. This ensures that the components are evenly distributed in the solution, which is beneficial for the uniform progress of the subsequent polymerization reaction.

[0020] In one possible implementation, acrylamide powder is first weighed and slowly added to deionized water, while a crosslinking monomer such as N,N-methylenebisacrylamide is added simultaneously. The mixture is then stirred at a moderate speed using a magnetic stirrer for several minutes until no visible particles are visible. This uniform distribution helps to avoid uneven polymerization caused by excessively high local concentrations, thereby improving the overall quality and stability of the gel.

[0021] In one possible implementation, an appropriate amount of initiator, such as ammonium persulfate, is added to the initial reaction solution to start the polymerization reaction, and the mixture is continuously stirred at a preset temperature to allow the monomers in the solution to gradually form a polymer network structure. This approach can improve the controllability and efficiency of the polymerization process.

[0022] For example, slowly adding the initiator solution dropwise to the reaction solution and maintaining a constant temperature and stirring in a water bath helps to generate and spread free radicals evenly, avoids reactions that are too fast or too slow, and is beneficial to the formation of a dense network structure, thereby improving the mechanical strength and durability of the gel.

[0023] For example, adjusting the temperature and stirring speed in the reaction environment when the polymerization reaction has progressed to a predetermined extent can further stabilize the polymer network structure and form a preliminary gel-like mixture, thereby optimizing the crosslinking density of the structure.

[0024] In one possible implementation, the temperature is lowered and stirring is slowed down when the solution begins to thicken. This helps to arrange the molecular chains in an orderly manner and form additional cross-linking points, which is beneficial to the elasticity of the gel and the improvement of its swelling properties. For example, in practice, this adjustment can prevent brittleness caused by overpolymerization and improve the applicability of the gel in biopharmaceutical applications, such as for the separation of glycoproteins.

[0025] In one possible implementation, the initial gel-like mixture is placed in a specific container and left to stand for a period of time to allow its internal structure to further cross-link and improve, ultimately obtaining a reaction solution that meets the requirements. This approach can lead to the final optimization of the gel structure and improved stability.

[0026] For example, transferring the mixture to a sealed container and allowing it to stand at room temperature for several hours helps the slow polymerization of the remaining monomers and the self-adjustment of the network, which is beneficial for obtaining a uniform gel for subsequent preparation processes, improving the overall reliability of the method and the quality of the output.

[0027] S3, an initiator solution and a catalyst are added to the reaction solution to carry out a polymerization reaction.

[0028] An initiator solution, obtained by pre-preparing ammonium persulfate solution, is added to the reaction solution. During addition, the reaction solution must be kept in a constant-temperature water bath to promote uniform dispersion of the initiator and initiation of the reaction. After the initiator solution is uniformly dispersed, tetramethylethylenediamine (TMD) catalyst is added. This catalyst accelerates the polymerization reaction and must be added slowly dropwise using a precise measuring tool to avoid uneven reaction caused by excessively high local concentrations. After the catalyst is added, the reaction solution is continuously stirred while maintaining a constant water bath temperature. This stirring ensures sufficient contact between the initiator and catalyst and all components in the reaction solution, thereby promoting uniform polymerization. After stirring for a certain period, the viscosity change of the reaction solution is observed. This viscosity change serves as an indicator of the polymerization progress. If the viscosity reaches a preset threshold, stirring is stopped, completing the initial stage of the polymerization reaction. The resulting product is used for subsequent gel preparation.

[0029] For example, during the process of adding the initiator solution to the reaction solution, the initiator solution is obtained by pre-preparing an ammonium persulfate solution. This method of obtaining the initiator ensures its stability and purity, because ammonium persulfate, as a common free radical initiator, can effectively decompose to generate free radicals in a water bath environment, thereby initiating the polymerization chain reaction. When adding the initiator, it is necessary to ensure that the reaction solution is in a water bath environment at a constant temperature to promote the uniform dispersion of the initiator and the initiation of the reaction. This can bring about the beneficial effect of uniform reaction and avoid gel heterogeneity caused by excessively rapid local polymerization.

[0030] For example, when preparing polyacrylamide gel, if the water bath temperature is controlled at around 20 degrees Celsius, the addition of the initiator solution can make the reaction start more controllable, thereby improving the mechanical strength and separation efficiency of the gel. This uniform dispersion effect can also support the addition of catalysts in subsequent steps, providing a stable reaction basis.

[0031] In one possible implementation, after the initiator solution is uniformly dispersed, a catalyst, tetramethylethylenediamine, is added. The catalyst is used to accelerate the polymerization reaction. This acceleration effect stems from the fact that tetramethylethylenediamine, as a reducing agent, can work synergistically with the redox system formed by ammonium persulfate. It needs to be slowly added to the reaction solution using a precise measuring tool to avoid excessively high local concentrations that could lead to uneven reactions. This approach can optimize the reaction rate and ensure that the polymerization process proceeds smoothly.

[0032] For example, in a mixed solution of borate ions and gel-forming materials, the slow addition of a catalyst can prevent bubble formation and local overheating, thereby obtaining a denser gel structure. This measure to avoid uneven reaction is directly linked to the uniform dispersion in the previous step and together supports the requirement for sufficient contact in the stirring step.

[0033] For example, after the catalyst is added, the reaction solution is continuously stirred while the water bath temperature is kept constant during the stirring process. The stirring is intended to ensure that the initiator and catalyst are in full contact with the components in the reaction solution, thereby promoting the uniformity of the polymerization reaction. This full contact can enhance the collision frequency between molecules and improve the polymerization efficiency. This can bring about the beneficial effect of uniform gel formation and avoid peak distortion in the separation experiment.

[0034] For example, in the preparation of gels using potassium borate buffer, continuous stirring combined with a constant temperature allows borate ions to be uniformly distributed in the polymer network, thereby improving the separation resolution of monosaccharide isomers. This benefit of promoting uniformity continues the stable foundation of the first two steps and provides reliable monitoring of the reaction progress for viscosity observation.

[0035] In one possible implementation, after stirring for a certain period of time, the viscosity change of the reaction solution is observed. The viscosity change serves as an indicator of the polymerization process. If the viscosity reaches a preset threshold, stirring is stopped, completing the initial stage of the polymerization reaction. The resulting product is then used for subsequent gel preparation. This observation method is based on the decrease in fluidity caused by the growth of molecular chains during polymerization, which can accurately determine the reaction endpoint. This approach can bring the beneficial effect of precisely controlling the degree of polymerization and preventing gel brittleness caused by over-polymerization.

[0036] For example, in the preparation of electrophoretic gels after sample pretreatment, when the viscosity change indicates the completion of the reaction, the resulting product can form a stable potassium borate gel, supporting the separation of mannose and galactose by capillary electrophoresis. This benefit of viscosity as an indicator integrates the uniformity and acceleration of the aforementioned steps, ultimately ensuring the overall success of the polymerization reaction and the practicality of the gel.

[0037] S4, freeze-dry the polymerization product to obtain a solid polymer product.

[0038] The polymerization mixture is initially filtered to remove unreacted impurities, yielding a clear reaction solution. This clarified reaction solution is then transferred to a freeze-drying apparatus, where the water in the solution gradually sublimates under low temperature, forming a dry intermediate. The freeze-drying apparatus is maintained under vacuum to ensure complete removal of residual water, resulting in a water-free solid polymer product. This solid polymer product is then removed from the freeze-drying apparatus, sealed, and stored for subsequent monosaccharide composition analysis to ensure the stability of the polymer product.

[0039] For example, when processing the mixture after the polymerization reaction, unreacted impurities are first removed by preliminary filtration. This effectively purifies the solution, prevents impurities from interfering with the subsequent drying process, and thus improves the purity of the final product. For example...

[0040] In one embodiment, the mixture is vacuum filtered using glass fiber filter paper, which traps impurities such as unpolymerized monomers. The resulting clarified reaction solution is more suitable for the drying stage, which helps ensure the homogeneity of the solid polymer product and reduces the potential risk of contamination.

[0041] In one possible implementation, this filtration can also prevent equipment clogging and extend the life of the freeze-drying equipment, because the clarified reaction liquid is less likely to form irregular ice crystals in a low-temperature environment, resulting in a more stable sublimation effect.

[0042] In one embodiment, the clarified reaction solution is transferred to a freeze-drying apparatus, where the water is gradually sublimated in a low-temperature environment to form a dry intermediate substance. The freeze-drying apparatus is a device that utilizes the principles of vacuum and low temperature. The process involves first rapidly freezing the solution into a solid state, and then heating it under vacuum to directly convert the ice into a gaseous state without passing through a liquid state. This preserves the structural integrity of the polymer and avoids degradation that may be caused by high-temperature drying.

[0043] For example, freeze-drying equipment typically includes a cold trap, a vacuum pump, and a heating plate. The reaction solution is placed in a tray and first cooled to -40 degrees Celsius to form ice. Then, the vacuum pump evacuates the air to a low-pressure state, and the heating plate slowly heats up to promote sublimation. This method is particularly suitable for heat-sensitive substances such as polymers, and can produce high-purity intermediates while maintaining their biological activity.

[0044] For example, maintaining a vacuum state for the dried intermediate material ensures that residual moisture is completely removed, resulting in a water-free solid polymer product. This prevents the product from absorbing moisture or degrading due to residual moisture, thereby improving storage stability.

[0045] In one possible implementation, pressure changes are monitored under vacuum. If the pressure stabilizes at a specified low value, it indicates that sublimation is complete. This helps to obtain a thoroughly dried product. For example, in the pharmaceutical field, this technique of thoroughly removing moisture extends the shelf life of the product and ensures accuracy in monosaccharide analysis because anhydrous products are less prone to hydrolysis.

[0046] In one embodiment, the solid polymer product is removed from the device and sealed for subsequent monosaccharide composition analysis. This isolates the product from air and humidity, maintains its stability, and prevents oxidation or contamination.

[0047] For example, vacuum-sealing products in aluminum foil bags and storing them in a drying oven provides technical benefits such as maintaining the long-term integrity of the polymer structure, facilitating direct use during capillary electrophoresis analysis, and ensuring the reliability of analytical results.

[0048] In one possible implementation, this preservation method can also be integrated with other quality control steps. For example, in the quality control of biopharmaceuticals, the sealed product can be directly used in the preparation of potassium borate buffer solution, thereby supporting accurate monosaccharide composition analysis and achieving the quality control target for the glycosyl moiety of glycoproteins.

[0049] S5 is a potassium borate buffer solution containing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate.

[0050] First, obtain the raw materials for preparing the buffer solution, including tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and anhydrous potassium tetraborate. Weigh them according to a preset ratio, ensuring the purity of the raw materials meets the requirements, to obtain an initial mixture. Transfer the initial mixture to a designated container, add an appropriate amount of deionized water to dissolve it, and continue stirring until the initial mixture is completely dissolved, forming a homogeneous solution. Adjust the volume of the homogeneous solution to a predetermined mark, ensuring the solution concentration reaches the target value, to obtain the prepared potassium borate buffer solution. Store the prepared potassium borate buffer solution in a designated environment to ensure its stability, so that it can be used in subsequent related experiments or separation processes, completing the preparation of the buffer solution.

[0051] For example, in the preparation of potassium borate buffer, the raw materials such as tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and anhydrous potassium tetraborate are first obtained. These raw materials are the core components of the buffer. Tris(hydroxymethyl)aminomethane acts as a pH adjuster to maintain the acid-base balance of the solution. EDTA acts as a chelating agent to bind metal ions and prevent interference. Anhydrous potassium tetraborate provides borate ions for complexation reactions. Weighing according to the preset ratio ensures the accurate proportion of each component. This provides stable buffering capacity and is beneficial to the accuracy of subsequent experiments.

[0052] In one possible implementation, these raw materials are weighed on a precision balance. For example, after weighing an appropriate amount of tris(hydroxymethyl)aminomethane, other components are added sequentially to form an initial mixture. This method avoids weighing errors, resulting in a uniform solution concentration. The beneficial effect is to improve the reliability of the buffer solution and prevent experimental deviations.

[0053] For example, after the initial mixture is transferred to a designated container, deionized water is added to dissolve it. This transfer process is carried out using a glass rod or pipette to prevent contamination. Stirring is continued until the mixture is completely dissolved to form a homogeneous solution. This results in sufficient molecular dispersion, which is beneficial to the overall stability of the buffer solution.

[0054] In one possible implementation, a magnetic stirrer is used to stir the mixture at room temperature, and the mixture is observed to gradually become clear. This stirring promotes the uniform combination of solute and solvent, and has the beneficial effect of eliminating particle residue, ensuring that the solution is free of precipitation. This is suitable for precision applications such as capillary electrophoresis and avoids clogging problems.

[0055] For example, adjusting the volume of a homogeneous solution to a predetermined mark involves using a volumetric flask to bring the volume to the target value, which provides precise concentration control and improves the reproducibility of the experiment.

[0056] In one possible implementation, the solution is slowly poured into a volumetric flask, rinsed with deionized water and the volume is replenished, shaken well and then the scale is checked. This adjustment can standardize the solution parameters and has the beneficial effect of enhancing the buffer capacity of the buffer solution, maintaining pH stability, and supporting the consistency of electrophoretic conditions during monosaccharide separation.

[0057] For example, the prepared potassium borate buffer solution is stored in a designated environment, such as in a sealed container in a refrigerator, to ensure stability. This provides long-term storage reliability and is beneficial for the continuity of subsequent experiments.

[0058] In one possible implementation, the preservation date is marked and the sample is placed in an environment of 4 degrees Celsius. This preservation prevents degradation or contamination and has the beneficial effect of maintaining the activity of borate ions, which can improve resolution when used in related separation processes and support accurate analysis of carbohydrate samples.

[0059] S6. The solid polymer product is added to the potassium borate buffer solution and allowed to stand at room temperature to dissolve, thereby obtaining a functionalized gel.

[0060] The freeze-dried solid polymer product was added to a prepared potassium borate buffer solution according to a predetermined ratio, ensuring full contact between the solid polymer product and the buffer solution to form a preliminary mixture. The preliminary mixture was then allowed to stand at room temperature, allowing the solid polymer product to gradually dissolve in the potassium borate buffer solution. Gentle shaking of the container during this process promoted uniform dissolution, resulting in a homogeneous solution. The homogeneous solution was then maintained at room temperature for an extended standing time to ensure complete dissolution of the solid polymer product, generating a gel with specific functionalities. The generated functional gel was then subjected to appearance and viscosity tests to confirm that it met the predetermined gel characteristics requirements for subsequent monosaccharide separation applications.

[0061] In one possible implementation, the freeze-dried solid polymer product is added to a pre-prepared potassium borate buffer solution in a preset ratio to ensure that the solid polymer product and the buffer solution are in full contact to form a preliminary mixture. This promotes the uniform distribution of polymer particles in the buffer solution and avoids local aggregation that leads to uneven dissolution.

[0062] For example, in a laboratory setting, a suitable amount of solid polymer product is slowly poured into a buffer solution and stirred to ensure that each part of the solid is immersed in the liquid. This process helps to stabilize the initial mixture because the solid polymer product begins to swell in the buffer solution, forming a more soluble structure, which provides a basis for subsequent dissolution and is beneficial to the overall uniformity of the gel.

[0063] Specifically, the preliminary mixture is placed at room temperature to allow the solid polymer product to gradually dissolve in the potassium borate buffer solution. During this process, the container can be gently shaken to promote uniform dissolution and form a homogeneous solution. This method utilizes the principle of molecular diffusion under room temperature conditions to allow potassium borate ions to gradually combine with the polymer chains.

[0064] For example, in practice, the mixture is placed in a constant temperature chamber at around 25 degrees Celsius, and the container is gently shaken every once in a while. This not only accelerates the dissolution process but also prevents the formation of precipitates, thereby obtaining a highly transparent solution. The beneficial effect is to improve the purity and separation efficiency of the gel, because a uniform solution can ensure the consistency of the final gel flow in capillary electrophoresis.

[0065] In one embodiment, the uniform solution is kept at room temperature and the standing time is extended to ensure that the solid polymer product is completely dissolved and a gel material with specific functions is generated. This method achieves complete dissolution through time control and avoids residual particles from affecting the gel performance.

[0066] For example, when preparing potassium borate gel, the solution is left to stand for several days, during which the change in liquid viscosity is observed. This helps the polymer to fully hydrate and form a network structure, which is beneficial for the borate ion functionalization of the gel, because a fully dissolved gel can better bind to monosaccharide samples and improve separation resolution.

[0067] For example, the generated functional gel material is subjected to appearance and viscosity tests to confirm that it meets the preset gel characteristic requirements for subsequent monosaccharide separation applications. This test can verify the quality of the gel.

[0068] For example, using a microscope to check for the absence of bubbles and measuring the flow resistance with a viscometer ensures that the gel operates stably in the electrophoresis apparatus. This is beneficial in optimizing the peak separation of monosaccharides such as mannose and galactose, because a qualified gel provides a uniform electrophoretic medium, thereby supporting the reliability of the overall preparation method.

[0069] S7. The derivatized monosaccharide sample and formamide are mixed in a specified ratio to form the sample to be tested. The functionalized gel is used to fill the capillary tube and the potassium borate buffer is used as the buffer at both ends. Capillary electrophoresis separation is performed under specified electrophoresis conditions.

[0070] The derivatized monosaccharide sample was mixed with formamide in a preset ratio to form a test mixture for subsequent separation. The test mixture was placed in the sample tank of a capillary electrophoresis apparatus, and a pre-prepared functionalized gel was used to fill the capillary, ensuring uniform gel distribution for effective separation. Pre-prepared potassium borate buffer was added to both ends of the capillary as the electrophoresis medium. The temperature of the incubator and the electrophoresis time were set, and the electrophoresis apparatus was started to achieve separation. During electrophoresis, the migration behavior of the monosaccharide components in the test mixture within the functionalized gel was monitored to complete the capillary electrophoretic separation process.

[0071] In one possible implementation, when mixing the derivatized monosaccharide sample with formamide in a preset ratio, the derivatized monosaccharide sample can be prepared in advance. This sample is a fluorescently labeled sugar chain obtained by reacting sugar molecules with fluorescent markers such as APTS. This can enhance the detection sensitivity of the sample in electrophoresis because the fluorescent labeling helps the laser-induced fluorescence detector capture the signal, thereby improving the separation resolution.

[0072] For example, mixing 10 μL of derivatized sample with 160 μL of formamide to form the test mixture ensures proper sample dilution, avoids excessive concentration leading to peak distortion, and facilitates uniform migration and clear separation of monosaccharide components during subsequent electrophoresis.

[0073] In one possible implementation, the mixture to be tested is placed in the sample tank of a capillary electrophoresis apparatus. When filling the capillary with a pre-prepared functionalized gel, it is necessary to ensure that the gel is uniformly distributed. This can be achieved by vacuum pump-assisted filling. The functionalized gel is a polymer network prepared by mixing borate ions with a gel-forming material. This network can form reversible complexes with sugar molecules, thereby regulating the migration speed and helping to distinguish the electrophoretic behavior of different monosaccharides.

[0074] For example, during the filling process, the gel solution is first injected into the capillary tube whose inner wall has been silanized, and then pressure is applied to solidify the gel uniformly. This can prevent the formation of air bubbles and improve separation efficiency because the uniform gel reduces the variability of migration paths, thereby improving the repeatability and accuracy of the overall analysis.

[0075] In one possible implementation, a pre-prepared potassium borate buffer solution is added to both ends of the capillary as an electrophoresis medium. Conditions such as the temperature of the thermostat and the electrophoresis time are set. When the electrophoresis apparatus is started, the potassium borate buffer solution provides a stable ionic environment, supporting the balance between electroosmotic flow and electrophoretic migration. This buffer solution is prepared by potassium borate and a buffer such as tris(hydroxymethyl)aminomethane, which can maintain a stable pH value and help prevent sample degradation.

[0076] For example, setting the temperature of the constant temperature chamber to 30 degrees Celsius and the electrophoresis time to 3000 seconds can optimize the separation of monosaccharides under an electric field. This is because a constant temperature reduces the Joule heating effect, and extending the electrophoresis time allows for the full separation of slow-migrating components, thereby obtaining a more complete electrophoretic pattern.

[0077] In one possible implementation, when monitoring the migration behavior of monosaccharide components in the test mixture in the functionalized gel during electrophoresis, a built-in detector of the instrument can be used to record fluorescence signals in real time. The migration behavior depends on the difference in affinity between monosaccharides and borate ions. This difference results in different sugars having unique retention times, which is helpful for identifying mixtures such as mannose and galactose.

[0078] For example, by observing the timing and height of peaks, the degree of monosaccharide separation can be analyzed. This allows for the early detection of anomalies such as peak overlap, and the adjustment of conditions to optimize the results. Real-time monitoring ensures the reliability of the separation process, ultimately completing the capillary electrophoresis separation process.

[0079] S21, the crosslinking monomer is N,N-dimethylacrylamide, 40g of acrylamide and 4ml of the crosslinking monomer are added to 200ml of deionized water.

[0080] Acrylamide and a crosslinking monomer, N,N-dimethylacrylamide, are mixed in a predetermined ratio. The acrylamide and crosslinking monomer are added to deionized water to form an initial mixed solution, ensuring complete dissolution to obtain a homogeneous solution. An appropriate amount of initiator and catalyst are added to the initial mixed solution, and the monomers in the mixed solution undergo polymerization by controlling the reaction conditions to form a preliminary polymer network structure. The preliminary polymer network structure is then further processed by adjusting the solution environment to promote further crosslinking and ensure the formation of a stable gel matrix. The stable gel matrix is ​​placed under predetermined conditions for static treatment to complete the final gel formation. The structural characteristics of the gel matrix are tested to ensure compliance with the requirements for gel preparation based on molecular synthesis.

[0081] In one possible implementation, the specific method of mixing acrylamide and crosslinking monomers in a preset ratio can be accomplished using laboratory stirring equipment.

[0082] For example, acrylamide powder is weighed out and slowly added to a container of deionized water while stirring to prevent clumping. This ensures uniform monomer distribution and avoids uneven polymerization caused by excessively high local concentrations, which is beneficial to the stability of subsequent reactions. Furthermore, from another perspective, if an automated batching system is used in industrial-scale production, the addition rate can be precisely controlled, thereby improving mixing efficiency and reducing human error. This method and the laboratory method complement each other, both aiming to obtain a uniform initial mixed solution to support the overall structural consistency of the gel.

[0083] For example, the specific process of adding appropriate amounts of initiator and catalyst to the initial mixed solution may involve slowly adding ammonium persulfate as an initiator using a burette, supplemented with tetramethylethylenediamine as a catalyst, and controlling the temperature by heating in a water bath. This can initiate a free radical polymerization reaction, forming a preliminary polymer network structure, which is beneficial to enhancing the mechanical strength of the gel. Considering multiple aspects, such as adjusting the amount of catalyst under different pH conditions, the reaction rate can be optimized and side reactions can be prevented. These aspects support each other to ensure that the polymerization process is controllable and efficient, thereby providing a solid foundation for subsequent crosslinking.

[0084] It should be noted that specific implementations of subsequent processing of the preliminary polymer network structure include adjusting the solution pH or adding additional crosslinking agents to promote further crosslinking of the network structure.

[0085] For example, adding borate buffer to stabilize the environment strengthens intermolecular bonds, forming a stable gel matrix that improves the gel's durability and elasticity. Additionally, from a support perspective, combining freeze-drying with the gel removes excess moisture, further enhancing matrix density. These methods work together to ensure the gel is less prone to deformation during application.

[0086] In one possible implementation, the stable gel matrix is ​​placed under preset conditions for static treatment. Specifically, it can be sealed and left to stand at room temperature for several days to complete the final gel formation, and the structural characteristics are then tested using a microscope or rheometer.

[0087] For example, observing the distribution of network pores to verify uniformity can confirm that the gel meets the preparation requirements based on molecular synthesis, which is beneficial to ensuring its reliable performance in fields such as electrophoresis.

[0088] For example, from another perspective, if temperature fluctuations are monitored during the settling process, uneven curing can be avoided. These examples support each other and highlight the key role of settling in optimizing gel structure, thereby achieving efficient gel preparation.

[0089] S22, using a glass syringe, 1.56 ml of ammonium persulfate solution was added to a beaker, followed by 50 μl of N,N,N',N'-tetramethylethylenediamine. The mixture was stirred in a water bath at 20°C for 5 hours.

[0090] When adding the initiator to the reaction solution, a syringe is used to precisely draw ammonium persulfate solution and slowly add it to the beaker, ensuring uniform distribution of the solution within the reaction system and avoiding uneven reaction caused by excessively high local concentrations. Immediately after adding the ammonium persulfate solution, an appropriate amount of the accelerator, tetramethylethylenediamine, is drawn using a glass syringe and added to the beaker, ensuring thorough mixing with the ammonium persulfate solution to form a homogeneous initiation system to promote reaction initiation. The beaker is placed in a constant-temperature water bath, with the water bath temperature controlled within a suitable range. The reaction solution is continuously stirred using a stirring device to ensure uniform distribution of the initiator and accelerator within the reaction system, maintaining the stability of the reaction process. During stirring, the viscosity changes of the reaction solution are observed periodically, and the stirring speed is adjusted accordingly to ensure sufficient contact between the components within the reaction system, thereby achieving optimized control of the reaction conditions in the preparation of gels based on molecular synthesis.

[0091] In one embodiment, when adding the initiator to the reaction solution, the ammonium persulfate solution is precisely drawn up with a glass syringe and slowly added to the beaker. This ensures that the solution is evenly distributed in the reaction system. This helps to avoid uneven reaction caused by excessively high local concentrations. For example, in the process of preparing a gel, if the solution is not evenly distributed, it will cause differences in the polymerization reaction rate, thereby affecting the overall structural stability of the gel. By adding it slowly, the ammonium persulfate can be evenly dispersed as an oxidant, promoting the uniform generation of free radicals, thereby improving the uniformity and mechanical strength of the gel.

[0092] In one embodiment, after adding the ammonium persulfate solution, the accelerator tetramethylethylenediamine is immediately added to the beaker to thoroughly mix with the ammonium persulfate to form a homogeneous initiation system. This effectively promotes the initiation of the reaction. For example, in the scenario of molecular synthesis of gels, tetramethylethylenediamine acts as a reducing agent and reacts with ammonium persulfate to generate free radicals. If the mixing is insufficient, it may lead to a delayed or incomplete reaction. However, by adding and mixing it immediately afterward, the free radical initiation process can be accelerated, thereby shortening the reaction time and improving the yield and purity of the gel.

[0093] In one embodiment, the beaker is placed in a constant-temperature water bath to control the temperature within a suitable range. The reaction solution is continuously stirred by a stirring device to ensure that the initiator and accelerator are evenly distributed and maintain reaction stability. This is beneficial for controlling reaction conditions. For example, in a water bath environment of about 20 degrees Celsius, stirring can prevent the formation of temperature gradients. Without continuous stirring, the reaction system may experience sedimentation or stratification. However, this method can maintain uniform contact between the components, thereby optimizing the degree of polymerization of the gel and reducing the formation of by-products, and improving the separation effect of the final product.

[0094] In one embodiment, the viscosity change of the reaction solution is observed periodically during stirring, and the stirring speed is adjusted accordingly to ensure sufficient contact between the components. This allows for optimized control of the reaction conditions. For example, appropriately increasing the stirring speed when the viscosity increases can prevent premature gel solidification. Ignoring viscosity changes may lead to uneven reaction or gel defects. However, by adjusting the method, sufficient cross-linking between molecules can be ensured, thereby obtaining a more stable structure and better separation performance in the preparation of gels based on molecular synthesis.

[0095] S31, in the preparation of the potassium borate buffer solution, weigh 0.6057g of tris(hydroxymethyl)aminomethane, 0.0584g of ethylenediaminetetraacetic acid, and 0.0233g of anhydrous potassium tetraborate and place them in a 100ml volumetric flask, dissolve them with deionized water, and then bring the volume to 100ml.

[0096] To prepare the potassium borate buffer solution, firstly, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and anhydrous potassium tetraborate were obtained from the pre-prepared reagents and weighed according to a specific ratio to ensure that the mass ratio of each component met the preparation requirements. The weighed substances were placed in a volumetric flask to initially form a mixture matrix. An appropriate amount of deionized water was added to the mixture matrix for initial dissolution. Slow stirring was used to ensure that all components were evenly dispersed in the solution, forming a preliminary solution with no obvious particle residue. Deionized water was then added to the volumetric flask to complete the volume adjustment, ensuring that the solution volume reached the preset standard, forming the final potassium borate buffer solution. The final potassium borate buffer solution was thoroughly mixed to ensure a uniform concentration distribution of all components. This buffer solution is used for subsequent gel preparation or capillary electrophoresis to establish the buffer environment and ensure the stability of the prepared potassium borate buffer solution.

[0097] For example, in the preparation of potassium borate buffer, tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA), and anhydrous potassium tetraborate are obtained from pre-prepared reagents. These components serve as the core substances of the buffer system. Tris(hydroxymethyl)aminomethane is used to maintain pH stability, EDTA acts as a chelating agent to prevent interference from metal ions, and anhydrous potassium tetraborate provides borate ions. Weighing them according to a specific ratio ensures that the buffer capacity of the solution is balanced. This helps the subsequent dissolution steps to proceed smoothly, avoids precipitation problems caused by uneven composition, and thus improves the overall reliability of the preparation.

[0098] In one possible implementation, an appropriate amount of deionized water is added to the formed mixed matrix for initial dissolution. The components are then dispersed uniformly by slow stirring. This stirring process is similar to standard laboratory procedures, which promotes intermolecular interactions, forms an initial solution, and ensures that there are no obvious particle residues. This results in a uniform ion distribution, which is beneficial to the stability of the buffer solution and the accuracy of subsequent volume adjustment operations. For example, in actual preparation, insufficient stirring may lead to excessively high local concentrations, affecting the separation effect of electrophoresis experiments. Uniform dispersion, on the other hand, can support the establishment of a buffer environment for potassium borate buffer in capillary electrophoresis.

[0099] Specifically, deionized water is added to the volumetric flask to bring the solution to a final volume. This process ensures that the solution volume reaches the preset standard, forming the final potassium borate buffer solution. This allows for precise concentration control, which helps maintain the osmotic pressure balance of the solution. For example, in gel preparation applications, precise volume can avoid colloidal instability caused by concentration deviations, thereby improving the accuracy of monosaccharide separation and connecting with the aforementioned uniform dispersion to support the integrity of the overall buffer system.

[0100] In one possible implementation, the final potassium borate buffer solution is thoroughly mixed to ensure a uniform concentration distribution of all components within the solution. This mixing process, similar to vortexing or magnetic stirring, eliminates any potential stratification and serves as a buffer environment for subsequent gel preparation or capillary electrophoresis. This ensures the stability of the potassium borate buffer solution preparation. For example, in electrophoresis experiments, a uniform concentration distribution prevents peak distortion, improves peak resolution, and connects with the volume adjustment operation, forming a complete chain from component acquisition to final application, thereby supporting the separation effect of monosaccharide mixed samples.

[0101] S41, 5.5g of the solid polymer product was added to 100ml of the prepared potassium borate buffer solution and allowed to stand at room temperature for three days to dissolve, thus obtaining the functionalized gel.

[0102] The synthesized solid polymer product was added to a prepared potassium borate buffer solution in a predetermined ratio, ensuring sufficient contact between the solid polymer product and the buffer solution to form a preliminary mixture. The preliminary mixture was placed at room temperature and allowed to stand, allowing the solid polymer product to gradually dissolve in the buffer solution. The degree of dissolution was monitored periodically to ensure no significant particle residue remained. During the dissolution process, ambient humidity and temperature were controlled to avoid interference from external factors. After a predetermined dissolution period, a homogeneous solution was obtained. The homogeneous solution was then thoroughly stirred to ensure uniform distribution of the dissolved substances, ultimately forming the target functionalized gel for subsequent separation applications.

[0103] In one possible implementation, the synthesized solid polymer product is added to a prepared potassium borate buffer solution in a predetermined ratio. This ensures that the solid polymer product and the buffer solution are in full contact to form a preliminary mixture. This contact helps the polymer molecules to be evenly distributed in the buffer solution, avoiding uneven dissolution caused by local aggregation. The beneficial effect is to improve the efficiency of subsequent dissolution and the stability of the gel.

[0104] For example, in a laboratory setting, by first preparing a buffer solution and then slowly adding the polymer product, the mixture can be observed to gradually exhibit a uniform emulsion state. This provides a good foundation for the next step of static dissolution and further supports the reliability of the overall preparation process.

[0105] For example, the preliminary mixture is placed at room temperature and kept still, allowing the solid polymer product to gradually dissolve in the buffer solution. During this process, the degree of dissolution of the mixture is observed periodically to ensure that there are no obvious particle residues. The purpose of this stilling method is to allow polymer molecules to slowly dissolve into the buffer solution through diffusion and hydration, avoiding foam or unstable structures that may be caused by rapid stirring. The beneficial effect is to obtain a purer dissolved product.

[0106] For example, in practice, if particle residue is observed, it can be gently shaken to aid dissolution without violating the principle of settling. This is directly related to the preliminary mixture formed in the previous step, because the uniformity of the preliminary mixture determines the accuracy of dissolution observation, thereby improving the overall quality of gel preparation.

[0107] In one possible implementation, the ambient humidity and temperature are controlled during the static dissolution process of the mixture to avoid interference from external factors in the dissolution process. After the dissolution time reaches a predetermined period, a uniform solution state is obtained. This control helps to maintain stable conditions for the dissolution reaction and prevents condensation caused by excessive humidity or polymer degradation caused by temperature fluctuations. The beneficial effect is to ensure the uniformity and repeatability of the solution state.

[0108] For example, by setting the humidity to 50% and the temperature to 25 degrees Celsius in a constant temperature chamber, the solution can be observed to change from initially turbid to transparent. This directly continues the dissolution observation results from the previous step and provides an ideal solution basis for final stirring, further enhancing the consistency of the preparation of functionalized gels.

[0109] For example, the homogeneous solution is thoroughly stirred to ensure a consistent distribution of the dissolved substances, ultimately forming the target functionalized gel for subsequent separation applications. This stirring further homogenizes the molecular distribution and eliminates any microscopic inhomogeneities, which improves the separation performance and durability of the gel.

[0110] For example, when using a magnetic stirrer, the gel can be observed to exhibit a stable gel-like structure, which is closely related to the homogeneous solution state obtained in the previous step. This is because only after the solution is homogeneous can stirring effectively form a high-quality gel, thereby achieving better monosaccharide separation in applications such as capillary electrophoresis.

[0111] S51, in the preparation of the derivatized monosaccharide sample, 10 μL of 0.1 M sugar aqueous solution, 2 μL of 0.1 M 8-aminopyrene-1,3,6-trisulfonic acid in 4.2 M acetic acid solution, and 2 μL of 1 M sodium cyanoborohydride in tetrahydrofuran mixture are incubated at 37°C for 15 hours and then diluted to 100 μL.

[0112] For the preparation of derivatized monosaccharide samples, a certain amount of sugar aqueous solution, an appropriate amount of 8-aminopyrene-1,3,6-trisulfonic acid acetic acid solution, and a tetrahydrofuran mixture of sodium cyanoborohydride are mixed according to a preset ratio to form an initial reaction mixture. The initial reaction mixture is incubated at a constant temperature to ensure complete reaction and obtain the derivatization product. An appropriate amount of solvent is added to the derivatization product for dilution, forming a diluted sample solution for subsequent detection. The diluted sample solution is mixed with formamide according to a preset ratio to prepare the sample to be tested, ensuring the sample is suitable for the detection conditions of a capillary electrophoresis instrument, thus completing the preparation process of the derivatized monosaccharide sample.

[0113] In the preparation of derivatized monosaccharide samples, the key starting step is to mix the sugar solution, the 8-aminopyrene-1,3,6-trisulfonic acid acetic acid solution, and the tetrahydrofuran mixture of sodium cyanoborohydride.

[0114] For example, the sugar solution may be a solution of monosaccharides such as mannose or galactose, the concentration of which is preset to ensure the material basis for subsequent reactions.

[0115] Specifically, 8-aminopyrene-1,3,6-trisulfonic acid is used as a derivatizing agent. The amino group in its molecular structure can specifically react with the aldehyde group at the reducing end of the monosaccharide. Sodium cyanoborohydride acts as a reducing agent in this process, stabilizing the generated Schiff base intermediate and reducing it to a stable secondary amine bond, thereby covalently attaching the fluorescent tag to the monosaccharide molecule. This mixing process aims to ensure sufficient contact between the reactants, creating a homogeneous system for the subsequent incubation reaction.

[0116] The initial reaction mixture was incubated at a constant temperature to drive and complete the aforementioned derivatization chemical reaction.

[0117] In one possible implementation, the isothermal environment is typically set to 37 degrees Celsius, close to physiological temperature. This temperature ensures that the reaction proceeds at a suitable rate while preventing the decomposition of sugar molecules or labeling reagents due to excessively high temperatures.

[0118] It is important to note that the incubation time needs to be sufficiently long, such as several hours, to ensure the reaction proceeds completely and the reducing end of the sugar molecule is adequately labeled. This step directly determines the yield and uniformity of the final derivatization product; insufficient yield or incomplete reaction will result in weak detection signals or the appearance of spurious peaks.

[0119] The derivatization reaction product obtained after incubation has a high concentration, and direct use for detection may exceed the instrument's linear range or cause capillary blockage. Therefore, dilution is necessary. The derivatization reaction product is diluted with an appropriate solvent, such as deionized water or a specific buffer solution, to increase its volume by a certain factor. This operation adjusts the reaction product to a suitable concentration range, forming a diluted sample solution. Dilution not only reduces the absolute concentration of the sample but also reduces interference from potential salts or unreacted reagents in the system, thereby ensuring a stable baseline and sharp peaks in the electrophoresis spectrum during subsequent detection, which improves the accuracy and reproducibility of the detection.

[0120] Finally, the diluted sample solution is mixed with formamide in a specific ratio to prepare a sample suitable for capillary electrophoresis. Formamide, as a denaturant and solvent, effectively dissolves the derivatized sugar sample and maintains it in a well-dispersed monomolecular state, preventing aggregation during sample introduction. Simultaneously, formamide's high density facilitates the smooth entry of the sample into the capillary due to pressure difference during electrophoresis. Ensuring the sample is suitable for the capillary electrophoresis detection conditions means that the prepared sample is matched to the electrophoresis system in terms of viscosity, conductivity, and compatibility. This guarantees that the derivatized monosaccharides can be effectively separated in the electric field and sensitively captured by the laser-induced fluorescence detector, ultimately completing the entire process from sample preparation to a detectable state.

[0121] S52, the derivatized monosaccharide sample is mixed with formamide at a ratio of 1:160 and placed on a capillary electrophoresis apparatus as the sample to be tested.

[0122] The derivatized monosaccharide sample is mixed with formamide at a preset ratio (volume ratio of monosaccharide sample to formamide) to ensure homogeneous mixing and form a suitable analytical solution. The solution is then placed in the sample tank of a capillary electrophoresis apparatus, ensuring no air bubbles are present in contact with the instrument to guarantee the stability of subsequent detection. Buffer solutions are prepared at both ends of the capillary electrophoresis apparatus using a pre-prepared potassium borate solution, ensuring the buffer concentration matches the chemical environment of the solution. The operating parameters of the capillary electrophoresis apparatus are adjusted to ensure the detection environment meets the separation requirements of the analytical solution, thereby achieving accurate detection of the derivatized monosaccharide sample.

[0123] For example, in the process of mixing the derivatized monosaccharide sample with formamide in a preset ratio, the derivatized monosaccharide sample can be prepared in advance. This sample is a fluorescently labeled product obtained by incubating a sugar solution with specific reagents such as APTS and NaCNBH3. The ratio is set as a volume ratio to ensure that the formamide, as a solvent, can fully dilute the sample, thereby reducing the sample viscosity and improving its fluidity in the capillary. This will result in a uniform mixing effect, which will help the accuracy of subsequent electrophoretic separation, because uneven mixing may lead to peak distortion or signal interference, thereby affecting the resolution of monosaccharide isomers.

[0124] In one possible implementation, when placing the mixture into the sample cell of the capillary electrophoresis apparatus, it is necessary to check that there are no air bubbles in the cell, which can be achieved by gently shaking or slowly injecting the mixture using a pipette.

[0125] For example, the presence of air bubbles can block the current path and cause electrophoresis to be interrupted. Ensuring that there are no air bubbles can maintain a stable electric field distribution. The benefit of doing so is to improve the repeatability and reliability of the detection, because stable sample injection allows the derivatized monosaccharides to migrate uniformly under the electric field, avoiding noise signals caused by air bubbles, thereby supporting accurate peak identification.

[0126] For example, when configuring the buffer solution at both ends of the capillary electrophoresis apparatus using a pre-prepared potassium borate solution, it can be prepared by weighing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate, dissolving and adjusting the volume. The concentration of this buffer solution needs to match the chemical environment of the mixture.

[0127] For example, potassium borate can provide borate ions to form complexes with monosaccharides, thereby enhancing separation selectivity. This has the beneficial effect of stabilizing the electrophoresis medium, because the matched concentration prevents pH drift or ionic strength inhomogeneity, thus ensuring effective separation of derivatized monosaccharides based on differences in charge-to-mass ratio, avoiding peak overlap problems caused by environmental mismatch.

[0128] In one possible implementation, when adjusting the operating parameters of the capillary electrophoresis apparatus, the temperature of the constant temperature chamber and the electrophoresis time can be set to meet the separation requirements of the mixture to be tested.

[0129] For example, controlling the temperature within a suitable range can optimize the molecular diffusion rate, while appropriate electrophoresis time allows different monosaccharides to migrate fully. The beneficial effect of doing so is to achieve accurate detection of derivatized monosaccharide samples, because optimized parameters can amplify the small differences between isomers, supporting the acquisition of quantitative information from peak area or migration time. This allows for cross-validation of separation efficiency in the analysis of mixed samples such as mannose and galactose in multiple directions, avoiding misjudgments caused by low resolution.

[0130] For example, the above process can be examined from multiple perspectives. For instance, regarding the mixing ratio, an inaccurate ratio may result in samples that are too dilute or too concentrated, affecting fluorescence intensity. On the other hand, regarding buffer preparation, the complexing effect of potassium borate can synergistically enhance selectivity with derivatized tags. These multiple aspects mutually support the robustness of the overall method because the steps of uniform mixing, bubble-free injection, buffer matching, and parameter adjustment ensure a complete chain from sample preparation to detection, which is beneficial for high-throughput monosaccharide analysis applications.

[0131] In one possible implementation, further examples illustrate the parameter adjustment aspect. For instance, when dealing with complex sugar mixtures, extending the electrophoresis time can better distinguish similar isomers. On the buffer side, using potassium borate can create a dynamic complexation equilibrium, which can improve separation sensitivity. These measures together support the accuracy of detection and avoid the limitations caused by the same mass-charge ratio in traditional methods, thus providing reliable data in practical applications such as glycomics research.

[0132] S61, the capillary electrophoresis conditions include a constant temperature chamber temperature of 30°C, a pre-electrophoresis voltage of 15kV, a pre-electrophoresis time of 180 seconds, a sample loading voltage of 2 to 6kV, a sample loading time of 18 seconds, an electrophoresis voltage of 15kV, and an electrophoresis time of 3000 seconds.

[0133] In capillary electrophoresis, the incubator temperature is preset to a suitable value to ensure sample stability during electrophoresis. Pre-electrophoresis voltage and time are also configured to activate the capillary wall and create a uniform electric field distribution. After pre-electrophoresis, the loading voltage is adjusted to an appropriate range, and the loading time is controlled to ensure the sample is uniformly loaded to the capillary initiation position, providing a good foundation for subsequent separation. After sample loading, the electrophoresis voltage is set to a stable value, and combined with the preset electrophoresis time, the sample is driven to separate within the capillary, causing different components to gradually stratify based on differences in charge and molecular weight. During separation, the incubator temperature is continuously monitored to maintain environmental parameter consistency, and the electrophoresis time is recorded to ensure the predetermined separation duration is achieved, thus enabling precise control of capillary electrophoresis conditions.

[0134] In capillary electrophoresis, the temperature of the incubator is preset to a suitable value to maintain sample stability. This setting can effectively prevent sample degradation or uneven separation caused by temperature fluctuations. For example, when processing sugar mixtures, keeping the temperature of the incubator constant can ensure that the migration rate of molecules in the electric field is stable, thereby improving the separation resolution.

[0135] In one possible implementation, the thermostat is first calibrated using a temperature sensor to ensure minimal deviation throughout the process. This results in the complete preservation of sample components. Simultaneously, the pre-electrophoresis voltage and time are configured to activate the capillary inner wall, creating a uniform electric field distribution environment. This activation process involves applying voltage to homogenize the charge on the inner wall, avoiding peak distortion caused by local electric field inhomogeneity. For example, when pre-electrophorizing potassium borate gel samples, appropriate voltage and time can optimize the surface characteristics of the inner wall, improving the uniformity of subsequent sample flow. This optimization leads to improved separation efficiency and lays the foundation for the sample loading process.

[0136] After pre-electrophoresis, adjust the loading voltage to an appropriate range and control the loading time to ensure that the sample is uniformly loaded to the starting position of the capillary.

[0137] In one possible implementation, a precision injection system is used to inject the sample. The voltage range is selected based on the sample viscosity to prevent overloading or underloading. For example, when processing APTS-labeled sugar samples, controlling the loading time can prevent the sample from spreading too quickly. This results in a uniform distribution of sample concentration at the starting position, providing a good foundation for subsequent separation. This foundation ensures that different components have the same starting conditions in the electric field, avoiding initial deviations from affecting the final separation results. For example, in polysaccharide analysis, this uniform loading can improve peak clarity, resulting in improved detection accuracy, and is connected to the electrophoresis-driven stage.

[0138] After the sample is loaded, the electrophoresis voltage is set to a stable value and combined with the preset electrophoresis time to drive the sample to separate in the capillary, so that different components gradually separate into layers according to differences in charge and molecular weight.

[0139] In one possible implementation, a stable voltage can generate a constant electric field that drives molecular migration, for example, in the separation of a mixed sample of mannose and galactose. The time setting allows for sufficient stratification, which can lead to improved component resolution. This stratification is based on the principle of electrophoresis, in which charged molecules move at different speeds in an electric field, with larger molecules migrating more slowly, thus achieving separation, improving analytical accuracy, and transitioning to the monitoring stage.

[0140] During the separation process, the temperature of the constant temperature chamber is continuously monitored to maintain the consistency of environmental parameters, and the electrophoresis time is recorded to ensure that the predetermined separation time is reached, so as to achieve precise control of capillary electrophoresis conditions.

[0141] In one possible implementation, an integrated monitoring module provides real-time feedback of temperature data and adjusts it to maintain stability. For example, in long-duration electrophoresis, this monitoring prevents sample denaturation caused by overheating, thus improving the reliability of the entire process. Time recording is precisely tracked by a timer to ensure the completion of separation. For example, in the processing of complex sugar samples, precise duration control optimizes peak separation, resulting in reproducible results. This overall control improves the accuracy and efficiency of the electrophoresis operation.

[0142] S62, the buffer solution at both ends of the capillary and the medium inside the pump and the capillary use the potassium borate buffer solution and the functionalized gel.

[0143] A suitable amount of solution is obtained from a pre-prepared potassium borate buffer solution and used as the buffer medium at both ends of the capillary. Simultaneously, the buffer solution is injected into the capillary to ensure the consistency of the medium throughout the electrophoresis channel. To ensure the consistency of the medium inside the capillary, the prepared functionalized gel is mixed with the potassium borate buffer solution at a predetermined ratio to form a homogeneous gel mixture, which is then filled into the capillary as the separation medium. After filling the gel mixture, the capillary is installed in the electrophoresis apparatus, connecting the buffer tanks at both ends to ensure that the potassium borate buffer solution and the gel mixture in the capillary form a continuous conductivity pathway. To ensure the stability of the conductivity pathway, the potassium borate buffer solution in the buffer tanks at both ends of the capillary is periodically replenished, and the state of the functionalized gel inside the capillary is checked to maintain a stable operating environment for the buffer solution at both ends of the capillary and the internal medium.

[0144] In one possible implementation, an appropriate amount of solution is obtained from a pre-prepared potassium borate buffer solution as a buffer medium at both ends of the capillary, which can maintain the ion balance during electrophoresis and avoid the decrease in separation efficiency caused by pH fluctuations.

[0145] For example, in the separation of carbohydrate samples, this buffer provides a stable borate ion environment, which facilitates the complexation reaction between sugar molecules and the label, thereby improving the accuracy of detection.

[0146] Specifically, the buffer solution is injected into the capillary to ensure the consistency of the medium throughout the electrophoresis channel. This reduces current instability caused by differences in interfacial resistance, and has the beneficial effects of extending the lifespan of the capillary and improving reproducibility. In practice, if the consistency of the buffer solution is guaranteed, the sample migration speed is more uniform, reducing the risk of peak distortion.

[0147] In one possible implementation, to meet the consistency requirements of the medium inside the capillary, the prepared functionalized gel is mixed with the potassium borate buffer solution in a preset ratio to form a uniform gel mixture medium. This mixing process achieves uniform dispersion of the gel by slow stirring, avoiding the formation of air bubbles that would affect the filling quality.

[0148] For example, in the analysis of glycan chain structures, this gel-mixed medium acts as a separation medium by providing a molecular sieving effect, helping sugar molecules of different sizes to be separated according to their molecular weight, thereby obtaining clear electrophoretic patterns.

[0149] Specifically, filling the inside of the capillary has the beneficial effect of enhancing separation resolution, because the network structure of the gel works synergistically with the ionic strength of the buffer to suppress electroosmotic interference and improve the retention time stability of small molecule compounds.

[0150] In one possible implementation, after filling the gel mixing medium, the capillary is installed into the electrophoresis apparatus and connected to the buffer tanks at both ends to ensure that the potassium borate buffer solution and the gel mixing medium in the capillary form a continuous electrical conductivity path. This connection method achieves seamless docking through a precision interface, avoiding contamination caused by leakage.

[0151] For example, in the detection of mixed samples of monosaccharides such as mannose and galactose, the role of this continuous conductivity pathway is to maintain a uniform electric field distribution. The beneficial effect is to reduce the temperature gradient caused by Joule heat accumulation, thereby maintaining the integrity and separation consistency of the sample.

[0152] Specifically, this approach can also optimize voltage application efficiency, shorten electrophoresis time, and improve the signal-to-noise ratio.

[0153] In one possible implementation, the potassium borate buffer solution in the buffer tanks at both ends of the capillary is periodically updated in advance to ensure the stability of the conductivity pathway. At the same time, the state of the functionalized gel in the capillary is checked to maintain a stable operating environment for the buffer solution at both ends of the capillary and the internal medium. This update is achieved by periodically replacing the buffer solution with fresh buffer solution to restore pH and ion concentration.

[0154] For example, in long-duration electrophoresis experiments, this protective function prevents ion depletion caused by buffer exhaustion, which has the beneficial effect of ensuring reproducibility of multiple runs and reducing baseline drift.

[0155] Specifically, checking the gel state includes observing changes in its viscosity and transparency. This practice can detect degradation problems early, thereby extending the overall system reliability and analytical accuracy.

[0156] To further verify the technical effects that the present invention can produce, experiments were conducted, as follows: Experimental Example 1: Experimental materials: The sample consisted of a mixture of mannose and galactose, 0.1 mol APTS (3-aminopropyltriethoxysilane) in 4.2 M acetic acid solution, 1 mol NaCNBH3 (sodium cyanoborohydride) in tetrahydrofuran solution, prepared potassium borate gel, and potassium borate buffer solution.

[0157] Experimental methods 1. Dissolve 100 μg of monosaccharide in 10 μL of water, add 2 μL of 0.1 mol APTS (3-aminopropyltriethoxysilane) in 4.2 M acetic acid solution, and then add 2 μL of 1 mol NaCNBH3 (sodium cyanoborohydride) in tetrahydrofuran mixture.

[0158] 2. Incubate at a constant temperature of 37℃ for 15 hours.

[0159] 3. Dilute the obtained liquid to 100 microliters.

[0160] 4. Loading: Add 9 μL of formamide to the 96-well plate as needed, then add 1-2 μL of the prepared sample to the wells containing the formamide. Perform capillary electrophoresis on a glycosylation analyzer using a two-stage sample loading model. Electrophoresis conditions: oven temperature 30℃; pre-electrophoresis voltage 15 kV; pre-electrophoresis time 180 sec; loading voltage 2-6 kV (depending on sample concentration); loading time 18 sec; electrophoresis voltage 15 kV; electrophoresis time 3000 sec. Use potassium borate buffer for both ends of the capillary, and potassium borate gel for both the pump and the capillary. 5. Identification method: Spiking method, that is, after the run is completed, add a certain sugar alone, run it again, and the peak with the change in peak height and peak area is the corresponding added sugar.

[0161] Experimental results The sample was a mixture of mannose and galactose. Potassium borate gum, mannose, and galactose mix Figure 2 As shown.

[0162] Regular POP7 gum, galactomannose mix, etc. Figure 3 As shown.

[0163] Experimental Example 2: Experimental materials: a mixed sample of ribose, xylose, and fructose; 0.1 mol APTS (3-aminopropyltriethoxysilane) in 4.2 M acetic acid solution; 1 mol NaCNBH3 (sodium cyanoborohydride) in tetrahydrofuran solution; prepared potassium borate gel; and potassium borate buffer solution.

[0164] The steps of a capillary electrophoresis-based method for separating monosaccharides are the same as those in Experimental Example 1.

[0165] Experimental results Potassium borate, ribose, xylose, fructose mix, etc. Figure 4 As shown.

[0166] Regular POP7 ribose, xylose, fructose mix, etc. Figure 5 As shown.

[0167] The above experiments further demonstrate that, compared to the existing liquid acrylamide method, the separating gel prepared with potassium borate can screen out more peaks and achieve more stable separation results for monosaccharide mixed reagents.

[0168] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a functionalized gel for capillary electrophoresis analysis, characterized in that, include: A gel-forming material and a borate ion source are provided, wherein the borate ions are derived from their potassium or ammonium salts. The gel-forming material includes acrylamide and a crosslinking monomer. The acrylamide and the crosslinking monomer are added to deionized water in a specified ratio to form a reaction solution. An initiator solution and a catalyst are added to the reaction solution to carry out a polymerization reaction. The polymerization product is freeze-dried to obtain a solid polymer product. A potassium borate buffer solution containing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate is prepared. The solid polymer product is added to the potassium borate buffer solution and allowed to dissolve at room temperature to obtain the functionalized gel. A sugar solution, a label molecule acetic acid solution, and a reducing agent mixture are mixed in a specified ratio and incubated at a specified temperature to obtain a derivatized monosaccharide sample. The derivatized monosaccharide sample is then diluted. The derivatized monosaccharide sample is mixed with formamide in a specified ratio to obtain a sample to be tested. The functionalized gel is used to fill a capillary tube, and the potassium borate buffer solution is used as the buffer solution at both ends of the capillary tube. Separation is performed on a capillary electrophoresis apparatus under specified electrophoresis conditions.

2. The method as described in claim 1, characterized in that, The provision of the gel-forming material and borate ion source includes: adding the acrylamide and the crosslinking monomer to deionized water in a specified ratio to form the reaction solution, wherein the crosslinking monomer is N,N-dimethylacrylamide; adding the initiator solution and the catalyst to the reaction solution, wherein the initiator solution is ammonium persulfate solution and the catalyst is N,N,N',N'-tetramethylethylenediamine; stirring the mixture in a water bath at a specified temperature for a specified time to carry out the polymerization reaction; and freeze-drying the polymerization product to obtain the solid polymer product.

3. The method as described in claim 1, characterized in that, The configuration comprises a potassium borate buffer solution containing tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate, and includes: weighing the tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and anhydrous potassium tetraborate in a specified ratio and placing them in a volumetric flask, dissolving them in deionized water, and then making up to a final volume to obtain the potassium borate buffer solution.

4. The method as described in claim 1, characterized in that, The step of adding the solid polymer product to the potassium borate buffer and allowing it to stand at room temperature to dissolve to obtain the functionalized gel includes: adding a specified amount of the solid polymer product to the potassium borate buffer and allowing it to stand at room temperature for a specified time to dissolve to obtain the functionalized gel; and using the functionalized gel to fill the capillary and the pump.

5. The method as described in claim 1, characterized in that, The process of mixing a sugar solution, a label molecule acetic acid solution, and a reducing agent in a specified ratio, incubating at a specified temperature to obtain a derivatized monosaccharide sample, and then diluting the derivatized monosaccharide sample includes: mixing a sugar solution, a label molecule acetic acid solution, and a reducing agent tetrahydrofuran in a specified ratio, wherein the label molecule is 8-aminopyrene-1,3,6-trisulfonic acid, and the reducing agent is sodium cyanoborohydride; incubating at a specified temperature for a specified time to obtain a reaction product; and diluting the reaction product to a specified volume to obtain the derivatized monosaccharide sample.

6. The method as described in claim 1, characterized in that, The process of mixing the derivatized monosaccharide sample with formamide in a specified ratio to obtain the sample to be tested, filling a capillary with the functionalized gel and using the potassium borate buffer as the buffer at both ends of the capillary, and separating the sample on a capillary electrophoresis apparatus under specified electrophoresis conditions includes: adding formamide to a reaction plate, adding the derivatized monosaccharide sample to the formamide and mixing them in a specified ratio to obtain the sample to be tested; setting the constant temperature chamber temperature, pre-electrophoresis voltage, pre-electrophoresis time, sample loading voltage, sample loading time, electrophoresis voltage, and electrophoresis time on the capillary electrophoresis apparatus as the specified electrophoresis conditions; and performing pre-electrophoresis, sample loading, and electrophoresis separation of the sample to be tested according to the specified electrophoresis conditions.

7. The method as described in claim 2, characterized in that, The step of adding the initiator solution and the catalyst to the reaction solution includes: using a glass syringe to draw a specified amount of the ammonium persulfate solution and add it to the reaction vessel, then drawing a specified amount of the N,N,N',N'-tetramethylethylenediamine and adding it to the reaction vessel, and stirring for a specified time in a water bath at a specified temperature to complete the polymerization reaction.

8. The method as described in claim 5, characterized in that, The step of mixing the sugar aqueous solution, the tagged molecule acetic acid solution, and the reducing agent tetrahydrofuran mixture in a specified ratio includes: dissolving a monosaccharide in water to obtain the sugar aqueous solution, adding the tagged molecule acetic acid solution, and then adding the reducing agent tetrahydrofuran mixture to obtain a mixture; and incubating the mixture at a specified temperature to obtain the reaction product.

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