High-flux monosaccharide separation and quantification method and kit based on gel electrophoresis

Abstract

The invention provides a high-flux monosaccharide separation and quantification method and kit based on gel electrophoresis. The separation and quantification method comprises the following steps: (1) providing a fluorescence-labeled monosaccharide sample to be detected; (2) injecting the monosaccharide sample to be detected into a capillary electrophoresis system, and performing first electrophoretic separation under a first separation system to obtain a first electrophoretogram; the first separation system comprises borate-free first separation gel and a first buffer solution; (3) cleaning a capillary tube of the capillary electrophoresis system, injecting the monosaccharide sample to be detected into the capillary tube again, and performing electrophoretic separation for the second time in a second separation system to obtain a second electrophoretogram; the second separation system comprises second separation gel containing borate and a second buffer solution; and (4) carrying out qualitative identification and quantitative calculation on the mixed monosaccharide according to the migration time difference and peak overlapping condition of each monosaccharide component in the first electrophoretogram and the second electrophoretogram.

Description

Technical Field

[0001] This application relates to the field of biochemical analysis, specifically to a high-throughput method and kit for the separation and quantification of monosaccharides based on gel electrophoresis. Background Technology

[0002] Carbohydrates play crucial roles in living organisms, including energy storage, structural support, cell recognition, and signal transduction. Monosaccharides, as the basic building blocks of carbohydrates, directly determine the spatial structure and biological functions of polysaccharides or oligosaccharides through their types, proportions, and linkage patterns. In drug development, glycosylation modifications affect the stability, half-life, and immunogenicity of protein drugs; in food science, monosaccharide composition is directly related to the sweetness, flavor, and nutritional value of food. Therefore, developing accurate and efficient methods for monosaccharide composition analysis is of great significance for in-depth research into the biological functions of carbohydrates and the development of related products.

[0003] Currently, capillary electrophoresis (CE) is widely used in monosaccharide analysis due to its advantages such as high separation efficiency, fast analysis speed, and low sample consumption. Early capillary electrophoresis often used free solution electrophoresis, achieving separation by forming complexes with monosaccharides using borate buffer. However, this method typically uses pressure injection, making multi-channel parallel analysis difficult and resulting in low throughput, which is insufficient for rapid screening of large-scale samples. In contrast, capillary gel electrophoresis (CGE) uses gel as the separation medium, combined with electro-injection, enabling multi-channel (e.g., 16-channel) parallel detection and significantly improving analytical throughput. However, since most common monosaccharides are pentose or hexose with very similar molecular weights, and some are isomers (such as glucose, galactose, and mannose), they often exhibit similar migration rates in traditional single-gel systems, easily leading to peak overlap and insufficient separation resolution, making it difficult to achieve accurate qualitative and quantitative analysis of all components in complex mixtures.

[0004] While some studies have utilized the complexation reaction of boric acid with the ortho-hydroxyl groups of monosaccharides to alter the migration sequence, the resolution improvement under a single separation mode remains limited. For example, two monosaccharides that can be separated in one gel system may overlap in another, and vice versa. There is a lack of analytical methods that can comprehensively utilize different separation mechanisms to effectively address the problem of overlapping peaks in complex monosaccharide mixtures. Summary of the Invention

[0005] To address the problems of low separation and overlapping chromatographic peaks that make qualitative and quantitative analysis difficult when analyzing complex monosaccharide mixtures using existing high-throughput capillary gel electrophoresis techniques, this application provides a high-throughput monosaccharide separation and quantification method and kit based on gel electrophoresis.

[0006] The first aspect of this application provides a high-throughput method for the separation and quantification of monosaccharides based on gel electrophoresis, comprising the following steps: (1) Provide a fluorescently labeled monosaccharide sample to be tested; (2) The monosaccharide sample to be tested is injected into the capillary electrophoresis system and the first electrophoretic separation is performed in the first separation system to obtain the first electrophoretic pattern; the first separation system includes a first separating gel without borate and a first buffer. (3) Clean the capillary tube of the capillary electrophoresis system and inject the monosaccharide sample to be tested back into the capillary tube. Perform a second electrophoretic separation in the second separation system to obtain a second electrophoretic pattern. The second separation system includes a second separating gel containing borate and a second buffer. (4) Based on the differences in migration time and peak overlap of each monosaccharide component in the first electrophoresis pattern and the second electrophoresis pattern, the mixed monosaccharide is qualitatively identified and quantitatively calculated.

[0007] This invention creatively employs a "two-phase gel continuous electrophoresis-data analysis" strategy. In the first separation system (non-boric acid system), monosaccharides are mainly separated based on differences in molecular size (molecular weight) under the molecular sieving effect of the gel. In the second separation system (boric acid system), borate ions undergo coordination reactions with the ortho-hydroxyl groups of monosaccharide molecules, forming negatively charged complexes. This complexation alters the charge-to-mass ratio and spatial configuration of the monosaccharides, causing significant migration differences in isomers that initially showed similar migration rates in the first system. Because the separation mechanisms of different monosaccharides differ in these two systems, their peak elution order and overlap patterns in the two spectra often differ. For example, monosaccharide A and monosaccharide B may co-elute (overlapping peaks) in the first system but may be completely separated in the second system; or monosaccharide A may overlap with monosaccharide C in the first system but overlap with monosaccharide D in the second system. By comparing and analyzing the spectral data from two electrophoresis sessions, and constructing a logical discriminant matrix based on differentiated migration behaviors, overlapping peaks can be decoupled, thereby accurately identifying and quantifying each monosaccharide in complex mixtures. This method significantly improves separation resolution without sacrificing the advantages of high throughput (multi-channel parallel processing).

[0008] Furthermore, the first separating gel is a linear polyacrylamide gel, and the first buffer solution contains Tris base and EDTA. The linear polyacrylamide gel has a uniform mesh structure, suitable for separation based on the molecular sieve effect, and is free of boric acid, ensuring the purity of the first-dimensional separation.

[0009] Furthermore, the second separating gel is a dextran gel, and the second buffer contains Tris base, borate, and EDTA. The dextran gel also possesses excellent molecular sieving properties, and when combined with the borate buffer, a boric acid complexation separation mechanism is introduced, constructing a second separation dimension orthogonal to the first system.

[0010] Furthermore, step (1) also includes adding an internal standard, namely stachyose, to the monosaccharide sample to be tested. Stachyose, as an internal standard, typically has a peak position outside the mixed monosaccharide region, which can be used to correct migration time drift between different electrophoresis cycles, thereby improving the accuracy of qualitative analysis and the precision of quantitative analysis.

[0011] Further, in step (1), the fluorescent labeling step includes: mixing the monosaccharide to be tested with labeling reagents and labeling buffer, and reacting at 70-80℃ for 50-70 minutes; after the reaction, purifying the labeled product using sugar purification magnetic beads. Fluorescent labeling (such as APTS labeling) endows monosaccharides with fluorescent properties for high-sensitivity detection, while introducing a negative charge to enable electrophoretic migration. The magnetic bead purification step effectively removes unreacted free dyes and salts, reduces baseline noise, and further improves detection sensitivity, making the detection of pmol-level trace monosaccharides possible.

[0012] A second aspect of this application provides a high-throughput monosaccharide separation and quantification kit based on gel electrophoresis, comprising: Labeling reagents; The first separation system includes a borate-free first separating gel and a first buffer solution; The second separation system includes a second separating gel containing borate and a second buffer solution.

[0013] This kit integrates the key reagents required for the method of this invention. Users can perform the above-mentioned biphasic gel continuous electrophoresis method by using a multichannel capillary electrophoresis instrument. The operation is simple and the results are reliable.

[0014] Furthermore, the kit also includes a monosaccharide internal standard, which is a stachyose solution.

[0015] Furthermore, the kit also includes a sample purification component, which includes sugar purification magnetic beads.

[0016] Furthermore, the first separating gel is a linear polyacrylamide gel dissolved in Tris-EDTA buffer; the first buffer is a buffer containing Tris base and EDTA.

[0017] Furthermore, the second separating gel is a dextran gel dissolved in a borate-containing buffer; the second buffer is a buffer containing Tris base, borate, and EDTA.

[0018] The present invention has the following beneficial effects: High separation resolution: By constructing two gel electrophoresis systems with completely different separation mechanisms (molecular sieve effect vs. molecular sieve + complexation effect), the problem of overlapping peaks of isomers or monosaccharides with similar molecular weights in a single gel system is effectively solved by utilizing the difference in migration behavior of different monosaccharides in the two-phase system, thus achieving precise separation of complex mixed monosaccharides.

[0019] High throughput and automation: Based on the capillary gel electrophoresis platform, it can achieve parallel detection of 16 channels or even more. Combined with automatic sample introduction and automatic cleaning procedures, it significantly improves analysis efficiency and is suitable for rapid screening of large batches of samples.

[0020] Extremely high sensitivity: Utilizing fluorescent labeling combined with electrosampling mode, the detection limit can reach the pmol or even fmol level, with extremely low sample consumption, offering significant advantages for the analysis of precious trace samples.

[0021] Accurate quantification: The introduction of internal standard correction and combined calculation logic of biphase spectrum overcomes the quantitative deviation caused by the overlap of single spectrum peaks, and significantly improves the quantitative accuracy of multi-component mixed systems. Attached Figure Description

[0022] Figure 1 The results of gel electrophoresis of type A monosaccharides and mixed sugars in Example 3 are shown.

[0023] Figure 2 The results of B-type gel electrophoresis for monosaccharides and mixed sugars in Example 3 are shown. Detailed Implementation

[0024] To facilitate understanding of this application, a more complete description will be provided below. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0025] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of the application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0028] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0029] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0030] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0031] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0032] The term "particle" as used in this application, or a substance with a defined particle size distribution, is not necessarily spherical in shape; it may be irregular and can be either primary or secondary particles. The particle size of irregular particles is calculated as the average of its maximum and minimum diameters.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only for explaining the invention and are not intended to limit its scope. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0034] In some preferred embodiments, the fluorescent labeling reagent is selected from APTS (trisodium 8-aminopyrene-1,3,6-trisulfonic acid), fluorescein isothiocyanate (FITC), or fluorescein, preferably APTS, because it contains three sulfonic acid groups, which can endow the monosaccharide with a high charge-to-mass ratio and good electrophoretic migration properties. The labeling reaction temperature can be selected from 70℃, 72℃, 75℃, 78℃, or 80℃; the reaction time can be selected from 50 minutes, 55 minutes, 60 minutes, 65 minutes, or 70 minutes.

[0035] In some preferred embodiments, the concentration of linear polyacrylamide in the first separating gel can be selected as 3%, 4%, 5%, 6%, or 8% (w / v); the concentration of dextran in the second separating gel can be selected as 3%, 4%, 5%, 6%, or 8% (w / v). The choice of gel concentration depends on the molecular weight range of the target monosaccharide; lower concentrations are suitable for larger molecules, while higher concentrations can improve the separation of smaller molecules.

[0036] In some preferred embodiments, the concentration of Tris base in the first and second buffer solutions can be selected from 30 mM, 35 mM, 40 mM, 45 mM or 50 mM; and the concentration of EDTA can be selected from 0.5 mM, 1 mM, 1.5 mM or 2 mM.

[0037] In some preferred embodiments, the absolute voltage values ​​of the first and second electrophoresis can be selected between 10kV, 12kV, 15kV, 18kV or 20kV; the electrophoresis temperature can be controlled at 20℃, 22℃, 25℃, 28℃ or 30℃.

[0038] Example 1: This example provides a high-throughput monosaccharide separation and quantification kit.

[0039] Reagent Kit A (Storage temperature -20℃): Labeling reagent: 100 μl of 10 mM APTS (trisodium 8-aminopyrene-1,3,6-trisulfonate) reagent dissolved in 3.2 mM sodium citrate buffer was placed in a 2 ml brown storage tube.

[0040] Labeling buffer: 100 μl of 100 mM sodium cyanoborohydride solution dissolved in dimethyl sulfoxide was placed in a 2 ml transparent storage tube.

[0041] Monosaccharide internal standard: 100 μl of 100 ng / μl stachyose solution was placed in a 2 ml transparent storage tube.

[0042] Reagent Kit B (Storage temperature 4℃): Type A separating gel (first separating gel): 60 ml of 5% linear polyacrylamide gel dissolved in 1x Tris-EDTA buffer, placed in a 60 ml transparent plastic bottle.

[0043] Type B separating gel (second separating gel): 60 ml of 5% dextran gel dissolved in 1x Tris-EDTA buffer containing 40 mM borate, and placed in a 60 ml transparent plastic bottle.

[0044] Type A buffer (first buffer): 60 ml of buffer containing 40 mM Tris base and 1 mM EDTA, packaged in a 60 ml transparent plastic bottle.

[0045] Type B buffer (second buffer): 60 ml of buffer containing 40 mM Tris base, 40 mM borate, and 1 mM EDTA, in a 60 ml transparent plastic bottle.

[0046] Example 2: This example provides a method for monosaccharide isolation and quantification using the kit described in Example 1.

[0047] Sample labeling: Take 1 μl of a mixed monosaccharide sample (containing galacturonic acid, glucuronic acid, glucose, mannose, fucose, arabinose, galactose, xylose, ribose, and rhamnose) with a concentration of 100 ng / μl, add 1 μl of monosaccharide internal standard (stachyose), 2 μl of labeling reagent, and 2 μl of labeling buffer. Label at 75℃ for 1 hour.

[0048] Sample purification: After labeling, add 25 μl of sugar purification magnetic beads, vortex to mix, and incubate for 1 min. Add 180 μl of acetonitrile, vortex to mix, briefly centrifuge, and place on a magnetic rack for adsorption. Discard the supernatant, add 100 μl of 94% acetonitrile, and purify three times. After drying the magnetic beads, add 12 μl of HD formamide to reconstitute. Take 10 μl and add to a clean 96-well PCR plate, ready for instrumentation.

[0049] Electrophoresis consumables installation: Install the following solutions at each position on the capillary electrophoresis apparatus: Position ① 15ml of 0.1M NaOH, Position ② 15ml of 0.1M hydrochloric acid, Position ③ 15ml of 18.2MΩ deionized water, Position ④ 15ml of type A separating gel, Position ⑤ 15ml of type B separating gel, Position ⑥ 15ml of type A buffer, Position ⑦ 15ml of type B buffer, Positions ⑧ and ⑨ 15mL of deionized water, and Position ⑩ is the sample tray.

[0050] Perform the electrophoresis procedure: (1) Cleaning: Inject 0.1M NaOH, 0.1M HCl and deionized water into the dispensing pump, dispensing block and 16-channel capillary tube in sequence, each step lasting 2 minutes.

[0051] (2) Electrophoresis of the first separation system: Rinse the capillary with type A separation gel for 5 min; move the capillary to type A buffer bath and start pre-electrophoresis (-15kv, 3 min, 25℃); inject the sample (1kv, 10s); electrophoretic separation (-15kv, 20 min, 25℃), and collect data to obtain the first electrophoresis pattern.

[0052] (3) Cleaning: The capillary tube is cleaned by sequentially injecting 0.1M NaOH, 0.1M HCl, and deionized water.

[0053] (4) Electrophoresis of the second separation system: Rinse the capillary with type B separating gel for 5 min; move the capillary to type B buffer tank and start pre-electrophoresis (-8kv, 3 min, 25℃); inject the sample (1kv, 10s); electrophoretic separation (-8kv, 30 min, 25℃), and collect data to obtain the second electrophoresis pattern.

[0054] Data Analysis: The software outputs the first electrophoresis pattern (Type A gel) and the second electrophoresis pattern (Type B gel).

[0055] Example 3: Separation effect and quantitative analysis Separation effect: Type A gel (first separation system) results: Galacturonic acid, glucuronic acid, glucose, and xylose can be directly separated. Mixtures of arabinose and ribose, fucose and rhamnose, and mannose and galactose were also separated.

[0056] B-type gel (second separation system) results: xylose and rhamnose can be completely separated; glucose, mannose, and fucose are mixed together; galactose and ribose are mixed together.

[0057] The comparison revealed that the two gums had different separation patterns for monosaccharides, and no completely overlapping combinations were found. Therefore, all 10 monosaccharides could be separated by joint analysis (see Table 1).

[0058] Table 1. Distribution of 10 monosaccharides in the two types of gums Quantitative calculation: Calculate the concentration of each monosaccharide based on the peak area and internal standard correction.

[0059] For overlapping peaks, the difference method is used for calculation. For example: Fucose concentration = Type A colloid peak area at 5° - Type B colloid peak area at 5°; Galactose concentration = Area of ​​type B gel peak 7 - Area of ​​type A gel peak 4 + Corresponding value of area of ​​type B gel peak 4.

[0060] The content of each monosaccharide was finally obtained (Table 2) [1].

[0061] Comparative Example 1: Detection of a Single Type A Separating Gel This comparative example only uses the type A separating gel and type A buffer from Example 1 for detection, and the steps are the same as in Example 2, but the electrophoresis step of the second separation system is omitted.

[0062] The results showed that mannose and galactose were mixed together in type A gel (peak number 7) and could not be distinguished; arabinose and ribose were mixed together (peak number 4) and could not be distinguished. Due to the lack of data on a second separation mechanism, these overlapping peaks could not be decoupled through joint analysis, leading to inaccurate quantitative results.

[0063] Comparative Example 2: Detection of a single type B separating gel [2] This comparative example only uses the type B separating gel and type B buffer from Example 1 for detection, and the steps are the same as in Example 2, but the electrophoresis step of the first separation system is omitted.

[0064] The results showed that glucose, mannose, and fucose were mixed together in the B-type gel (peak number 6) and could not be distinguished; galactose and ribose were mixed together (peak number 7) and could not be distinguished. Similarly, precise quantification of all monosaccharides could not be achieved.

[0065] This embodiment employs capillary gel electrophoresis, with data acquired using a laser-induced fluorescence detector (LIF). The main test parameters include the relative migration time (relative to the internal standard stachyose) and peak area of ​​each monosaccharide component. Qualitative analysis is based on the relative migration time under different separation systems; quantitative analysis uses the internal standard method, calculating the concentration based on the ratio of each component's peak area to the internal standard's peak area.

[0066] Data from Examples 1-3 show that it is difficult to achieve complete baseline separation of 10 monosaccharides using a single separation system.

[0067] In type A gels (non-boric acid system), separation is mainly based on the molecular sieve effect. Since mannose and galactose are both hexoses with the same molecular weight and small difference in spatial volume, they co-elute (both peak number 7) and cannot be distinguished. Similarly, arabinose and ribose also overlap.

[0068] In type B gels (containing boric acid), a boric acid complexation mechanism was introduced. The stability of the complexes formed by borate ions and the ortho-hydroxyl groups of monosaccharides is significantly affected by the monosaccharide configuration. Data shows that mannose and galactose, which overlapped in type A gels, were separated in type B gels (mannose peak number 6, galactose peak number 7). However, new overlapping peaks also appeared in type B gels, such as the co-eluenting of glucose, mannose, and fucose (peak number 6).

[0069] The core advantage of this invention lies in solving the overlap problem by utilizing the "orthogonality" of the two separation mechanisms. A comparison of the data in Table 1 shows that the overlap patterns of type A and type B gels are completely different. For example, fucose and rhamnose overlap in type A gel (number 5), but separate in type B gel (numbers 5 and 6). Glucose, mannose, and fucose overlap in type B gel (number 6), but separate in type A gel (numbers 5, 6, and 7).

[0070] By combining the data from two electrophoresis sessions, a two-dimensional logical discrimination matrix can be constructed. For substances that overlap in one system, separation data from the other system can be used for "decoupling." For example, when calculating fucose content, the difference between peak 5 of type A gel and peak 5 of type B gel can eliminate the interference of rhamnose. Ultimately, the qualitative identification and quantitative calculation of 10 monosaccharides were successfully achieved through the combined analysis method, solving the quantitative bias problem caused by insufficient resolution in single gel systems.

[0071] Comparative Examples 1 and 2 further validated the limitations of a single system. Comparative Example 1 used only Type A gels, resulting in the merging of mannose and galactose measurements, making it impossible to determine their individual contents. Comparative Example 2 used only Type B gels, leading to the merging of glucose, mannose, and fucose measurements. This indicates that accurate quantification of complex monosaccharide mixtures cannot be achieved without the combined analytical step of biphasic gel electrophoresis.

[0072] The method provided by this invention significantly improves the separation resolution of the system by continuously running two gel electrophoresis methods with different mechanisms on the same instrument without increasing additional hardware costs. This method not only retains the high throughput and high sensitivity of capillary gel electrophoresis but also effectively overcomes its weakness in separating isomers, making it highly practical.

[0073] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A high-throughput monosaccharide separation and quantification method based on gel electrophoresis, characterized in that, Includes the following steps: (1) Provide a fluorescently labeled monosaccharide sample to be tested; (2) The monosaccharide sample to be tested is injected into the capillary electrophoresis system and the first electrophoretic separation is performed in the first separation system to obtain the first electrophoretic pattern; the first separation system includes a first separating gel without borate and a first buffer. (3) Clean the capillary tube of the capillary electrophoresis system and inject the monosaccharide sample to be tested back into the capillary tube. Perform a second electrophoretic separation in the second separation system to obtain a second electrophoretic pattern. The second separation system includes a second separating gel containing borate and a second buffer. (4) Based on the differences in migration time and peak overlap of each monosaccharide component in the first electrophoresis pattern and the second electrophoresis pattern, the mixed monosaccharide is qualitatively identified and quantitatively calculated.

2. The method according to claim 1, characterized in that, The first separating gel is a linear polyacrylamide gel, and the first buffer solution contains Tris base and EDTA.

3. The method according to claim 1, characterized in that, The second separating gel is a dextran gel, and the second buffer contains Tris base, borate, and EDTA.

4. The method according to claim 1, characterized in that, Step (1) also includes the step of adding an internal standard to the monosaccharide sample to be tested, wherein the internal standard is stachyose.

5. The method according to claim 1, characterized in that, In step (1), the fluorescent labeling step includes: Mix the monosaccharide to be tested with the labeling reagent and labeling buffer, and react at 70-80℃ for 50-70 minutes; After the reaction was completed, the labeled product was purified using sugar purification magnetic beads.

6. A high-throughput monosaccharide separation and quantification kit based on gel electrophoresis, characterized in that, include: Labeling reagents; The first separation system includes a borate-free first separating gel and a first buffer solution; The second separation system includes a second separating gel containing borate and a second buffer solution.

7. The reagent kit according to claim 6, characterized in that, The kit also includes a monosaccharide internal standard, which is a stachyose solution.

8. The reagent kit according to claim 6, characterized in that, The kit also includes a sample purification component, which includes sugar purification magnetic beads.

9. The reagent kit according to claim 6, characterized in that, The first separating gel is a linear polyacrylamide gel dissolved in Tris-EDTA buffer; the first buffer is a buffer containing Tris base and EDTA.

10. The reagent kit according to claim 6, characterized in that, The second separating gel is a dextran gel dissolved in a borate-containing buffer; the second buffer is a buffer containing Tris base, borate, and EDTA.

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