Low-electroendosmosis agarose and preparation method thereof

Abstract

The invention discloses low-electroendosmosis agarose and a preparation method thereof. Comprising the following steps: adding peroxydisulfate and an accelerant into an agar suspension, adjusting pH and temperature, and reacting under an ultraviolet condition; adding a trapping agent for reaction until excessive sulfate free radicals are removed, adding p-benzoquinone or superoxide dismutase for continuous reaction, washing and filtering, preparing a suspension, adding peroxymonosulfate and an accelerant, and adjusting the reaction pH and temperature for reaction; finally, L-histidine is added for a reaction; and after the reaction is finished, washing, filtering and drying. The low-added-value agar is converted into the high-added-value agarose, the sulfate group content of the prepared low-electroendosmosis agarose is smaller than or equal to 0.14%, the electroendosmosis is smaller than or equal to 0.14%, the gel strength (1.5%, w / v) is larger than or equal to 1295 g / cm < 2 >, and the yield is larger than or equal to 92.4%; and a good electrophoretic separation effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of agarose, and particularly to a low electroendosmosis agarose and a preparation method thereof. Background Art

[0002] At present, the commonly used techniques for preparing agarose mainly include polyethylene glycol method, acetylation method, CPC method, DMSO method, sodium iodide method, ammonium sulfate method, chitosan method, ion exchange method, chelating agent method, enzyme method, etc. The principle of these methods is mainly to separate sulfated agar from agar. Sulfated agar and agarose are the two main components of agar. The precipitation method mainly utilizes the difference in solubility properties of sulfated agar and agarose in salt and alcohol solutions to achieve the purpose of separating agarose. Representative ones are the EDTA-Na2 method and the polyethylene glycol method. The ion chromatography method mainly utilizes an anion exchange resin to adsorb acidic polysaccharides and does not adsorb neutral polysaccharide agarose, thereby achieving the purpose of separating agarose. However, there are certain drawbacks in the production of agarose by the above methods. Since the traditional methods are all homogeneous treatment processes, agar solutions are produced by treating seaweed with traditional alkali treatment processes. The concentration of agar in this solution is 1.0 to 1.5% (heating agar to dissolve to obtain 1.0 to 1.5% agar). After treatment with EDTA-Na2, polyethylene glycol or anion exchange resin, the yield of agarose is relatively low. In addition, by using the traditional means of separating sulfated agar, even when combined with multiple separation processes, it is difficult to reduce the sulfate group content to 0.15%, and a large amount of ion exchange medium is required. The process is cumbersome, costly, and has a low yield.

[0003] In recent years, a new preparation technology for agarose by the hydrogen peroxide method has emerged. It mainly uses the hydroxyl radicals generated by hydrogen peroxide to desulfurize agar powder. The hydrogen peroxide method desulfurizes agar in a heterogeneous ethanol-water system. Since agar is easily swollen by water absorption, it is impossible to stir, which affects mass transfer and heat transfer. Therefore, it is necessary to desulfurize agar in an ethanol-water system. Since hydrogen peroxide is prone to release oxygen during the reaction, and ethanol is volatile at high temperatures, there are defects such as easy explosion in the ethanol reaction system, making it difficult to achieve large-scale production. In addition, using hydrogen peroxide can reduce the sulfate group content to 0.34%. If hydroxyl radicals are used for cyclic desulfurization multiple times, the final sulfate group content can be reduced to 0.1%, but the gel strength will be lower than 200 g / cm 2 (1.5% w / v). Overcoming the problem of excessive decomposition of agar is required when using hydroxyl radicals for desulfurization. Currently, there is no technology in the existing art that can solve this problem. Summary of the Invention

[0004] To solve the problems of low productivity and production efficiency in the traditional agarose extraction process, and to solve the problems of easy explosion and strength reduction in the desulfurization of the hydrogen peroxide-ethanol system, the present invention develops a heterogeneous system desulfurization process. The agar concentration participating in purification is increased through the heterogeneous system; the problem of reducing the agarose yield by separating sulfur agar in the traditional homogeneous purification process is solved through the desulfurization process; the problem of strength reduction by hydroxyl radicals is solved by introducing singlet oxygen and superoxide anion radicals for selective desulfurization. The final obtained agarose has a sulfate group content < 0.15% and an electroendosmosis < 0.15. This agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0005] To achieve the above object, the present invention provides a method for preparing low electroendosmosis agarose, which is characterized by comprising the following steps:

[0006] Step 1: Add persulfate to the agar suspension, add the first promoter for reaction, adjust the reaction pH, reaction temperature, and ultraviolet intensity, and react to generate sulfate radicals and superoxide anion radicals;

[0007] 2S2O8 2- +2H2O→SO4· - +3S04 2- +O2· - +4H + ;

[0008] Persulfate will generate sulfate radicals and superoxide anion radicals under alkaline conditions,

[0009] Step 2: Add a scavenger (methanol or tert-butanol) to the above reaction system for reaction to remove excess sulfate radicals (otherwise, the excess sulfate radicals will degrade agarose), then add p-benzoquinone or superoxide dismutase for reaction, and wash and filter the reacted agar suspension multiple times to obtain high electroendosmosis agarose;

[0010] Step 3: Redisperse the obtained high electroendosmosis agarose in water to obtain a high electroendosmosis agarose suspension; successively add peroxysulfate and a second promoter to the obtained high electroendosmosis agarose suspension for reaction, adjust the reaction pH and temperature, and react to generate singlet oxygen; preferably, the second promoter is any one of benzoquinone, phenol, TiO2, rose bengal, and urea;

[0011] Step 4: Add L-histidine to the above reaction system to quench singlet oxygen, and wash and filter the reacted agarose suspension multiple times to obtain low electroendosmosis agarose;

[0012] Further, in step 1, the mass concentration of the agar suspension is 5% - 20% w / v.

[0013] Further, in step 1, the persulfate is at least one of sodium persulfate, potassium persulfate, and ammonium persulfate; the addition amount of the persulfate is 0.005 - 0.025 mmol / L; the first promoter is any one of graphene, carbon nanotubes, and biochar; the addition amounts of the graphene, carbon nanotubes, or biochar are 0.1 - 0.5 g / L, 0.2 - 1.0 g / L, and 0.5 - 5.0 g / L respectively.

[0014] Further, in step 1, the reaction pH is 9 - 12, the reaction temperature is 50 - 70 °C, the ultraviolet intensity is 20–100 mW / cm 2 , and the reaction time is 1 - 3 h.

[0015] Further, in step 2, the addition amounts of methanol or tert-butanol are 1 - 5 mol / L and 0.05 - 0.5 mol / L respectively; the reaction time after adding the scavenger is 0.5 - 2 h, and the addition amounts of p-benzoquinone or superoxide dismutase are 50 - 200 μmol / L and 1 - 100 U / mL respectively; the reaction time after adding p-benzoquinone or superoxide dismutase is 15 - 60 min.

[0016] Further, in step 3, the concentration of the high electroendosmosis agarose suspension is 5% - 20% w / v, the monopersulfate is potassium peroxymonosulfate compound salt, sodium bisulfate, or ammonium monopersulfate; the addition amount of the monopersulfate is 1 - 5 mmol / L.

[0017] Further, in step 3, the addition amount of any one of p-benzoquinone, phenol, TiO₂, rose bengal, and urea is 50 - 200 μmol / L, 1 - 5 mmol / L, 1 - 3 g / L, 10 - 50 μmol / L, and 1 - 5 mmol / L respectively;

[0018] the reaction pH is 5 - 9, the reaction temperature is 30 - 40 °C, and the reaction time is 0.5 - 2 h.

[0019] Further, in step 4, the addition amount of L-histidine is 1 - 5 mmol / L, and the reaction time is 30 - 60 min.

[0020] Further, in step 4, the prepared low electroendosmosis agarose satisfies any of the following conditions:

[0021] The sulfate group content < 0.15%;

[0022] The electroendosmosis < 0.15;

[0023] The gel strength at 1.5% > 1200 g / cm2 ;

[0024] Yield > 90%.

[0025] The present invention also protects the low electroendosmosis agarose prepared by the said preparation method.

[0026] The present invention reacts in the aqueous suspension of agar powder without heating and dissolution, and the concentration of agar powder can be as high as 20%.

[0027] The content of sulfate group of the low electroendosmosis agarose prepared by the preparation method of the present invention is ≤ 0.14%, the electroendosmosis is ≤ 0.14, and the gel strength (1.5%, w / v) ≥ 1295 g / cm 2 , and the yield is ≥ 92.4%;

[0028] Preparing the prepared low electroendosmosis agarose into an electrophoresis gel can effectively separate DNAs with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. Description of the Drawings

[0029] Figure 1 is the gel electrophoresis diagram of the agarose prepared in Example 1 for DNAs with different molecular weights.

[0030] Figure 2 is the gel electrophoresis diagram of the agarose prepared in Example 2 for DNAs with different molecular weights.

[0031] Figure 3 is the gel electrophoresis diagram of the agarose prepared in Example 3 for DNAs with different molecular weights.

[0032] Figure 4 is the gel electrophoresis diagram of the agarose prepared in Example 4 for DNAs with different molecular weights.

[0033] Figure 5 is the gel electrophoresis diagram of the agarose prepared in Example 5 for DNAs with different molecular weights.

[0034] Figure 6 is the gel electrophoresis diagram of the agarose prepared in Example 6 for DNAs with different molecular weights.

[0035] Figure 7 is the gel electrophoresis diagram of the agarose prepared in Example 7 for DNAs with different molecular weights.

[0036] Figure 8 is the gel electrophoresis diagram of the agarose prepared in Example 8 for DNAs with different molecular weights.

[0037] Figure 9It is the gel electrophoresis diagram of the agarose prepared in Example 9 for DNAs with different molecular weights.

[0038] Figure 10 It is the gel electrophoresis diagram of the agarose prepared in Example 10 for DNAs with different molecular weights.

[0039] Figure 11 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 1 for DNAs with different molecular weights.

[0040] Figure 12 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 2 for DNAs with different molecular weights.

[0041] Figure 13 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 3 for DNAs with different molecular weights.

[0042] Figure 14 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 4 for DNAs with different molecular weights.

[0043] Figure 15 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 5 for DNAs with different molecular weights.

[0044] Figure 16 It is the gel electrophoresis diagram of the agarose prepared in Comparative Example 6 for DNAs with different molecular weights.

[0045] Figures 1-16 In [description], the separation effect diagrams of the DNA Marker with molecular weights of 2000, 1000, 800, 600, 400, and 200 bp from top to bottom in the DL2002 lane.

[0046] In the DL2502 lane, the separation effect diagrams of the DNA Marker with molecular weights of 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp from top to bottom.

[0047] In the DL10003 lane, the separation effect diagrams of the DNA Marker with molecular weights of 15000, 8000, 5000, 3000, 2000, and 1000 bp from top to bottom. Specific Embodiments

[0048] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention. For those technical or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0049] A preparation method of low electroendosmosis agarose:

[0050] Step 1: Add persulfate to the agar suspension, add the first promoter to react, adjust the reaction pH, reaction temperature and ultraviolet intensity, and react to generate sulfate radicals and superoxide anion radicals;

[0051] Step 2: Add a scavenger (methanol or tert-butanol) to the above reaction system to react to remove excessive sulfate radicals, then add p-benzoquinone or superoxide dismutase to react, and wash and filter the reacted agar suspension multiple times to obtain high electroendosmosis agarose;

[0052] Step 3: Redisperse the obtained high electroendosmosis agarose in water to obtain a high electroendosmosis agarose suspension; sequentially add peroxymonosulfate and a second promoter to the obtained high electroendosmosis agarose suspension to react, adjust the reaction pH and temperature, and react to generate singlet oxygen; preferably, the second promoter is any one of benzoquinone, phenol, TiO2, rose bengal, urea;

[0053] Step 4: Add L-histidine to the above reaction system to quench singlet oxygen, and wash and filter the reacted agarose suspension multiple times to obtain low electroendosmosis agarose;

[0054] Further, in step 1, the mass concentration of the agar suspension is 5% - 20% w / v.

[0055] Further, in step 1, the persulfate is at least one of sodium persulfate, potassium persulfate, ammonium persulfate; the addition amount of the persulfate is 0.005 - 0.025 mmol / L; the first promoter is any one of graphene, carbon nanotubes, biochar; the addition amounts of graphene, carbon nanotubes or biochar are 0.1 - 0.5 g / L, 0.2 - 1.0 g / L, 0.5 - 5.0 g / L respectively.

[0056] Further, in step 1, the reaction pH is 9 - 12, the reaction temperature is 50 - 70 °C, and the ultraviolet intensity is 20–100 mW / cm 2, The reaction time is 1 to 3 h.

[0057] Further, in step 2, the addition amounts of methanol or tert-butanol are 1 to 5 mol / L and 0.05 to 0.5 mol / L respectively; the reaction time after adding the scavenger is 0.5 to 2 h, and the addition amounts of p-benzoquinone or superoxide dismutase are 50 to 200 μmol / L and 1 to 100 U / mL respectively; the reaction time after adding p-benzoquinone or superoxide dismutase is 15 to 60 min.

[0058] Further, in step 3, the concentration of the high electroendosmosis agarose suspension is 5% - 20% w / v, and the persulfate is potassium peroxymonosulfate compound salt, sodium bisulfate, or ammonium peroxymonosulfate; the addition amount of the persulfate is 1 to 5 mmol / L.

[0059] Further, in step 3, the addition amount of any one of p-benzoquinone, phenol, TiO₂, rose bengal, and urea is 50 to 200 μmol / L, 1 to 5 mmol / L, 1 to 3 g / L, 10 to 50 μmol / L, and 1 to 5 mmol / L respectively;

[0060] The reaction pH is 5 to 9, the reaction temperature is 30 to 40 °C, and the reaction time is 0.5 to 2 h.

[0061] Further, in step 4, the addition amount of L-histidine is 1 to 5 mmol / L, and the reaction time is 30 to 60 min.

[0062] Example 1: Preparation of low electroendosmosis agarose (graphene + sodium persulfate + TiO₂)

[0063] It includes the following steps:

[0064] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 10% (w / v); the persulfate is sodium persulfate at 0.01 mmol / L, and the first promoter is graphene at 0.3 g / L; the reaction conditions are: pH 10, 60 °C, ultraviolet intensity 60 mW / cm 2 , and react for 2 h;

[0065] Step 2: The scavenger is tert-butanol at 0.3 mol / L, and react for 1 h; the addition amount of p-benzoquinone is 150 μmol / L and react for 30 min;

[0066] Step 3: The concentration of the high electroendosmosis agarose suspension is 10% (w / v); the persulfate is potassium peroxymonosulfate compound salt at 3 mmol / L, and the second promoter is TiO₂ at 2 g / L; the reaction conditions are pH 7, 35 °C, and react for 1 h;

[0067] Step 4: The addition amount of L-histidine is 3 mmol / L, and react for 45 min.

[0068] The prepared low electroendosmosis agarose was tested: the sulfonic acid group content was 0.12%, the electroendosmosis was 0.13, and the gel strength (1.5%, w / v) was 1355 g / cm 2 , and the yield was 93.7%;

[0069] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of agarose was 1.5 (w / v). The results are shown in Figure 1 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0070] Example 2: Preparation of low electroendosmosis agarose (carbon nanotubes + potassium persulfate + rose bengal)

[0071] It includes the following steps:

[0072] Step 1: As in the above steps. Specifically, the concentration of the agar suspension was 15% (w / v); the persulfate was potassium persulfate at 0.02 mmol / L, and the first promoter was carbon nanotubes at 0.6 g / L; the reaction conditions were: pH 11, 55 °C, and the ultraviolet intensity was 80 mW / cm 2 , and the reaction was carried out for 2.5 h;

[0073] Step 2: The capture agent was methanol at 3 mol / L for 1.5 h; SOD at 50 U / mL for 20 min;

[0074] Step 3: The concentration of the high electroendosmosis agarose suspension was 15% (w / v); the monopersulfate was sodium bisulfate at 2 mmol / L, and the second promoter was rose bengal at 30 μmol / L; the reaction conditions were: pH 8, 40 °C, and the reaction was carried out for 1.5 h;

[0075] Step 4: L-histidine at 2 mmol / L for 30 min.

[0076] The prepared low electroendosmosis agarose was tested: the sulfonic acid group content was 0.11%, the electroendosmosis was 0.12, and the gel strength (1.5%, w / v) was 1410 g / cm 2 , and the yield was 92.8%;

[0077] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of agarose was 1.5% (w / v). The results are shown in Figure 2It can be seen that this agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0078] Example 3: Preparation of low electroendosmosis agarose (biochar + ammonium persulfate + urea)

[0079] It includes the following steps:

[0080] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 5% (w / v); the persulfate is ammonium persulfate at 0.025 mmol / L, and the first promoter is biochar at 2.0 g / L; the reaction conditions are: pH 12, 70 °C, ultraviolet intensity 100 mW / cm 2 , and react for 3 h;

[0081] Step 2: The scavenger is tert-butanol at 0.5 mol / L and reacts for 2 h; p-benzoquinone at 200 μmol / L and reacts for 60 min;

[0082] Step 3: The concentration of the high electroendosmosis agarose suspension is 5% (w / v); the monopersulfate is ammonium monopersulfate at 4 mmol / L, and the second promoter is urea at 3 mmol / L; the reaction conditions are: pH 5, 30 °C, and react for 2 h;

[0083] Step 4: L-histidine at 5 mmol / L and reacts for 60 min.

[0084] The prepared low electroendosmosis agarose was tested: the sulfate group content was 0.14%, the electroendosmosis was 0.14, the gel strength (1.5%, w / v) was 1400 g / cm 2 , and the yield was 92.4%;

[0085] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5 (w / v), and the results are shown in Figure 3 It can be seen that this agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0086] Example 4: Preparation of low electroendosmosis agarose (graphene + potassium persulfate + phenol)

[0087] It includes the following steps:

[0088] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 20% (w / v); the persulfate is potassium persulfate at 0.015 mmol / L; the first promoter is graphene at 0.5 g / L; the reaction conditions are: pH 9, 50 °C, ultraviolet intensity 20 mW / cm 2 , react for 1 h;

[0089] Step 2: The capturer is methanol at 5 mol / L and react for 0.5 h; SOD is 100 U / mL and react for 15 min;

[0090] Step 3: The concentration of the high electroendosmosis agarose suspension is 20% (w / v); the monopersulfate is potassium peroxymonosulfate compound salt at 1 mmol / L, and the second promoter is phenol at 1 mmol / L; the reaction conditions are: pH 9, 40 °C, react for 0.5 h;

[0091] Step 4: L-histidine at 1 mmol / L reacts for 30 min.

[0092] The prepared low electroendosmosis agarose is tested: the sulfate group content is 0.13%, the electroendosmosis is 0.13, and the gel strength (1.5%, w / v) is 1422 g / cm 2 , and the yield is 93.9%;

[0093] The prepared low electroendosmosis agarose is made into an electrophoresis gel for electrophoresis. The concentration of the agarose is 1.5% (w / v), and the results are shown in Figure 4 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0094] Example 5: Preparation of low electroendosmosis agarose (carbon nanotubes + sodium persulfate + benzoquinone)

[0095] It includes the following steps:

[0096] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 12% (w / v); the persulfate is sodium persulfate at 0.005 mmol / L; the first promoter is carbon nanotubes at 0.2 g / L; the reaction conditions are: pH 10, 65 °C, ultraviolet intensity 40 mW / cm 2 , react for 1.5 h;

[0097] Step 2: The capturer is tert-butanol at 0.05 mol / L and react for 0.5 h; p-benzoquinone is 50 μmol / L and react for 15 min;

[0098] Step 3: The concentration of the high electroendosmosis agarose suspension is 12% (w / v); the persulfate is sodium bisulfate at 5 mmol / L; the second promoter is benzoquinone at 200 μmol / L; the reaction conditions are: pH 6, 35 °C, reaction for 1.5 h;

[0099] Step 4: React with 4 mmol / L of L-histidine for 45 min.

[0100] The prepared low electroendosmosis agarose was tested: the sulfate group content was 0.12%, the electroendosmosis was 0.12, the gel strength (1.5%, w / v) was 1355 g / cm 2 , and the yield was 93.1%;

[0101] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v), and the results are shown in Figure 5 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0102] Example 6: Preparation of low electroendosmosis agarose (biochar + ammonium persulfate + rose bengal)

[0103] Including the following steps:

[0104] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 18% (w / v); the persulfate is ammonium persulfate at 0.02 mmol / L, and the first promoter is biochar at 5.0 g / L; the reaction conditions are: pH 11, 60 °C, ultraviolet intensity 70 mW / cm 2 , and the reaction is for 2 h;

[0105] Step 2: React with 2 mol / L of methanol as the capturer for 1 h; react with 20 U / mL of SOD for 30 min;

[0106] Step 3: The concentration of the high electroendosmosis agarose suspension is 18% (w / v); the persulfate is ammonium monopersulfate at 3 mmol / L, and the second promoter is rose bengal at 10 μmol / L; the reaction conditions are: pH 7, 30 °C, reaction for 1 h;

[0107] Step 4: React with 3 mmol / L of L-histidine for 45 min.

[0108] The prepared low electroendosmosis agarose was tested: the sulfate group content was 0.11%, the electroendosmosis was 0.11, the gel strength (1.5%, w / v) was 1450 g / cm 2 , and the yield was 94.1%;

[0109] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v). The results are shown in Figure 6 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0110] Example 7: Preparation of low electroendosmosis agarose (graphene + ammonium persulfate + benzoquinone)

[0111] It includes the following steps:

[0112] Step 1: As in the above steps. Specifically, the concentration of the agar suspension was 8% (w / v); the persulfate was ammonium persulfate at 0.02 mmol / L, and the first promoter was graphene at 0.1 g / L; the reaction conditions were: pH 10, 50 °C, ultraviolet intensity 30 mW / cm 2 , and the reaction was carried out for 1.5 h;

[0113] Step 2: The capturer was tert-butanol at 0.2 mol / L for 1 h; p-benzoquinone at 100 μmol / L for 20 min;

[0114] Step 3: The concentration of the high electroendosmosis agarose suspension was 8% (w / v); the monopersulfate was potassium peroxymonosulfate compound salt at 2 mmol / L, and the second promoter was benzoquinone at 150 μmol / L; the reaction conditions were: pH 6, 30 °C, and the reaction was carried out for 1 h;

[0115] Step 4: L-histidine at 2 mmol / L for 30 min.

[0116] The prepared low electroendosmosis agarose was tested: the sulfate group content was 0.13%, the electroendosmosis was 0.13, the gel strength (1.5%, w / v) was 1382 g / cm 2 , and the yield was 92.6%;

[0117] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v). The results are shown in Figure 7 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0118] Example 8: Preparation of low electroendosmosis agarose (carbon nanotubes + sodium persulfate + urea)

[0119] It includes the following steps:

[0120] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 16% (w / v); the persulfate is sodium persulfate at 0.015 mmol / L, and the first promoter is carbon nanotubes at 0.8 g / L; the reaction conditions are: pH 11, 60 °C, ultraviolet intensity 50 mW / cm 2 , react for 2 h;

[0121] Step 2: The capturer is methanol at 4 mol / L and reacts for 1 h; SOD is 30 U / mL and reacts for 25 min;

[0122] Step 3: The concentration of the high electroendosmosis agarose suspension is 16% (w / v); the monopersulfate is sodium bisulfate at 4 mmol / L, and the second promoter is urea at 5 mmol / L; the reaction conditions are: pH 8, 35 °C, react for 1.5 h;

[0123] Step 4: L-histidine at 4 mmol / L reacts for 45 min.

[0124] The prepared low electroendosmosis agarose is tested: the sulfate group content is 0.12%, the electroendosmosis is 0.12, and the gel strength (1.5%, w / v) is 1442 g / cm 2 , and the yield is 93.6%;

[0125] The prepared low electroendosmosis agarose is made into an electrophoresis gel for electrophoresis. The concentration of the agarose is 1.5% (w / v), and the results are shown in Figure 8 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0126] Example 9: Preparation of low electroendosmosis agarose (biochar + potassium persulfate + TiO2)

[0127] It includes the following steps:

[0128] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 14% (w / v); the persulfate is potassium persulfate at 0.005 mmol / L, and the first promoter is biochar at 3.0 g / L; the reaction conditions are: pH 9, 70 °C, ultraviolet intensity 90 mW / cm 2 , react for 3 h;

[0129] Step 2: The capturer is tert-butanol at 0.4 mol / L and reacts for 1.5 h; p-benzoquinone at 180 μmol / L reacts for 45 min;

[0130] Step 3: The concentration of the high electroendosmosis agarose suspension is 14% (w / v); the persulfate is 5 mmol / L of ammonium monopersulfate, and the second promoter is 1 g / L of TiO2; the reaction conditions are: pH 5, 40 °C, and the reaction is carried out for 0.5 h;

[0131] Step 4: React with 5 mmol / L of L-histidine for 60 min.

[0132] The prepared low electroendosmosis agarose was tested: the sulfuric acid group content was 0.14%, the electroendosmosis was 0.14, and the gel strength (1.5%, w / v) was 1295 g / cm 2 , and the yield was 92.6%;

[0133] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v), and the results are shown in Figure 9 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

[0134] Example 10: Preparation of low electroendosmosis agarose (graphene + potassium persulfate + rose bengal)

[0135] It includes the following steps:

[0136] Step 1: As in the above steps, specifically, the concentration of the agar suspension is 17% (w / v); the persulfate is 0.025 mmol / L of potassium persulfate, and the first promoter is 0.4 g / L of graphene; the reaction conditions are: pH 12, 65 °C, and the ultraviolet intensity is 100 mW / cm 2 , and the reaction is carried out for 2.5 h;

[0137] Step 2: React with 1 mol / L of methanol as the capture agent for 0.5 h; react with 80 U / mL of SOD for 15 min;

[0138] Step 3: The concentration of the high electroendosmosis agarose suspension is 17% (w / v); the persulfate is 5 mmol / L of potassium monopersulfate complex salt, and the second promoter rose bengal is 50 μmol / L; the reaction conditions are: pH 7, 35 °C, and the reaction is carried out for 2 h;

[0139] Step 4: React with 3 mmol / L of L-histidine for 45 min.

[0140] The prepared low electroendosmosis agarose was tested: the sulfuric acid group content was 0.10%, the electroendosmosis was 0.10, and the gel strength (1.5%, w / v) was 1550 g / cm 2 , and the yield was 94.8%;

[0141] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v). The results are shown in Figure 10 . It can be seen that this agarose can effectively separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0142] Comparative Example 1:

[0143] Step parameters: In Step 1, only 0.015 mmol / L of sodium persulfate was added, and no first promoter was added.

[0144] Others were the same as in Example 1.

[0145] Test indicators: The sulfonic acid group content was 0.29%, the electroendosmosis was 0.27, the gel strength (1.5%, w / v) was 1320 g / cm 2 , and the yield was 91.5%;

[0146] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v). The results are shown in Figure 11 . It can be seen that this agarose can separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, but there is a trailing phenomenon starting from 750bp.

[0147] Comparative Example 2: Omit the persulfate treatment in Step 4

[0148] Step parameters:

[0149] In Step 3, only a high electroendosmosis agarose suspension was used, without persulfate and promoter;

[0150] The remaining steps were the same as in Example 1.

[0151] Test indicators: The sulfonic acid group content was 0.38%, the electroendosmosis was 0.32, the gel strength (1.5%, w / v) was 1255 g / cm 2 , and the yield was 92.3%;

[0152] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of the agarose was 1.5% (w / v). The results are shown in Figure 12 . It can be seen that this agarose can separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, but there is a trailing phenomenon starting from 750bp.

[0153] Comparative Example 3: Using methanol to replace L-histidine

[0154] Step parameters:

[0155] In step 4, replace L-histidine with 5 mol / L methanol;

[0156] The remaining steps are the same as those in Example 1.

[0157] Test indicators: Sulfate group content 0.15%, electroendosmosis 0.15, gel strength (1.5%, w / v) 1185 g / cm 2 , yield 89.2%;

[0158] Prepare the prepared low electroendosmosis agarose into an electrophoresis gel for electrophoresis. The concentration of agarose is 1.5% (w / v). The results are shown in Figure 13 . It can be seen that this agarose can separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, but there is a trailing phenomenon starting from 1000 bp.

[0159] Comparative Example 4: The addition amount of persulfate exceeds the scope of the claims

[0160] Step parameters:

[0161] In step 1, the addition amount of sodium persulfate is 0.03 mmol / L (exceeding 0.005 - 0.025 mmol / L);

[0162] The remaining steps are the same as those in Example 1.

[0163] Test indicators: Sulfate group content 0.14%, electroendosmosis 0.15, gel strength (1.5%, w / v) 1123 g / cm 2 (Over-oxidation causes damage to the agarose structure), yield 88.8%;

[0164] Prepare the prepared low electroendosmosis agarose into an electrophoresis gel for electrophoresis. The concentration of agarose is 1.5% (w / v). The results are shown in Figure 14 . It can be seen that this agarose can separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, but there is a trailing phenomenon starting from 1000 bp.

[0165] Comparative Example 5:

[0166] Step parameters:

[0167] In step 3, the reaction pH is 4;

[0168] The remaining steps are the same as those in Example 1.

[0169] Test indicators: The sulfate group content is 0.26%, the electroendosmosis is 0.24 (too strong acidity inhibits the generation of singlet oxygen), and the gel strength (1.5%, w / v) is 1255 g / cm 2 , and the yield is 92.3%;

[0170] The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of agarose was 1.5% (w / v). The results are shown in Figure 15 . It can be seen that this agarose can separate DNA with molecular weights of 5000 bp, 3000 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, but there is a trailing phenomenon starting from 1500 bp.

[0171] Comparative Example 6:

[0172] Step 1: The concentration of the agar suspension is 10% (w / v);

[0173] Step 2: Hydrogen peroxide is 2% (v / v), the reaction temperature is 40 °C, the reaction pH is 9.0, and the reaction is carried out for 2 h; after the reaction is completed, it is washed with deionized water until there is no residual hydrogen peroxide, then filtered and dried.

[0174] Step 3: The concentration of the agar suspension is 10% (w / v);

[0175] Step 4: Hydrogen peroxide is 2% (v / v), the reaction temperature is 40 °C, the reaction pH is 9.0, and the reaction is carried out for 2 h; after the reaction is completed, it is washed with deionized water until there is no residual hydrogen peroxide, then filtered and dried.

[0176] Step 5: The concentration of the agar suspension is 10% (w / v);

[0177] Step 6: Hydrogen peroxide is 2% (v / v), the reaction temperature is 40 °C, the reaction pH is 9.0, and the reaction is carried out for 2 h; after the reaction is completed, it is washed with deionized water until there is no residual hydrogen peroxide, then filtered and dried.

[0178] Test indicators: The sulfate group content is 0.1%, the electroendosmosis is 0.1, and the gel strength (1.5%, w / v) is 135 g / cm 2 , and the yield is 52%;

[0179] In this example, hydroxyl radicals were used for cyclic desulfurization multiple times. The prepared low electroendosmosis agarose was made into an electrophoresis gel for electrophoresis. The concentration of agarose was 1.5% (w / v). The results are shown in Figure 16It can be seen that this agarose can separate DNA with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. However, affected by the low gel strength, the gel is close to the melting state, and DNA diffusion results in a high electrophoresis background.

[0180] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A preparation method of low electroendosmosis agarose, characterized in that, It includes the following steps: Step 1: Add persulfate to the agar suspension, add the first promoter and react, adjust the reaction pH, reaction temperature and ultraviolet intensity, and react to generate sulfate radicals and superoxide anions; Step 2: Add a scavenger to the above reaction system to react to remove excessive sulfate radicals, then add p-benzoquinone or superoxide dismutase to react, and wash and filter the reacted agar suspension multiple times to obtain high electroendosmosis agarose; preferably, the scavenger is methanol or tert-butanol; Step 3: Redisperse the obtained high electroendosmosis agarose in water to obtain a high electroendosmosis agarose suspension; sequentially add peroxysulfate and the second promoter to the obtained high electroendosmosis agarose suspension to react, adjust the reaction pH and temperature, and react to generate singlet oxygen; preferably, the second promoter is any one of benzoquinone, phenol, TiO2, rose bengal, urea; Step 4: Add L-histidine to the above reaction system to quench singlet oxygen, and wash and filter the reacted agarose suspension multiple times to obtain low electroendosmosis agarose.

2. The preparation method of a low electroendosmosis agarose according to claim 1, wherein, In Step 1, the mass concentration of the agar suspension is 5% - 20% w / v.

3. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that, In Step 1, the persulfate is at least one of sodium persulfate, potassium persulfate, ammonium persulfate; the addition amount of persulfate is 0.005 - 0.025 mmol / L; the first promoter is any one of graphene, carbon nanotubes, biochar; the addition amounts of graphene, carbon nanotubes or biochar are 0.1 - 0.5 g / L, 0.2 - 1.0 g / L, 0.5 - 5.0 g / L respectively.

4. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that, In Step 1, the reaction pH is 9 - 12, the reaction temperature is 50 - 70 °C, the ultraviolet intensity is 20–100 mW / cm 2 , and the reaction time is 1 - 3 h.

5. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that, In Step 2, the addition amounts of methanol or tert-butanol are 1 - 5 mol / L, 0.05 - 0.5 mol / L respectively; the reaction time after adding the scavenger is 0.5 - 2 h, the addition amounts of p-benzoquinone or superoxide dismutase are 50 - 200 μmol / L, 1 - 100 U / mL respectively; the reaction time after adding p-benzoquinone or superoxide dismutase is 15 - 60 min.

6. The preparation method of a low electroendosmosis agarose according to claim 1, wherein, In Step 3, the concentration of the high electroendosmosis agarose suspension is 5% - 20% w / v, the peroxysulfate is potassium peroxymonosulfate compound salt, sodium peroxymonosulfate, ammonium peroxymonosulfate; the addition amount of peroxysulfate is 1 - 5 mmol / L.

7. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that, In Step 3, the addition amounts of any one of benzoquinone, phenol, TiO2, rose bengal, urea are 50 - 200 μmol / L, 1 - 5 mmol / L, 1 - 3 g / L, 10 - 50 μmol / L, 1 - 5 mmol / L respectively; The reaction pH is 5 - 9, the reaction temperature is 30 - 40 °C, and the reaction time is 0.5 - 2 h.

8. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that In Step 4, the addition amount of L-histidine is 1 - 5 mmol / L, and the reaction time is 30 - 60 min.

9. The preparation method of a low electroendosmosis agarose according to claim 1, characterized in that In Step 4, the prepared low electroendosmosis agarose satisfies any one of the following conditions: Sulfate group content < 0.15%; Electroendosmosis < 0.15; Gel strength at 1.5% > 1200 g / cm 2 ; Yield > 90%.

10. A low electroendosmosis agarose, characterized in that, It is prepared by the preparation method described in any one of claims 1 - 9.

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