A method for preparing a cationic-enzyme activity test paper by beating pretreatment

Abstract

The present invention discloses a method for preparing a cationic - enzyme activity test paper by beating pretreatment. The method comprises the following steps: (1) The pulp board wetted with water is disintegrated and dispersed at a concentration of 1 - 8 wt% to obtain disintegrated pulp I; (2) The concentration of the disintegrated pulp I is adjusted to 3 - 18 wt%, and it is added to a PFI refiner for beating pretreatment to obtain beaten pretreatment pulp I; (3) A sodium hydroxide solution and a (3 - chloro - 2 - hydroxypropyl) trimethyl ammonium chloride solution are mixed and stirred at room temperature to obtain mixed solution I; (4) The concentration of the beaten pretreatment pulp I is adjusted to 1 - 15 wt%, and the mixed solution I is added to obtain cationic pulp fibers I; (5) The cationic pulp fibers I, the disintegrated pulp I, and a wet strength agent are mixed and stirred, and then sheet - formed, and after drying, a cationic - enzyme activity test paper sheet is obtained. The method of the present invention has the advantages of simple process, low cost, environmental protection, high applicability, and good enzyme activity detection effect, etc.

Description

Technical Field

[0001] The present invention relates to a method for preparing a cationic - enzyme activity test paper by beating pretreatment, belonging to the field of biomass - based functional paper materials. Background Art

[0002] In recent years, some cationic polymers with medium or high charge density have been widely used in the papermaking process to improve the retention rate of fillers and fines, and to promote the formation of strong binding forces between pulp fibers to improve the strength properties of paper. However, due to environmental protection and resource utilization factors, cationic polyelectrolytes are highly toxic to fish. These polymers will bind to the negatively charged gill surface and hinder respiration, seriously affecting the living environment of aquatic organisms. The use of biomass - based polymers to replace chemical synthesis products has received increasing attention. In addition, biomass - based cationic derivatives also have certain application prospects in the fields of paper functional materials, wastewater treatment, and molecular cell biology.

[0003] Plant fiber raw materials, as extremely abundant renewable plant resources in nature, have cellulose, hemicellulose, and a small amount of lignin as their main chemical components. For different types of paper with different required properties, an appropriate amount of hemicellulose in the pulp is retained during pulping. The presence of hemicellulose is beneficial to the beating of pulp fibers, and it promotes the increase of the accessibility of pulp fibers and the improvement of paper properties (such as tearing strength, tensile strength, etc.). However, the highly regular structure of pulp fibers makes them not easily hydrolyzed by chemical reagents. Affected by factors such as the molecular structure, aggregated state structure, and fiber morphological structure of pulp fibers, the functionalizable chemical groups are generally difficult to reach. Usually, only regions such as the amorphous region of the fiber and the surface of partially accessible crystalline regions are easy to react with reaction reagents, and the fiber accessibility is relatively low. Therefore, it is particularly important to adopt appropriate pretreatment methods to improve the accessibility of pulp fibers, enhance the reaction performance of pulp fibers, and increase the modification efficiency of pulp fiber cationization. However, beating pretreatment can not only promote the water absorption and swelling of pulp fibers, the fibrillation and fibrillation of fibers, but also destroy the fiber crystalline region and reduce the fiber crystallinity, exposing more free hydroxyl groups on the fiber, increasing fiber accessibility. The fibrillation of fibers is beneficial to the binding of fibers, increasing the binding force between fibers, improving the strength properties such as the wet strength of the paper sheet, and having the potential to prepare paper sheets with high wet strength, strong absorbency, and high cationicity, which has a good promoting effect on the research of enzyme activity test paper.

[0004] Regarding the characteristics of cationic fibers, they are currently mainly applied in fields such as papermaking, textiles, and wastewater treatment, and no one has studied them in the field of molecular cell biology. Enzyme activity test paper is a positively charged functional paper in the field of molecular cell biology that can detect the activity of biological enzymes. Biological enzymes are extremely important biological catalysts. Under the efficient catalysis of enzymes, chemical reactions in living organisms can proceed efficiently and specifically under extremely mild conditions. Therefore, a functional paper material that can detect the activity of biological enzymes in a timely manner is of great significance in the field of molecular cell biology. Currently, enzyme activity test paper is prepared by mixing cationic resin and chemical pulp fibers in a certain proportion and stirring evenly to form a cationic mixed suspension, and then using wet preparation technology to sheet the mixed suspension to obtain a cellulose functional paper with ion exchange ability. However, the production process of this enzyme activity test paper is relatively cumbersome and complex, with a high production cost, and the finished product effect is somewhat lacking. We consider using the method of cationic modification of pulp fibers to prepare cationic pulp fibers, and then using wet preparation technology to sheet pure cationic pulp fibers to obtain a cationic fiber functional paper made of pure fibers. Therefore, a green and efficient preparation process for enzyme activity test paper has important research significance and application value in the field of molecular cell biology. Summary of the Invention

[0005] The present invention uses softwood pulp fibers as raw materials, improves the accessibility of the fibers through beating pretreatment, then obtains cationic fibers after etherification graft modification, and finally obtains a cationic-enzyme activity test functional paper by sheet making the cationic fibers in different proportions to solve the above problems.

[0006] On the one hand, the present invention provides a method for preparing a cationic-enzyme activity test paper using beating pretreatment, and the method includes the following steps:

[0007] (1) Add the pulp board fully wetted with water to a fiber defibrator, defibrate and disperse it at a concentration of 1-8 wt% for 10-30 min to obtain defibrated pulp Ⅰ;

[0008] (2) Adjust the concentration of defibrated pulp Ⅰ to 3-18 wt% to obtain defibrated pulp Ⅱ, add defibrated pulp Ⅱ to a PFI refiner and perform beating pretreatment at 2000-8000 revs to obtain beaten pretreatment pulp Ⅰ;

[0009] (3) Mix and stir a sodium hydroxide solution and a (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride solution at room temperature for 3-20 min to obtain mixed solution Ⅰ;

[0010] (4) Adjust the concentration of the beaten and pretreated pulp I to 1-15 wt%, obtaining the beaten and pretreated pulp II. Add the mixed solution I to the beaten and pretreated pulp II to obtain the mixture I. Heat the mixture I at 25-90 °C for 30-360 min. After the reaction ends, let it stand for 10-45 min, and then wash, filter, and dry to obtain the cationic pulp fiber I;

[0011] (5) Mix and stir the cationic pulp fiber I, the disintegrated pulp I, and the wet strength agent for 1-20 min to obtain the mixed pulp. Make the mixed pulp into a sheet by papermaking and dry it to obtain the cationic-enzyme activity test paper sheet.

[0012] In the above technical solution, further, in step (1), the pulp sheet is a softwood pulp sheet.

[0013] In the above technical solution, further, the molar ratio of (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride to the pulp sheet is 0.5-6:1, where the molar amount of the pulp sheet is calculated based on the anhydroglucose unit AGU.

[0014] In the above technical solution, further, in step (3), in the mixed solution I, the molar ratio of sodium hydroxide to (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride is 0.5-5:1.

[0015] In the above technical solution, further, in step (4), the washing uses a mixed solution of ethanol and water with a mass ratio of 1:1.

[0016] In the above technical solution, further, in step (5), the cationic pulp fiber I accounts for 15-100% of the total mass of the cationic pulp fiber I and the disintegrated pulp I.

[0017] In the above technical solution, further, in step (5), the mass ratio of the wet strength agent to the pulp sheet is 0-30 kg:1 t.

[0018] In the above technical solution, further, the wet strength agent is polyamide epichlorohydrin resin PAE.

[0019] On the other hand, the present invention provides a cationic-enzyme activity test paper prepared by the method of preparing a cationic-enzyme activity test paper using beating pretreatment as described above.

[0020] The present invention also provides an application of the cationic-enzyme activity test paper in testing the enzyme activity of rTaq DNA polymerase.

[0021] The beneficial effects of the present invention are:

[0022] The raw materials of the present invention are widely sourced. The method of beating pretreatment is used to improve the accessibility of pulp fibers while promoting the formation of strong binding forces between pulp fibers to enhance the strength properties of paper. A green and efficient method of physically and chemically activating etherification reagents is adopted to increase the grafting rate of direct cationic modification of pulp fibers, preparing a cationic-enzyme activity test paper with high cationicity, high wet strength, and excellent selective absorbency, and capable of efficiently detecting the activity of rTaq DNA polymerase widely used in the field of molecular cell biology. It is a preparation method of special paper-based functional materials with simple production process, low cost, environmental friendliness, and excellent performance. Description of the Drawings

[0023] Figure 1 It is a scatter plot of the enzyme activity test results of Example 1. a is 15wt% cationic fiber, b is 20wt% cationic fiber, and c is 25wt% cationic fiber;

[0024] Figure 2 It is a scatter plot of the enzyme activity test results of Example 2. a is 50wt% cationic fiber, b is 75wt% cationic fiber, and c is 100wt% cationic fiber;

[0025] Figure 3 It is a scatter plot of the enzyme activity test results of Example 3. a is 25wt% cationic fiber + 15kg / t PAE, and b is 50wt% cationic fiber + 15kg / t PAE;

[0026] Figure 4 It is a scatter plot of the enzyme activity test results of Example 4. a is 4500 revs + 15kg / t PAE, b is 5000 revs + 15kg / t PAE, c is 4500 revs + 30kg / t PAE, and a is 5000 revs + 30kg / t PAE. Detailed Description of the Invention

[0027] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0028] The following pulp fibers are softwood pulp boards with a moisture content of 5.71%;

[0029] The following CHPTAC solution is an aqueous solution of CHPTAC with a mass fraction of 65%.

[0030] Example 1

[0031] (1) Add 150 g of pulp board fully wetted with water to a fiber defibrator, and defibrate and disperse it at a concentration of 4wt% for 15 min to obtain uniformly dispersed defibrated pulp I;

[0032] (2) Adjust the concentration of the defibrated pulp I to 10 wt% to obtain the defibrated pulp II. Add the defibrated pulp II to a PFI refiner and perform beating pretreatment at 4000 revs to obtain the beaten pretreatment pulp I;

[0033] (3) Mix and stir the sodium hydroxide solution and the (3-chloro-2-hydroxypropyl) trimethylammonium chloride (CHPTAC) solution at room temperature for 10 min to obtain the mixed solution I;

[0034] (4) Adjust the concentration of the beaten pretreatment pulp I to 3 wt% to obtain the beaten pretreatment pulp II. Add the mixed solution I to the beaten pretreatment pulp II with a concentration of 3 wt% to form the mixture I. Heat the mixture I at 70 °C for 120 min. After the reaction ends, let it stand for 30 min first, then wash and filter it with an ethanol-water mixed solution with a mass ratio of 1:1, and dry it to obtain the cationic pulp fiber I;

[0035] (5) Mix and stir the cationic pulp fiber I with the defibrated pulp I at ratios of 15%, 20%, and 25% of the total mass of the cationic pulp fiber I and the defibrated pulp I for 5 min to obtain the mixed pulp I. Add the mixed pulp I to a rapid Kajaani sheet former for sheet forming, and dry it to obtain the cationic-enzyme activity test sheet.

[0036] Test the enzyme activity of rTaq DNA polymerase:

[0037] Add rTaq DNA polymerase to the 3 dNTP substrate mixed solution labeled with ³H-dTTP to catalyze the synthesis of a large molecular double-stranded DNA labeled with the isotope tritium ( 3 ³H) to form a mixed solution containing 3 ³H-DNA; Drop the mixed solution containing 3 ³H-DNA onto the surface of the cationic-enzyme activity test sheet. After the reaction ends, obtain the enzyme activity test paper; Rinse the enzyme activity test paper in three steps: the first step is sodium hydrogen phosphate (Na₂HPO₄), the second step is alcohol (C₂H₅OH), and the third step is water (H₂O). Then put it into a liquid scintillation counter to detect the radioactive ΔCPM value of the test paper. According to the definition of enzyme activity, complete the activity determination of rTaq DNA polymerase from the ΔCPM value of the test paper.

[0038] Two groups of parallel experiments were carried out for the determination, and the results are shown in Table 1 and Figure 1 as follows.

[0039] Table 1 Enzyme activity test results of the cationic-enzyme activity test paper in Example 1

[0040]

[0041]

[0042] As shown in Table 1, with the increase of the blending ratio of cationic fibers, the ΔCPM value of the test paper and the activity of DNA polymerase increase, indicating that the wet strength and absorbency of the test paper will increase with the increase of the addition amount of cationic fibers. From Figure 1 it can be seen that in the use of cation-enzyme activity test paper, there is a strong linear correlation between the dilution amount of DNA polymerase and the radioactive ΔCPM, indicating that the test paper has good applicability for the detection of DNA polymerase activity.

[0043] Example 2

[0044] (1) Add 150 g of pulp board fully wetted with water to a fiber disperser, and disperse it at a concentration of 4 wt% for 15 min to obtain uniformly dispersed pulp I;

[0045] (2) Adjust the concentration of pulp I to 10 wt% to obtain pulp II. Add pulp II to a PFI refiner for 4000 revs of beating pretreatment to obtain pretreated beaten pulp I;

[0046] (3) Mix and stir the sodium hydroxide solution and (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) solution at room temperature for 10 min to obtain mixed solution I;

[0047] (4) Adjust the concentration of pretreated beaten pulp I to 3 wt% to obtain pretreated beaten pulp II. Add mixed solution I to pretreated beaten pulp II with a concentration of 3 wt% to form mixture I. Heat mixture I at 70 °C for 120 min. After the reaction, let it stand for 30 min first, then wash and filter with an ethanol-water mixed solution with a mass ratio of 1:1, and dry to obtain cationic pulp fiber I;

[0048] (5) Mix and stir cationic pulp fiber I with pulp I at ratios of 50%, 75%, and 100% of the total mass of cationic pulp fiber I and pulp I for 5 min to obtain mixed pulp I. Add mixed pulp I to a rapid Kajaani paper former for papermaking, and dry to obtain cation-enzyme activity test paper.

[0049] Using the same test method as in Example 1, two sets of parallel experiments were carried out for determination, and the determination results are shown in Table 2 and Figure 2 as follows.

[0050] Table 2 Enzyme activity test results of the enzyme activity test paper in Example 2

[0051]

[0052]

[0053] As can be seen from Table 2, the integrity of the cationic paper sheet is very good under this condition, providing good assurance for the detection of enzyme activity. As the dosage of cationic fiber increases, the radioactive ΔCPM value of the enzyme activity test paper sheet also increases. When 100% cationic fiber is used, the radioactive ΔCPM value detected from the sample paper and the activity of DNA polymerase are the highest. From Figure 2 it can be seen that as the dosage of cationic fiber increases, the linear correlation between the dilution dosage of DNA polymerase and the radioactivity ΔCPM also reaches its peak when 100% cationic fiber is used. All these conclusions indicate that by using the present invention, the paper sheet can have high wet strength and strong absorbability for nucleic acids without using wet strength agents. The excellent properties of the paper sheet of the present invention have good applicability and practicality for the detection of DNA polymerase activity.

[0054] Example 3

[0055] (1) Add 150 g of pulp board fully wetted with water to a fiber defibrator, and defibrate and disperse it at a concentration of 4 wt% for 15 min to obtain uniformly dispersed defibrated pulp Ⅰ;

[0056] (2) Adjust the concentration of defibrated pulp Ⅰ to 10 wt% to obtain defibrated pulp Ⅱ. Add defibrated pulp Ⅱ to a PFI refiner at a concentration of 10 wt% and perform beating pretreatment for 4000 revs to obtain pretreated beaten pulp;

[0057] (3) Mix and stir a sodium hydroxide solution and a (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) solution at room temperature for 10 min to obtain mixed solution Ⅰ;

[0058] (4) Adjust the concentration of pretreated beaten pulp Ⅰ to 3 wt% to obtain pretreated beaten pulp Ⅱ. Add mixed solution Ⅰ to pretreated beaten pulp Ⅱ at a concentration of 3% to form mixture Ⅰ. Heat mixture Ⅰ at 70 °C for 120 min. After the reaction ends, let it stand for 30 min first, then wash, filter with ethanol-water with a mass ratio of 1:1, and dry to obtain cationic pulp fiber Ⅰ;

[0059] (5) Mix and stir cationic pulp fiber Ⅰ with defibrated pulp Ⅰ and PAE at a ratio of 25% and 50% of the total mass of cationic pulp fiber Ⅰ and defibrated pulp Ⅰ for 30 min to obtain mixed pulp Ⅰ. Add mixed pulp Ⅰ to a rapid Kajaani paper sheet former for sheet forming, and dry to obtain a cationic - enzyme activity test paper sheet.

[0060] Using the same test method as in Example 1, two sets of parallel experiments were carried out for determination, and the determination results are shown in Table 3 and Figure 3 as follows.

[0061] Table 3 Enzyme Activity Test Results of Cation-Enzyme Activity Test Paper in Example 3

[0062]

[0063] As can be seen from Table 3, in terms of the addition amount of cationic fibers, the higher the content of cationic fibers in the cation-enzyme activity test paper sheet, the higher the radioactive ΔCPM value of the test paper sheet and the activity of the detected DNA polymerase. From Figure 3 it can be seen that after adding the wet strength agent PAE, with the increase in the dosage of cationic fibers, the linear correlation of the enzyme activity test paper sheet also increases, which further demonstrates the importance of the dosage of cationic fibers in the enzyme activity test.

[0064] Example 4

[0065] (1) Add 150 g of pulp board fully wetted with water to a fiber defibrator, defibrate and disperse it at a concentration of 4% for 15 min to obtain uniformly dispersed defibrated pulp I;

[0066] (2) Adjust the concentration of defibrated pulp I to 10 wt% to obtain defibrated pulp II. Add defibrated pulp II to a PFI refiner at a concentration of 10% and perform beating pretreatment at 4500 revs and 5000 revs to obtain beaten pretreatment pulp I;

[0067] (3) Mix and stir the sodium hydroxide solution and (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) solution at room temperature for 10 min to obtain mixed solution III;

[0068] (4) Adjust the concentration of beaten pretreatment pulp I to 3 wt% to obtain beaten pretreatment pulp II. Add mixed solution I to beaten pretreatment pulp I with a concentration of 3% to form mixture I. Heat mixture I at 70 °C for 120 min. After the reaction ends, let it stand for 30 min first, then wash, filter with ethanol-water with a mass ratio of 1:1, and dry to obtain cationic pulp fiber I;

[0069] (5) Mix and stir cationic pulp fiber I and PAE for 30 min to obtain mixed pulp. Add the mixed pulp to a rapid Kajaani paper sheet former for sheet forming, and dry to obtain cation-enzyme activity test paper sheet.

[0070] Using the same test method as in Example 1, two groups of parallel experiments were carried out for determination, and the determination results are shown in Table 4 and Figure 4 as follows.

[0071] Table 4 Enzyme Activity Test Results of Cation-Enzyme Activity Test Paper in Example 4

[0072]

[0073]

[0074] As can be seen from Table 4, as the beating revolutions increase, the radioactive ΔCPM value of the test paper also increases. Under the action of PAE, the test paper sheet has excellent wet strength, ensuring the integrity and accuracy of the paper sheet during the detection process. However, from the four groups of data, the radioactive ΔCPM value of the test paper is relatively low, and the detected DNA polymerase activity is not high either. This indicates that when PAE is added, while the test paper sheet has high wet strength, its absorbability for nucleic acids is reduced. From Figure 4 it can be seen that although the addition of PAE further increases the linear correlation between the dilution dosage of DNA polymerase and the radioactive ΔCPM, it also inhibits the absorbability of the test paper sheet for nucleic acids.

[0075] The above embodiments are only the preferred embodiments of the present invention and are not limitations on the implementation manners. The protection scope of the present invention should be subject to the scope defined by the claims. Based on the above description, other different forms of changes or variations can also be made. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing a cationic-enzyme activity test paper by beating pretreatment, which is characterized in that: The method includes the following steps: (1) Add the wet pulp board with water to a fiber defibrator, defibrate and disperse it at a concentration of 1-8 wt% for 10-30 min to obtain defibrated pulp I; (2) Adjust the concentration of defibrated pulp I to 3-18 wt% to obtain defibrated pulp II. Add defibrated pulp II to a PFI refiner and perform beating pretreatment at 2000-8000 revs to obtain beaten pretreatment pulp I; (3) Mix and stir a sodium hydroxide solution and a (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride solution at room temperature for 3-20 min to obtain mixed solution I; (4) Adjust the concentration of beaten pretreatment pulp I to 1-15 wt% to obtain beaten pretreatment pulp II. Add mixed solution I to beaten pretreatment pulp II to obtain mixture I. Heat mixture I at 25-90 °C for 30-360 min. After the reaction ends, let it stand for 10-45 min, then wash, filter, and dry to obtain cationic pulp fiber I; (5) Mix and stir cationic pulp fiber I, defibrated pulp I, and a wet strength agent for 1-20 min to obtain mixed pulp. Sheet the mixed pulp and dry it to obtain a cationic-enzyme activity test paper sheet; The molar ratio of the (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride to the pulp board is 0.5-6:1, where the molar amount of the pulp board is calculated based on anhydroglucose unit (AGU); The wet strength agent is polyamide epichlorohydrin resin (PAE).

2. The method for preparing a cationic - enzyme activity test paper by using beating pretreatment according to claim 1, wherein: In step (1), the pulp board is a softwood pulp board.

3. The method for preparing a cationic - enzyme activity test paper by using beating pretreatment according to claim 1, wherein: In step (3), in the mixed solution I, the molar ratio of sodium hydroxide to the (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride is 0.5-5:

1.

4. The method for preparing a cationic - enzyme activity test paper by using beating pretreatment according to claim 1, wherein: In step (4), the washing uses a mixed solution of ethanol and water with a mass ratio of 1:

1.

5. The method for preparing a cationic-enzyme activity test paper by using beating pretreatment according to claim 1, wherein: In step (5), the cationic pulp fiber I accounts for 15-100% of the total mass of the cationic pulp fiber I and the defibrated pulp I.

6. The method for preparing a cationic-enzyme activity test paper by using beating pretreatment according to claim 1, wherein: In step (5), the mass ratio of the wet strength agent to the pulp board is 0-30 kg:1 t.

7. A cationic-enzyme activity test paper prepared by the method for preparing a cationic-enzyme activity test paper using beating pretreatment according to any one of claims 1-6.

8. An application of the cationic-enzyme activity test paper according to claim 7 in testing the enzyme activity of rTaq DNA polymerase.

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