CN116041779B - PH response type macroporous hydrogel loaded with nucleic acid lysate, and preparation method and application thereof - Google Patents
PH response type macroporous hydrogel loaded with nucleic acid lysate, and preparation method and application thereof Download PDFInfo
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 102
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 56
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 56
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 56
- 239000006166 lysate Substances 0.000 title claims abstract description 46
- 230000004044 response Effects 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 47
- 229920001661 Chitosan Polymers 0.000 claims abstract description 46
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 44
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 44
- 239000000661 sodium alginate Substances 0.000 claims abstract description 44
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 44
- 239000011259 mixed solution Substances 0.000 claims abstract description 41
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 26
- 238000002791 soaking Methods 0.000 claims abstract description 22
- 238000004108 freeze drying Methods 0.000 claims abstract description 21
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims description 55
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical group [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 19
- 239000001110 calcium chloride Substances 0.000 claims description 13
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 12
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- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
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Abstract
The invention provides a pH response type macroporous hydrogel loaded with nucleic acid lysate, and a preparation method and application thereof, wherein the preparation method comprises the following steps: step 1, dispersing carboxymethyl chitosan, sodium alginate and polyethylene glycol in water to obtain a mixed solution; step 2, adding a cross-linking agent into the mixed solution, solidifying to obtain hydrogel, washing, and freeze-drying to obtain freeze-dried hydrogel; and 3, soaking the freeze-dried hydrogel in the nucleic acid lysate, and taking out the freeze-dried hydrogel after the soaking is finished to obtain the pH response type macroporous hydrogel loaded with the nucleic acid lysate. The hydrogel carrier prepared by the method can achieve compression deformation of 60-80%, has good toughness and stability, and the hydrogel has a three-dimensional network porous structure inside, and has potential advantages as a nucleic acid lysate carrier. The invention overcomes the defects of inconvenient carrying, high cost, time consumption, environmental protection and the like existing in the prior nucleic acid lysate storage.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a pH response type macroporous hydrogel loaded with nucleic acid lysate, and a preparation method and application thereof.
Background
Nucleic acid detection has been widely used in various fields such as disease diagnosis, food safety detection and environmental monitoring as a technique with good specificity and high sensitivity. The traditional laboratory detection method has the defects of high cost, complex operation, high requirement on samples, time consumption, portability and the like, and is difficult to apply to places with limited resources. Compared with the method, point-of-CARE TESTING (POCT) is taken as a detection technology with low cost, simple and convenient operation, rapidness, portability and no need of professional operators, can realize detection in a short time in the fields of families, bedside, clinics and the like, and has definite practicability in developing the research of the rapid and simple extraction technology of nucleic acid. The rapid extraction of nucleic acid is a first step for realizing rapid detection of nucleic acid, and the standard rapid extraction process of nucleic acid comprises sample cleavage, washing and elution, wherein sample cleavage is the most important step in rapid extraction of nucleic acid, but different cleavage reagents need different storage and operation methods, and at present, the rapid extraction of nucleic acid usually needs a brown reagent bottle for low-temperature and light-proof storage, and has the defects of inconvenient carrying, complex operation, high cost, time consumption, environmental protection and the like. Therefore, it is important to develop a method for storing a nucleic acid cleavage reagent, which is easy to carry, simple to operate, low in cost and environment-friendly.
Disclosure of Invention
The invention aims to provide a pH response type macroporous hydrogel loaded with nucleic acid lysate, a preparation method and application thereof, and the material can be used as a carrier for storing the nucleic acid lysate to realize the release of the nucleic acid lysate.
The invention is realized by the following technical scheme:
a preparation method of pH response type macroporous hydrogel loaded with nucleic acid lysate comprises the following steps:
step1, dispersing carboxymethyl chitosan, sodium alginate and polyethylene glycol in water to obtain a mixed solution;
Step 2, adding a cross-linking agent into the mixed solution, solidifying to obtain hydrogel, washing, and freeze-drying to obtain freeze-dried hydrogel;
And 3, soaking the freeze-dried hydrogel in the nucleic acid lysate, and taking out the freeze-dried hydrogel after the soaking is finished to obtain the pH response type macroporous hydrogel loaded with the nucleic acid lysate.
Preferably, the step 1 specifically comprises: preparing carboxymethyl chitosan aqueous solution and sodium alginate aqueous solution respectively, adding the carboxymethyl chitosan aqueous solution into the sodium alginate aqueous solution, mixing, adding polyethylene glycol, and mixing to obtain a mixed solution.
Preferably, in step 1, the carboxymethyl chitosan has a degree of carboxymethyl of 80% or more.
Preferably, in step 1, the polyethylene glycol has a molecular weight of 400-6000MW.
Preferably, in the step 1, the mass of the polyethylene glycol accounts for 1% -5% of the mass of the mixed solution.
Preferably, in the step 1, the mass ratio of the carboxymethyl chitosan to the sodium alginate to the polyethylene glycol is (1-3) to (1-5).
Preferably, in step 2, the crosslinking agent is calcium chloride.
Preferably, in step 3, the freeze-dried hydrogel is soaked in the nucleic acid lysate for 3-5 hours, and then taken out for freeze drying.
The pH response type macroporous hydrogel loaded with the nucleic acid lysate is obtained by the preparation method.
The pH response type macroporous hydrogel loaded with the nucleic acid lysate is applied to nucleic acid extraction.
Compared with the prior art, the invention has the following beneficial effects:
The carboxymethyl chitosan, the sodium alginate and the polyethylene glycol adopted by the preparation method are all linear polysaccharides which are wide in source, low in cost, safe and nontoxic, the crosslinking reaction can be completed by only adding the crosslinking agent in the preparation process, and the pH responsive macroporous hydrogel can be obtained by directly washing the surface superfluous crosslinking agent with deionized water after solidification, so that the preparation process is green and the operation is simple. The pH response type macroporous hydrogel carrying the nucleic acid lysate has good pH response, stable structure in acid and alkali environments, good swelling performance in neutral environments, and capability of releasing the nucleic acid lysate after swelling, so that the pH response type macroporous hydrogel can be used as an effective carrier of the nucleic acid lysate without being stored in a dark place. The hydrogel carrier prepared by the method can achieve compression deformation of 60-80%, has good toughness and stability, and the hydrogel has a three-dimensional network porous structure inside, and has potential advantages as a nucleic acid lysate carrier. The invention overcomes the defects of inconvenient carrying, high cost, time consumption, environmental protection and the like existing in the prior nucleic acid lysate storage, is a high-efficiency and convenient production mode, and has important social significance and application and popularization value.
Furthermore, the molecular weight of polyethylene glycol has a larger influence on the pore diameter of the hydrogel, and the invention selects proper molecular weight, so that the obtained hydrogel has proper macroporous structure, can be used as a carrier for loading nucleic acid lysate, and can ensure proper mechanical property.
Drawings
FIG. 1A is an SEM image of hydrogels obtained in comparative examples 1 (a), 2 (b), 3 (c), 1 (a), 2 (b), and 3 (c); b is the average pore diameter of the hydrogels obtained in comparative examples 1 to 3 (a) and examples 1 to 3 (B).
FIG. 2A shows the compression set properties of the hydrogels obtained in examples 1 to 3, and B shows the infrared spectra of the hydrogels obtained in examples 2 to 3.
The hydrogels of examples 1-3 in FIG. 3 had swelling properties at pH 1.2 (A), pH 7.4 (B) and pH 13 (C).
FIG. 4 shows the results of nucleic acid extraction from the hydrogels of examples 1-3.
Detailed Description
For a further understanding of the present invention, the present invention is described below in conjunction with the following examples, which are provided to further illustrate the features and advantages of the present invention and are not intended to limit the claims of the present invention.
The preparation method of the pH response type macroporous hydrogel loaded with the nucleic acid lysate comprises the following steps:
Step 1, preparing carboxymethyl chitosan (CMCS) aqueous solution;
step 2, preparing Sodium Alginate (SA) aqueous solution;
step 3, adding the carboxymethyl chitosan aqueous solution into the sodium alginate aqueous solution, rapidly stirring, and performing ultrasonic degassing to obtain a mixed solution 1;
step 4, adding a certain amount of polyethylene glycol into the mixed solution 1, rapidly stirring, and performing ultrasonic degassing to obtain a mixed solution 2;
Step 5, pouring the mixed solution 2 into a mould, adding a cross-linking agent, taking out the obtained hydrogel after solidification, flushing with deionized water, and freeze-drying for standby to obtain freeze-dried hydrogel;
And 6, soaking the freeze-dried hydrogel prepared in the step 5 in the nucleic acid lysate for several hours by a dry soaking method, and freeze-drying to obtain the pH-responsive macroporous hydrogel loaded with the nucleic acid lysate.
In step 1: the carboxymethyl chitosan is used with the carboxymethyl chitosan degree of carboxymethylation not less than 80%, the mass concentration of the carboxymethyl chitosan in the carboxymethyl chitosan water solution is preferably 3%, and the carboxymethyl chitosan is rapidly stirred for 1h and uniformly dispersed.
In step 2: the mass concentration of sodium alginate in the sodium alginate aqueous solution is preferably 3%, and the sodium alginate aqueous solution is rapidly stirred for 1h to be uniformly dispersed.
In step 3: in the stirring process, pouring the carboxymethyl chitosan aqueous solution into the sodium alginate aqueous solution, wherein the volume ratio of the carboxymethyl chitosan aqueous solution to the sodium alginate aqueous solution is preferably 1:2, stirring for 2 hours, and then performing ultrasonic degassing to form a uniform mixed solution.
In step 4: adding polyethylene glycol into the mixed solution 1, wherein the molecular weight of the selected polyethylene glycol is 6000MW, preferably the mass of the polyethylene glycol accounts for 5% of the total mass of the mixed solution 1, stirring, and performing ultrasonic degassing to form a uniform mixed solution. In the mixed solution, the mass ratio of carboxymethyl chitosan to sodium alginate to polyethylene glycol is 1:2:5.
In step 5: calcium chloride is selected as a cross-linking agent, the concentration of the prepared calcium chloride aqueous solution is preferably 5%, the cross-linking reaction temperature is 20-30 ℃ (room temperature), the curing is preferably carried out for 12 hours, deionized water is used for washing 5-6 times after the curing, and the freeze drying is carried out for 24 hours for standby.
In step 6: and (3) soaking the freeze-dried hydrogel prepared in the step (5) in the nucleic acid lysate for 3-5h by adopting a dry soaking method, and freeze-drying for 24h.
The pH response type macroporous hydrogel loaded with the nucleic acid lysate, which is prepared by the method, has the thickness of 1mm-5mm. It can be used in nucleic acid extraction.
Example 1
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
And 3, pouring the carboxymethyl chitosan aqueous solution prepared in the step 1 into the sodium alginate aqueous solution prepared in the step 2 in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
And 4, weighing 2g of polyethylene glycol with the molecular weight of 400, adding the polyethylene glycol into the mixed solution in the step 3, stirring for 1h, and performing ultrasonic degassing for 30min to form a uniform mixed solution, wherein the mass of the polyethylene glycol accounts for 5% of the total mass of the mixed solution.
And 5, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 5%, then carrying out a crosslinking reaction with the mixed solution in the step 4, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing for 5 times with deionized water after curing, and freeze-drying for 24 hours for standby.
And 6, soaking the freeze-dried hydrogel prepared in the step 5 in a nucleic acid lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
Step 7, firstly, placing the hydrogel carrier prepared in the step 6 on an EP pipe orifice, secondly, dripping 200 mu L of saliva sample on the hydrogel carrier, wherein the hydrogel carrier has obvious pH response characteristic, the swelling rate of the saliva sample in the neutrality is obviously increased, then, the saliva sample permeates the hydrogel carrier and flows into the EP pipe, 10 mu LTE buffer solution is added for eluting, and then, PCR amplification is carried out on the saliva sample, and detection is carried out by gel electrophoresis.
Example 2
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
And 3, pouring the carboxymethyl chitosan aqueous solution prepared in the step 1 into the sodium alginate aqueous solution prepared in the step 2 in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
And step 4, weighing 2g of polyethylene glycol with the molecular weight of 2000, adding the polyethylene glycol into the mixed solution in the step 3, stirring for 1h, and performing ultrasonic degassing for 30min to form a uniform mixed solution, wherein the mass of the polyethylene glycol accounts for 5% of the total mass of the mixed solution.
And 5, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 5%, carrying out a crosslinking reaction with the mixed solution in the step 4, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing with deionized water for 5 times after curing, and freeze-drying for 24 hours for standby.
And 6, soaking the freeze-dried hydrogel prepared in the step 5 in the lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
Step 7, firstly, placing the hydrogel carrier prepared in the step 6 on an EP pipe orifice, secondly, dripping 200 mu L of saliva sample on the hydrogel carrier, wherein the hydrogel carrier has obvious pH response characteristic, the swelling rate of the saliva sample in the neutrality is obviously increased, then, the saliva sample permeates the hydrogel carrier and flows into the EP pipe, 10 mu LTE buffer solution is added for eluting, and then, PCR amplification is carried out on the saliva sample, and detection is carried out by gel electrophoresis.
Example 3
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
Step 3: and (3) pouring the carboxymethyl chitosan aqueous solution prepared in the step (1) into the sodium alginate aqueous solution prepared in the step (2) in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
Step 4: 2g of polyethylene glycol with the molecular weight of 6000 is weighed and added into the mixed solution in the step 3, the mass of the polyethylene glycol accounts for 5 percent of the total mass of the mixed solution, the mixed solution is stirred for 1h, and ultrasonic degassing is carried out for 30min, so that a uniform mixed solution is formed.
And 5, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 5%, carrying out a crosslinking reaction with the mixed solution in the step 4, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing with deionized water for 5 times after curing, and freeze-drying for 24 hours for standby.
And 6, soaking the freeze-dried hydrogel prepared in the step 5 in the lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
Step 7, firstly, placing the hydrogel carrier prepared in the step 6 on an EP pipe orifice, secondly, dripping 200 mu L of saliva sample on the hydrogel carrier, wherein the hydrogel carrier has obvious pH response characteristic, the swelling rate of the saliva sample in the neutrality is obviously increased, then, the saliva sample permeates the hydrogel carrier and flows into the EP pipe, 10 mu LTE buffer solution is added for eluting, and then, PCR amplification is carried out on the saliva sample, and detection is carried out by gel electrophoresis.
Comparative example 1
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
Step 3: and (3) pouring the carboxymethyl chitosan aqueous solution prepared in the step (1) into the sodium alginate aqueous solution prepared in the step (2) in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
And 4, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 3%, carrying out a crosslinking reaction with the mixed solution in the step 3, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing with deionized water for 5 times after curing, and freeze-drying for 24 hours for standby.
And 5, soaking the freeze-dried hydrogel prepared in the step 4 in the lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
Comparative example 2
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
Step 3: and (3) pouring the carboxymethyl chitosan aqueous solution prepared in the step (1) into the sodium alginate aqueous solution prepared in the step (2) in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
And 4, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 5%, carrying out a crosslinking reaction with the mixed solution in the step 3, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing with deionized water for 5 times after curing, and freeze-drying for 24 hours for standby.
And 5, soaking the freeze-dried hydrogel prepared in the step 4 in the lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
Comparative example 3
Step 1, weighing 0.4g of carboxymethyl chitosan, dissolving in 13.3ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare carboxymethyl chitosan aqueous solution with the mass concentration of 3%.
And 2, weighing 0.8g of sodium alginate, dissolving in 26.7ml of deionized water, and rapidly stirring for 1h to uniformly disperse to prepare a sodium alginate aqueous solution with the mass concentration of 3%.
Step 3: and (3) pouring the carboxymethyl chitosan aqueous solution prepared in the step (1) into the sodium alginate aqueous solution prepared in the step (2) in the stirring process, mixing the carboxymethyl chitosan aqueous solution and the sodium alginate aqueous solution according to the volume ratio of 1:2, stirring for 2h, and performing ultrasonic degassing for 30min to form a uniform mixed solution.
And 4, weighing 5g of calcium chloride, dissolving in 100ml of deionized water, stirring and dispersing uniformly to prepare a calcium chloride solution with the mass concentration of 7%, carrying out a crosslinking reaction with the mixed solution in the step 3, curing for 12 hours at the temperature of 20-30 ℃ (room temperature), flushing with deionized water for 5 times after curing, and freeze-drying for 24 hours for standby.
And 5, soaking the freeze-dried hydrogel prepared in the step 4 in the lysate for 4 hours by adopting a dry soaking method, and freeze-drying for 24 hours for standby.
In the above examples, the method for testing the pH response performance of the hydrogel was: the swelling properties of the pH responsive macroporous hydrogels were measured in PBS buffer solutions at three different pH (pH 1.2, 7.4, 13, respectively) to evaluate the pH responsiveness of the composite hydrogels. The freeze-dried hydrogel with certain mass is weighed, placed in three PBS buffer solutions with different pH values respectively, incubated for 5 hours at room temperature (25 ℃), the swelled hydrogel is taken out every 1 hour, and excessive water on the surface is wiped off by filter paper and weighed. The swelling ratio was calculated using the following formula: swelling ratio= (Ws-Wd)/Wd, where Wd and Ws are the weight of the composite hydrogel after lyophilization and the weight of the hydrogel after swelling, respectively.
To examine the effect of a pH-responsive macroporous hydrogel material loaded with a nucleic acid lysate prepared in the present invention as a carrier on nucleic acid extraction performance, the present invention tested and obtained the physicochemical properties of hydrogels in examples 1-3 (FIGS. 1 and 2) and the responsiveness to different pH buffers (FIG. 3) and the verification of the nucleic acid extraction performance of a hydrogel carrier loaded with a nucleic acid lysate (FIG. 4). The following is a detailed analysis in conjunction with the accompanying drawings and experimental data.
In FIG. 1, SEM pictures of hydrogels of comparative examples 1-3 and examples 1-3 were shown in the order of (a) - (f), the hydrogels prepared without PEG and having calcium chloride concentrations of 3%, 5% and 7%, respectively, and the hydrogels prepared with molecular weights of 400, 2000 and 6000 and calcium chloride concentrations of 5% were shown in examples 1-3, respectively. The results show that: the surface of the composite hydrogel prepared by using PEG with different molecular weights is porous. As can be seen from examination of panels A SEM (a) - (f), the three-dimensional porous network structure was more pronounced after the addition of the pore-forming agent PEG relative to the hydrogels of comparative examples 1-3, and was more advantageous for release as a reservoir for nucleic acid lysates. Average pore size results fig. 1B shows: the larger the calcium chloride concentration, the smaller the pore size, the larger the average pore size after PEG addition, and the composite hydrogel pore size tends to increase gradually as the molecular weight of PEG increases.
The results in fig. 2 a show that: the compression deformation of the composite hydrogel obtained by the invention can reach 60% -80%, which shows that the composite hydrogel carrier prepared by the invention has better strength and toughness. The infrared plot of fig. 2B shows: the carboxyl group of CMCS and the carbonyl group of SA and the hydroxyl group of PEG were successfully complexed on the hydrogel, indicating successful preparation of the composite hydrogel.
The results of fig. 3 show that: the composite hydrogel prepared in example 3 has obvious pH response characteristics, the swelling ratio in the buffer solution with pH of 7.4 is far greater than that in the buffer solutions with pH of 1.2 and 13, and the swelling balance can be reached quickly within 2 hours, which indicates that the composite hydrogel can swell quickly when meeting neutral samples and release the samples.
The results of fig. 4 show: the presence of the band of interest in the 200. Mu.L saliva sample extracted from the composite hydrogel prepared using PEG with molecular weight 6000 in step 4 of example 3 between 200-300bp demonstrates that the composite hydrogel loaded with the nucleic acid cleavage reagent can extract DNA from the saliva sample and can be detected by nucleic acid electrophoresis. The composite hydrogels prepared in examples 1 and 2 failed to extract DNA from saliva samples, and the extraction effect was not achieved because the average pore diameters of the hydrogels of examples 1 and 2 were not large enough.
In summary, in the preparation process of the pH response type macroporous hydrogel loaded with the nucleic acid lysate, the PEG is added to change the pore structure of the composite hydrogel, so that the pH response performance of the composite hydrogel is affected, and the purpose of extracting nucleic acid by taking the composite hydrogel as a lysate carrier is achieved; finally, the pH response macroporous hydrogel loaded with the nucleic acid lysate is successfully prepared. The preparation method is simple to operate, low in cost and rapid in gel formation, the prepared hydrogel carrier has a three-dimensional porous structure, the problems of complex preparation, inconvenient carrying, high cost, time consumption and environmental protection in the storage process of the existing lysate are solved, the prepared composite hydrogel carrier has obvious pH response characteristics, and the prepared composite hydrogel carrier has good application prospect and potential application value in the application field of nucleic acid detection by using the composite hydrogel carrier as a carrier.
Claims (7)
1. The application of the pH response type macroporous hydrogel loaded with the nucleic acid lysate in nucleic acid extraction is characterized in that the preparation method of the pH response type macroporous hydrogel loaded with the nucleic acid lysate comprises the following steps:
step 1, dispersing carboxymethyl chitosan, sodium alginate and polyethylene glycol in water to obtain a mixed solution; the molecular weight of the polyethylene glycol is 6000 MW;
Step 2, adding a cross-linking agent into the mixed solution, solidifying to obtain hydrogel, washing, and freeze-drying to obtain freeze-dried hydrogel;
And 3, soaking the freeze-dried hydrogel in the nucleic acid lysate, and taking out the freeze-dried hydrogel after the soaking is finished to obtain the pH response type macroporous hydrogel loaded with the nucleic acid lysate.
2. The use according to claim 1, wherein step 1 is specifically: preparing carboxymethyl chitosan aqueous solution and sodium alginate aqueous solution respectively, adding the carboxymethyl chitosan aqueous solution into the sodium alginate aqueous solution, mixing, adding polyethylene glycol, and mixing to obtain a mixed solution.
3. The use according to claim 1, wherein in step 1, the degree of carboxymethyl chitosan is 80% or more.
4. The use according to claim 1, wherein in step 1, the polyethylene glycol accounts for 1% -5% of the mass of the mixed solution.
5. The application of claim 1, wherein in the step 1, the mass ratio of carboxymethyl chitosan to sodium alginate to polyethylene glycol is (1-3) (1-5).
6. The use according to claim 1, wherein in step 2, the cross-linking agent is calcium chloride.
7. The use according to claim 1, wherein in step 3, the freeze-dried hydrogel is taken out after being immersed in the nucleic acid lysate for 3-5 hours and freeze-dried.
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| CN113372578A (en) * | 2021-06-04 | 2021-09-10 | 江南大学 | preparation method of pH response type carboxymethyl chitosan/sodium alginate hydrogel spheres |
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| CN113372578A (en) * | 2021-06-04 | 2021-09-10 | 江南大学 | preparation method of pH response type carboxymethyl chitosan/sodium alginate hydrogel spheres |
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