CN111272615B - Gel particle size distribution detection method - Google Patents
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- 238000009826 distribution Methods 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 239000007863 gel particle Substances 0.000 title claims abstract description 21
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 238000004043 dyeing Methods 0.000 claims abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 39
- 239000000499 gel Substances 0.000 claims description 26
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 21
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 21
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 19
- 229950003937 tolonium Drugs 0.000 claims description 18
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical group [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000010586 diagram Methods 0.000 claims description 10
- 239000003086 colorant Substances 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 6
- 230000004323 axial length Effects 0.000 claims description 4
- 230000001788 irregular Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 238000010186 staining Methods 0.000 claims 3
- 238000007619 statistical method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 238000002316 cosmetic surgery Methods 0.000 description 5
- 238000000149 argon plasma sintering Methods 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000038 blue colorant Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
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Abstract
The invention provides a gel particle size distribution detection method, which mainly comprises the steps of sample pretreatment, gel dyeing treatment, gel color separation treatment, final treatment, microscopic detection and data statistical analysis.
Description
Technical Field
The invention belongs to the field of gel particle size detection, and particularly relates to a method for detecting the particle size distribution of crosslinked sodium hyaluronate gel for plastic surgery.
Background
The main components of the crosslinked sodium hyaluronate gel for plastic surgery are crosslinked sodium hyaluronate and free sodium hyaluronate. Sodium hyaluronate is a linear polysaccharide composed of disaccharide repeating structural units formed by connecting D-glucuronic acid and N-acetyl-D-glucosamine through beta- (1-3) glycosidic bond. Each disaccharide unit is linked to another disaccharide unit by a β - (1-4) glycosidic bond. Under the action of a cross-linking agent, the sodium hyaluronate is cross-linked to obtain the cross-linked sodium hyaluronate gel. The cross-linked hyaluronic acid is formed into transparent block solid gel, and the free sodium hyaluronate is high-viscosity liquid, so that the particle size detection of the cross-linked sodium hyaluronate gel for plastic surgery mainly aims at the cross-linked sodium hyaluronate gel particles. The size distribution of the gel particle size directly influences the in-vivo degradation effect of the product, the pushing force during injection and the uniformity of the product, so the size of the gel particle size belongs to an important technical parameter of the crosslinked sodium hyaluronate gel for plastic surgery, and the detection method is directly related to the accuracy of the detection of the gel particle size.
The existing method for detecting the particle size distribution of the crosslinked sodium hyaluronate gel for plastic surgery is mainly determined by a third method (light scattering method) of a 0982 particle size and particle size distribution method according to the pharmacopoeia of the people's republic of China (four parts) (2015 edition). The light scattering method wet method measurement needs that the sample to be detected is uniformly dispersed in the liquid phase in the detection process, but does not describe how to keep the particles to be detected uniformly dispersed in the liquid phase. The sedimentation degree of the crosslinked sodium hyaluronate gel in a liquid phase can be caused by different treatment methods, and the light scattering method has high detection professionality and high detection cost.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides the gel particle size distribution detection method which has the advantages of low cost, simple operation, convenient detection and high accuracy, and can observe and record the shape of gel particles.
The aim of the invention is realized by the following technical scheme:
the gel particle size distribution detection method is characterized by comprising the following steps of:
1) Sample pretreatment, namely taking a gel sample, adding the gel sample into 10-20 times of PBS solution for vibration cleaning, and cleaning for 5-20min each time and 5-10 times to obtain a pretreated sample;
2) Taking a pretreated sample, adding the pretreated sample into a centrifuge tube, and adding a coloring agent for dyeing; color separation treatment is carried out by adopting 70% -95% ethanol; 5% -10% of glycerol is adopted for final treatment, and a final treated sample is obtained;
3) Microscopic detection: taking a final treated sample, carrying out data detection by using a microscope, and taking an axial length measurement value of a long axis of the gel as the particle size of irregular particles;
4) Data statistics: and counting the measured data, establishing a data normal distribution diagram, and calculating the gel particle size distribution according to the normal distribution diagram.
Preferably, the coloring agent is selected from toluidine blue coloring agents, wherein the mass fraction of toluidine blue in the toluidine blue coloring agents is as follows: 0.1% -1%.
Preferably, in the step 2), after the dyeing treatment by adding the dyeing agent, a color separation treatment is required.
Preferably, after the pretreatment, the sample is dyed, PBS solution is added, the sample is oscillated for 30-60s, the sample is kept stand for 5-10min, and after the supernatant is discarded, color separation treatment is carried out.
Preferably, the specific process of the color separation treatment is as follows: adding 70% -95% ethanol, oscillating for 30-60s, centrifuging at 800-1500rpm for 5-10min, and discarding supernatant.
Preferably, the specific process of the final treatment with 5% -10% glycerol is: adding 5% -10% glycerol, oscillating for 30-60s, centrifuging at 800-1500rpm for 5-10min, removing supernatant, adding 5% -10% glycerol, and oscillating for 30-60s to obtain final treated sample.
Preferably, the gel is selected from crosslinked sodium hyaluronate gels.
The invention has the beneficial effects that for the prior art:
according to the detection method, the sample pretreatment can effectively remove the interference of free sodium hyaluronate in the sample to be detected, the toluidine blue coloring agent can effectively carry out deep coloring on gel particles of the sample to be detected, 70% -95% ethanol can consolidate and strengthen the coloring effect of the sample, 5% -10% glycerol can effectively simulate the external environment of crosslinked sodium hyaluronate gel in the sample to be detected, and the size of the particles of the sample to be detected is ensured to have no obvious change in the detection process.
The detection method involves less detection reagents including toluidine blue stain, ethanol and glycerol only. The detection method has the advantages of low cost, simple operation, convenient detection and high accuracy, and can be effectively applied and popularized to the production and detection of enterprise samples.
Drawings
FIG. 1 is a graph showing the effect of the 0.2% toluidine blue stain of example 1 of the present invention;
FIG. 2 is a graph showing the effect of the microscope at a magnification of 40 in example 1 of the present invention;
FIG. 3 is a normal distribution chart of the particle size distribution of the data statistical gel in example 1 of the present invention;
FIG. 4 is a graph showing the effect of the 0.2% toluidine blue stain of example 2 of the present invention;
FIG. 5 is a graph showing the effect of the microscope at a magnification of 40 in example 2 of the present invention;
FIG. 6 is a normal distribution chart of the particle size distribution of the data statistical gel in example 2 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1.
The embodiment provides a gel particle size distribution detection method, which comprises the following steps:
1) Sample pretreatment, namely taking a crosslinked sodium hyaluronate gel sample, adding the crosslinked sodium hyaluronate gel sample into a 20-time PBS solution for vibration cleaning, and cleaning for 10min each time and 5 times to obtain a pretreated sample;
2) 60mg of the pretreated sample is taken, added into an EP centrifuge tube with 1.5mL, 1mL of 0.2% toluidine blue colorant is added, and the mixture is oscillated by an oscillator for 45s and centrifuged at 1000rpm for 5min, and the supernatant is discarded; adding 1mL of PBS solution, oscillating for 45s, standing for 5min, and discarding supernatant until 0.5mL of solution remains; adding 1mL of 95% ethanol, oscillating for 45s, carrying out color separation, centrifuging at 1000rpm for 5min, and discarding the supernatant until 0.5mL remains in the solution; adding 1mL of 10% glycerol, oscillating for 45s, centrifuging at 1000rpm for 5min, and discarding the supernatant until 0.5mL of solution remains; 10% glycerol 1mL was added and the mixture was shaken for 45s to obtain final treated samples. The preparation method of the toluidine blue coloring agent comprises the following steps: with 70% ethanol 10mL, 20mg of toluidine blue was added to prepare 0.2% toluidine blue stain, see fig. 1;
3) Microscopic detection: taking a final treated sample, performing microscopic observation and particle size measurement under a 40-fold mirror, taking the axial length measurement value of a long axis of the gel as the particle size of irregular particles, and referring to FIG. 2;
4) Data statistics: and counting the measured data, establishing a data normal distribution diagram, and calculating the gel particle size distribution according to the normal distribution diagram. The statistical results of the data are shown in table 1, and the normal distribution diagram is shown in fig. 3:
TABLE 1 gel particle size distribution interval
| Confidence interval | |X-µ|≤0.675σ | |X-µ|≤σ | |X-µ|≤1.29σ | |X-µ|≤1.645σ |
| Interval probability | 50.03% | 68.27% | 80.30% | 90.00% |
| Sample 1 | 1078.38~1802.64 | 904.02~1977.00 | 753.80~2127.22 | 557.98~2323.04 |
| Sample 2 | 675.82~1295.88 | 523.55~1445.16 | 397.94~1573.76 | 230.29~1741.41 |
| Sample 3 | 735.91~1233.36 | 616.15~1353.12 | 512.97~1456.30 | 378.48~1590.79 |
| Sample 4 | 452.39~874.89 | 350.68~976.60 | 263.05~1064.23 | 148.82~1178.46 |
| Sample 5 | 981.90~1595.85 | 834.10~1743.65 | 706.77~1870.98 | 540.78~2036.98 |
| Sample 6 | 610.95~1172.09 | 475.86~1307.18 | 359.47~1423.57 | 207.76~1575.28 |
| Sample 7 | 581.03~929.10 | 497.24~1012.90 | 425.04~1085.09 | 330.94~1179.19 |
| Sample 8 | 422.40~763.87 | 340.19~846.07 | 269.37~916.90 | 177.05~1009.22 |
Example 2.
The embodiment provides a gel particle size distribution detection method, which comprises the following steps:
1) Sample pretreatment, namely taking a crosslinked sodium hyaluronate gel sample, adding the crosslinked sodium hyaluronate gel sample into a 20-time PBS solution for vibration cleaning, and cleaning for 10 times every 20 minutes to obtain a pretreated sample;
2) 60mg of the pretreated sample is taken, added into an EP centrifuge tube with 1.5mL, 1mL of 0.2% toluidine blue colorant is added, and the mixture is oscillated by an oscillator for 45s and centrifuged at 1000rpm for 5min, and the supernatant is discarded; adding 1mL of PBS solution, oscillating for 45s, standing for 5min, and discarding supernatant until 0.5mL of solution remains; adding 1mL of 10% glycerol, and oscillating for 45s to obtain a final treated sample; the preparation method of the toluidine blue coloring agent comprises the following steps: with 10mL of 95% ethanol, 20mg of toluidine blue was added to prepare 0.2% toluidine blue stain, see fig. 4;
3) Microscopic detection: taking a final treated sample, performing microscopic observation and particle size measurement under a 40-fold mirror, taking the axial length measurement value of a long axis of the gel as the particle size of irregular particles, and referring to FIG. 5;
4) Data statistics: and counting the measured data, establishing a data normal distribution diagram, and calculating the gel particle size distribution according to the normal distribution diagram. The statistical results of the data are shown in table 2, and the normal distribution diagram is shown in fig. 6:
TABLE 2 gel particle size distribution interval
| Confidence interval | |X-µ|≤0.675σ | |X-µ|≤σ | |X-µ|≤1.29σ | |X-µ|≤1.645σ |
| Interval probability | 50.03% | 68.27% | 80.30% | 90.00% |
| Sample 1 | 924.25~2083.72 | 645.12~2362.85 | 387.46~2620.51 | 91.15~2916.82 |
| Sample 2 | 669.70~1199.05 | 542.27~1326.49 | 424.63~1444.12 | 289.35~1597.40 |
| Sample 3 | 482.26~1121.06 | 328.48~1274.84 | 186.52~1416.80 | 23.28~1580.04 |
| Sample 4 | 648.39~1145.73 | 528.66~1265.46 | 418.14~1375.98 | 291.04~1503.08 |
| Sample 5 | 541.94~895.83 | 456.74~981.03 | 378.09~1059.68 | 287.65~1150.12 |
Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described specific embodiments and application fields, which are merely illustrative, instructive, and not restrictive. Modifications and substitutions without the need for inventive labor will be apparent to those of ordinary skill in the art from the teachings of this specification without departing from the scope of the invention, which is defined by the appended claims.
Claims (7)
1. The gel particle size distribution detection method is characterized by comprising the following steps of:
1) Sample pretreatment: adding a gel sample into 10-20 times of PBS solution for vibration cleaning, and cleaning for 5-20min each time and 5-10 times to obtain a pretreated sample;
2) Taking a pretreated sample, adding the pretreated sample into a centrifuge tube, and adding a coloring agent for dyeing; 5% -10% of glycerol is adopted for final treatment, and a final treated sample is obtained;
3) Microscopic detection: taking a final treated sample, carrying out data detection by using a microscope, and taking an axial length measurement value of a long axis of the gel as the particle size of irregular particles;
4) Data statistics: and counting the measured data, establishing a data normal distribution diagram, and calculating the gel particle size distribution according to the normal distribution diagram.
2. The detection method according to claim 1, wherein the staining agent is selected from toluidine blue staining agents, and the mass fraction of toluidine blue in the toluidine blue staining agents is as follows: 0.1% -1%.
3. The method according to claim 1, wherein in the step 2), a color separation treatment is performed after the dyeing treatment by adding a coloring agent.
4. The method according to claim 1, wherein the sample after pretreatment is subjected to dyeing treatment, a PBS solution is added, the sample is oscillated for 30-60s, the sample is left for 5-10min, and the supernatant is discarded and then subjected to color separation treatment.
5. The method of claim 3, wherein the specific process of the color separation process is: adding 70% -95% ethanol, oscillating for 30-60s, centrifuging at 800-1500rpm for 5-10min, and discarding supernatant.
6. The method according to claim 1, wherein the specific process of performing the final treatment with 5% -10% glycerol is: adding 5% -10% glycerol, oscillating for 30-60s, centrifuging at 800-1500rpm for 5-10min, removing supernatant, adding 5% -10% glycerol, and oscillating for 30-60s to obtain final treated sample.
7. The method of claim 1, wherein the gel is selected from the group consisting of cross-linked sodium hyaluronate gels.
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| CN113804592B (en) * | 2021-09-24 | 2024-06-18 | 中国石油化工股份有限公司 | Detection method for gel particles in PAN spinning solution |
| CN114112816A (en) * | 2021-11-30 | 2022-03-01 | 山东泰山生力源集团股份有限公司 | Method for measuring particle size of microcapsule |
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