CN117964828A - Microsphere preparation method applied to glycosylated hemoglobin detection reagent - Google Patents
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- CN117964828A CN117964828A CN202410017838.5A CN202410017838A CN117964828A CN 117964828 A CN117964828 A CN 117964828A CN 202410017838 A CN202410017838 A CN 202410017838A CN 117964828 A CN117964828 A CN 117964828A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 29
- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 28
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 28
- 239000004005 microsphere Substances 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000002245 particle Substances 0.000 claims abstract description 74
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 28
- 239000004793 Polystyrene Substances 0.000 claims abstract description 26
- 229920002223 polystyrene Polymers 0.000 claims abstract description 26
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical group C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 21
- 239000008367 deionised water Substances 0.000 claims abstract description 19
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 19
- 238000003756 stirring Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 17
- 238000010926 purge Methods 0.000 claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 15
- 239000003995 emulsifying agent Substances 0.000 claims abstract description 12
- 239000003999 initiator Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims description 24
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical group [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 13
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 9
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 9
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 8
- XESUCHPMWXMNRV-UHFFFAOYSA-M sodium;2-ethenylbenzenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C1=CC=CC=C1C=C XESUCHPMWXMNRV-UHFFFAOYSA-M 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims 6
- 239000002105 nanoparticle Substances 0.000 abstract description 5
- 230000006911 nucleation Effects 0.000 abstract description 4
- 238000010899 nucleation Methods 0.000 abstract description 4
- 238000001308 synthesis method Methods 0.000 abstract description 3
- 238000010556 emulsion polymerization method Methods 0.000 abstract description 2
- 238000001000 micrograph Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 239000011246 composite particle Substances 0.000 description 4
- 239000011162 core material Substances 0.000 description 3
- 239000011258 core-shell material Substances 0.000 description 3
- 239000011257 shell material Substances 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000003631 expected effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a microsphere preparation method applied to glycosylated hemoglobin detection reagent, relates to the technical field of modified polystyrene nanoparticle synthesis methods for biological immunodiagnosis, and aims to solve the problems that in the prior art, an emulsion polymerization method is generally adopted to prepare carboxylated polystyrene nanoparticles, the process is easily affected by conditions such as rotating speed and temperature, the nucleation period of particles is long, and the particle size of a product is easily distributed in multiple stages. The preparation method of the target carboxyl particle C comprises the following steps: step one: putting deionized water into a three-neck flask, adding an emulsifying agent, and stirring for 20-40 minutes at room temperature under nitrogen purging; step two: heating the three-neck flask to 40-70 ℃ through water bath, adding styrene monomer after the temperature is balanced, and continuously stirring for 20-40 minutes; step three: and (3) heating the mixture in the three-neck flask to 60-80 ℃ again, adding an initiator after the temperature is balanced, and reacting for 7 hours to obtain the polystyrene seed particles C.
Description
Technical Field
The invention relates to the technical field of a synthesis method of modified polystyrene nano-particles for biological immunodiagnosis, in particular to a preparation method of microspheres applied to glycosylated hemoglobin detection reagents.
Background
The first composite particle with the micro-nano core-shell structure is formed by coating shell materials with different components, structures and sizes on the surface of a micro-nano core material through a certain acting force. With the continuous development of the micro-nano core-shell particles, the composite particles formed by combining two particles at the micro-nano level through physical or chemical action can be called as micro-nano core-shell structure composite particles. The core and shell materials of the structural particles may be selected and combined as desired to provide the composite particles with properties different from the single components of the core and shell layers. The microsphere has relatively uniform size, good optical, chemical, electrical, catalytic, magnetic and mechanical properties, can be used as an effective carrier of substances such as medicines, antigens, antibodies and the like, is widely applied to the fields of biological medicine chemical industry and diagnosis, has good practical technical prospect, but the existing synthesis method of modified polystyrene nano-particles for biological immunity diagnosis has the following problems:
1. In the prior art, the carboxylated polystyrene nano particles are prepared by an emulsion polymerization method, the process is easily influenced by conditions such as rotating speed, temperature and the like, the nucleation period of particles is long, and the particle size of the product is easily distributed in multiple stages;
2. In the polymerization process, as the reaction system is an oil-in-water system, functional groups such as carboxyl are added into the particles in the nucleation process, so that the quantity of carboxyl embedded in the particles is easy to increase, and the carboxyl content on the surface of the product is low;
3. Batch repetition is difficult, often with large batch-to-batch variation, and surface modification is not easy, and problems arise when it is desired to change the surface properties.
Disclosure of Invention
The invention aims to provide a microsphere preparation method applied to glycosylated hemoglobin detection reagent, which solves the problem that a common test tube proposed in the background art is easy to pollute a sample by foreign substances.
In order to achieve the above purpose, the present invention provides the following technical solutions: the microsphere of the glycosylated hemoglobin detection reagent is a target carboxyl particle C, and the preparation method of the target carboxyl particle C comprises the following steps:
Step one: putting deionized water into a three-neck flask, adding an emulsifying agent, and stirring for 20-40 minutes at room temperature under nitrogen purging;
step two: heating the three-neck flask to 40-70 ℃ through water bath, adding styrene monomer after the temperature is balanced, and continuously stirring for 20-40 minutes;
Step three: heating the mixture in the three-neck flask to 60-80 ℃ again, adding an initiator after the temperature is balanced, and reacting for 7 hours to obtain polystyrene seed particles C;
Step four: mixing the prepared polystyrene seed particles C with deionized water, adding into a three-neck flask, and purging with nitrogen for 30-50 minutes in an oil bath environment at 40-60 ℃;
Step five: adding styrene monomer and acrylic acid into the mixture of the three-neck flask, heating to 60-80 ℃ and continuously reacting for 0.5-2 hours;
Step six: and then adding an initiator into the three-neck flask for 0.5-2 hours of reaction, and continuing the reaction for 5-9 hours to obtain target carboxyl particles C.
In the preparation method of the microsphere of the glycosylated hemoglobin detection reagent, the target carboxyl particle C comprises styrene as a monomer, and the emulsifier is composed of sodium dodecyl sulfate and sodium vinylbenzenesulfonate in a proportion of 1:1.
In the microsphere preparation method of the glycosylated hemoglobin detection reagent, the initiator is potassium persulfate.
In the first step, 300mL of deionized water is taken, 500mL of a three-necked flask is taken, 0.2g of sodium dodecyl sulfate and 0.2g of sodium vinylbenzenesulfonate are taken as emulsifying agents, and the stirring time is 30 minutes at room temperature under nitrogen purging.
In the preparation method of the microsphere of the glycosylated hemoglobin detection reagent, in the second step, the three-neck flask is heated to 60 ℃ through a water bath, 40g of added styrene monomer is added, and the stirring time is 30 minutes.
In the method for preparing the microsphere of the glycosylated hemoglobin detection reagent, in the third step, the temperature of the mixture in the three-neck flask is raised to 75 ℃ again, and the initiator added after the temperature is balanced is 10mL of aqueous solution (10 wt%) of potassium persulfate.
In the method for preparing the microsphere of the glycosylated hemoglobin detection reagent, in the fourth step, the prepared polystyrene seed particles C are 200mL, the deionized water added into the three-neck flask is 100mL, and the nitrogen purging time is 40 minutes.
In the fifth step, 10g of styrene monomer and 1.5mL of acrylic acid are taken, and the temperature is raised to 75 ℃ to continue the reaction for 1 hour.
In the method for preparing the microsphere of the glycosylated hemoglobin detection reagent, in the sixth step, the initiator is 2mL of potassium persulfate aqueous solution (10 wt%) and the reaction time is 1 hour, and the continuous reaction time is 7 hours.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains good comparison of the relative performance of the target carboxyl particle C and the contrast reagent, achieves the expected effect, avoids the problems that the particle nucleation period is long, the particle size of the product is easy to be distributed in multiple stages, the quantity of carboxyl embedded in the particle is increased, the carboxyl content on the surface of the product is lower, the batch repetition is difficult, the difference between batches is large, and the surface modification is difficult, and the replacement of domestic raw materials can be realized in the follow-up process.
Drawings
FIG. 1 is a scanning electron microscope image of polystyrene seed particles C of the present invention;
FIG. 2 is a scanning electron microscope image of polystyrene seed particles A of the present invention;
FIG. 3 is a scanning electron microscope image of polystyrene seed particles B of the present invention;
FIG. 4 is a scanning electron microscope image of the target carboxyl particles A of the present invention;
FIG. 5 is a scanning electron microscope image of the target carboxyl particles B of the present invention;
fig. 6 is a scanning electron microscope image of the target carboxyl particles C of the present invention.
FIG. 7 is a diagram showing the on-machine data of a glycosylated hemoglobin kit prepared from target carboxyl groups A, B, and C according to the present invention;
FIG. 8 is a graph showing comparison of performance data of a glycosylated hemoglobin reagent prepared from particles C according to the present invention with that of a control reagent.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Example 1
A500 mL three-necked flask was charged with 300mL deionized water, 0.2g of sodium dodecyl sulfate as an emulsifier, 0.2g of sodium vinylbenzenesulfonate and stirred at room temperature for 20 minutes under a nitrogen purge. Heating to 40 ℃ in a water bath, adding 40g of styrene monomer after balancing the temperature, and stirring for 20 minutes. The mixture was again warmed to 60℃and 10mL of an aqueous solution of potassium persulfate (10 wt%) was added after the temperature was equilibrated, and the reaction was carried out for 7 hours to obtain polystyrene seed particles C having a particle diameter of 91.64nm.
200ML of the prepared polystyrene seed particles C are mixed with 100mL of deionized water and then added into a 500mL three-necked flask, under the oil bath environment of 40 ℃, nitrogen is purged for 30 minutes, 10g of styrene monomer and 1.5mL of acrylic acid are added, the temperature is raised to 60 ℃ for continuous reaction for 0.5 hour, then 2mL of potassium persulfate aqueous solution (10 wt%) is added, and the target carboxyl particles C with the particle size of 120.6nm can be obtained after the reaction is carried out for 5 hours.
Example 2
A500 mL three-necked flask was charged with 300mL deionized water, 0.2g of sodium dodecyl sulfate as an emulsifier, 0.2g of sodium vinylbenzenesulfonate and stirred at room temperature for 40 minutes under a nitrogen purge. Heating to 70 ℃ in a water bath, adding 40g of styrene monomer after balancing the temperature, and stirring for 40 minutes. The mixture was again warmed to 80℃and 10mL of an aqueous solution of potassium persulfate (10 wt%) was added after the temperature was equilibrated, and the reaction was carried out for 7 hours to obtain polystyrene seed particles C having a particle diameter of 89.84nm.
200ML of the prepared polystyrene seed particles C are mixed with 100mL of deionized water and then added into a 500mL three-necked flask, under the oil bath environment of 60 ℃, nitrogen is purged for 50 minutes, 10g of styrene monomer and 1.5mL of acrylic acid are added, the temperature is raised to 80 ℃ for continuous reaction for 2 hours, then 2mL of potassium persulfate aqueous solution (10 wt%) is added, and the target carboxyl particles C with the particle size of 115.4nm can be obtained after the reaction is carried out for 9 hours.
Example 3
A500 mL three-necked flask was charged with 300mL deionized water, 0.2g of sodium dodecyl sulfate as an emulsifier, 0.2g of sodium vinylbenzenesulfonate and stirred at room temperature for 30 minutes under a nitrogen purge. Heating to 60 ℃ in a water bath, adding 40g of styrene monomer after balancing the temperature, and stirring for 30 minutes. The mixture was again warmed to 75℃and 10mL of an aqueous solution of potassium persulfate (10 wt%) was added after the temperature was equilibrated, and the reaction was carried out for 7 hours to obtain polystyrene seed particles C having a particle diameter of 90.74nm.
200ML of the prepared polystyrene seed particles C are mixed with 100mL of deionized water and then added into a 500mL three-necked flask, under the oil bath environment of 50 ℃, nitrogen is purged for 40 minutes, 10g of styrene monomer and 1.5mL of acrylic acid are added, the temperature is raised to 75 ℃ for continuous reaction for 1 hour, then 2mL of potassium persulfate aqueous solution (10 wt%) is added, and the target carboxyl particles C with the particle size of 117.4nm can be obtained after 7 hours of reaction.
Example 4
A500 mL three-necked flask was charged with 300mL deionized water, 0.4g of sodium dodecyl sulfate as an emulsifier was added thereto, and the mixture was stirred at room temperature for 30 minutes under a nitrogen purge. Heating to 60 ℃ in a water bath, adding 40g of styrene monomer after balancing the temperature, and stirring for 30 minutes. The mixture was again warmed to 75℃and 10mL of an aqueous solution of potassium persulfate (10 wt%) was added after the temperature was equilibrated, and the reaction was carried out for 7 hours to obtain polystyrene seed particles A having a particle diameter of 92.22nm.
200ML of the prepared polystyrene seed particles A are mixed with 100mL of deionized water and then added into a 500mL three-necked flask, under the oil bath environment of 50 ℃, nitrogen is purged for 40 minutes, 10g of styrene monomer and 1.5mL of acrylic acid are added, the temperature is raised to 75 ℃ for continuous reaction for 1 hour, then 2mL of potassium persulfate aqueous solution (10 wt%) is added, and the target carboxyl particles A with the particle size of 122.9nm can be obtained after 7 hours of reaction.
Example 5
A500 mL three-necked flask was charged with 300mL deionized water, 0.4g of sodium vinylbenzenesulfonate as an emulsifier was added thereto, and the mixture was stirred at room temperature for 30 minutes under a nitrogen purge. Heating to 60 ℃ in a water bath, adding 40g of styrene monomer after balancing the temperature, and stirring for 30 minutes. The mixture was again warmed to 75℃and 10mL of an aqueous solution of potassium persulfate (10 wt%) was added after the temperature was equilibrated, and the reaction was carried out for 7 hours to obtain polystyrene seed particles B having a particle diameter of 91.51nm.
200ML of the prepared polystyrene seed particles B are mixed with 100mL of deionized water and then added into a 500mL three-necked flask, under the oil bath environment of 50 ℃, nitrogen is purged for 40 minutes, 10g of styrene monomer and 1.5mL of acrylic acid are added, the temperature is raised to 75 ℃ for continuous reaction for 1 hour, then 2mL of potassium persulfate aqueous solution (10 wt%) is added, and the target carboxyl particles B can be obtained after 7 hours of reaction, wherein the particle size of the target carboxyl particles B is 120.9nm.
By comparing the results of example 1, example 2 and example 3, the polystyrene seed particles C obtained after stirring the mixture at room temperature under nitrogen purging for 30 minutes, heating the mixture to 60 ℃ in a water bath, setting the stirring time to 30 minutes, and heating the mixture again to 75 ℃ and reacting for 7 hours have the particle size closest to that of the control reagent.
Then, under the oil bath environment of 50 ℃, nitrogen is purged for 40 minutes, the temperature is raised to 75 ℃ for continuous reaction for 1 hour, and the particle sizes of the target carboxyl particles C obtained after the reaction is carried out for 7 hours are closest to those of the control reagent.
Thus, the control tests of example 4 and example 5 were performed with the same control temperature, stirring time, purging time and reaction time.
The results of the synthesized target carboxyl groups A, B, and C were not significantly different from each other as shown in FIGS. 1 to 6 by the particles synthesized in example 3, example 4, and example 5, and the glycosylated hemoglobin kit was prepared using the three kinds of particles, respectively, and compared with the control kit.
The glycosylated hemoglobin kit is prepared by using the target carboxyl particles a, B and C of examples 3,4 and 5, and the data of the on-machine is as follows, and as can be seen from the data fig. 7, the background sensitivity of the particles a is higher, the background sensitivity of the particles B is slightly lower, and the particles C are closer to the control batch reagent, so that the performance of the glycosylated hemoglobin reagent prepared by the particles C is better than the performance of the glycosylated hemoglobin sample prepared by the particles C in 30 cases of comparison, and the expected effect is achieved.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
1. A method for preparing microspheres applied to glycosylated hemoglobin detection reagent is characterized in that: the microsphere of the glycosylated hemoglobin detection reagent is a target carboxyl particle C, and the preparation method of the target carboxyl particle C comprises the following steps:
Step one: putting deionized water into a three-neck flask, adding an emulsifying agent, and stirring for 20-40 minutes at room temperature under nitrogen purging;
step two: heating the three-neck flask to 40-70 ℃ through water bath, adding styrene monomer after the temperature is balanced, and continuously stirring for 20-40 minutes;
Step three: heating the mixture in the three-neck flask to 60-80 ℃ again, adding an initiator after the temperature is balanced, and reacting for 7 hours to obtain polystyrene seed particles C;
Step four: mixing the prepared polystyrene seed particles C with deionized water, adding into a three-neck flask, and purging with nitrogen for 30-50 minutes in an oil bath environment at 40-60 ℃;
Step five: adding styrene monomer and acrylic acid into the mixture of the three-neck flask, heating to 60-80 ℃ and continuously reacting for 0.5-2 hours;
Step six: and then adding an initiator into the three-neck flask for 0.5-2 hours of reaction, and continuing the reaction for 5-9 hours to obtain target carboxyl particles C.
2. The method for preparing the microsphere applied to the glycosylated hemoglobin detection reagent according to claim 1, wherein the method comprises the following steps: the target carboxyl particle C comprises styrene as a monomer, and the emulsifier is composed of sodium dodecyl sulfate and sodium vinylbenzenesulfonate in a proportion of 1:1.
3. The method for preparing the microsphere applied to the glycosylated hemoglobin detection reagent according to claim 2, wherein: the initiator is potassium persulfate.
4. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the first step, 300mL of deionized water is taken, a three-necked flask is 500mL, an emulsifier is 0.2g of sodium dodecyl sulfate and 0.2g of sodium vinylbenzenesulfonate, and stirring time at room temperature under nitrogen purging is 30 minutes.
5. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the second step, the three-neck flask is heated to 60 ℃ through a water bath, 40g of added styrene monomer is added, and the stirring time is 30 minutes.
6. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the third step, the temperature of the mixture in the three-neck flask is raised to 75 ℃ again, and the initiator added after the temperature is balanced is 10mL of aqueous solution (10 wt%) of potassium persulfate.
7. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the fourth step, the prepared polystyrene seed particles C are 200mL, the deionized water added into the three-neck flask is 100mL, and the nitrogen purging time is 40 minutes.
8. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the fifth step, 10g of styrene monomer and 1.5mL of acrylic acid are taken, and the temperature is raised to 75 ℃ to continue the reaction for 1 hour.
9. A method for preparing a microsphere for use in a glycosylated hemoglobin measurement reagent according to claim 3, wherein: in the sixth step, the initiator was 2mL of an aqueous potassium persulfate solution (10 wt%) and the reaction time was 1 hour, and the reaction time was continued for 7 hours.
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