CN113088276B - Fluorescent microsphere quality control product of flow cytometer and preparation method thereof - Google Patents
Fluorescent microsphere quality control product of flow cytometer and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 152
- 238000003908 quality control method Methods 0.000 title claims abstract description 66
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 230000008961 swelling Effects 0.000 claims abstract description 45
- 239000006185 dispersion Substances 0.000 claims abstract description 32
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 22
- 238000002390 rotary evaporation Methods 0.000 claims abstract description 22
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 15
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 14
- 239000011259 mixed solution Substances 0.000 claims abstract description 13
- 230000010355 oscillation Effects 0.000 claims abstract description 8
- 239000000047 product Substances 0.000 claims description 49
- 238000000034 method Methods 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 238000001704 evaporation Methods 0.000 claims description 7
- 230000008020 evaporation Effects 0.000 claims description 7
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 claims description 6
- 239000004793 Polystyrene Substances 0.000 claims description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 5
- 229920002223 polystyrene Polymers 0.000 claims description 5
- 239000002736 nonionic surfactant Substances 0.000 claims description 3
- FASDKYOPVNHBLU-ZETCQYMHSA-N pramipexole Chemical compound C1[C@@H](NCCC)CCC2=C1SC(N)=N2 FASDKYOPVNHBLU-ZETCQYMHSA-N 0.000 claims description 2
- 229960003089 pramipexole Drugs 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims 1
- 239000000975 dye Substances 0.000 description 31
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 3
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- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000009835 boiling Methods 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000001044 red dye Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- 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
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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
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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Abstract
The invention discloses a fluorescent microsphere quality control product of a flow cytometer and a preparation method thereof, wherein the fluorescent microsphere quality control product comprises 0.3 g-1 g of blank microsphere, 100 mL-250 mL of swelling medium, 2 mg-10 mg of hydrophobic dye and 20 mL-50 mL of swelling agent, the blank microsphere with the forward scattering light variation coefficient smaller than 3% is weighed to be 0.3 g-1 g, the blank microsphere is dissolved in the swelling medium with the mass fraction of 1-5%, the ultrasonic crushing instrument with the ultrasonic intensity of 50-100% is utilized for carrying out ultrasonic treatment for 1 min-5 min, 2 mg-10 mg of hydrophobic dye is weighed, and after the hydrophobic dye is dissolved in the swelling agent with the volume of 20 mL-50 mL, the mixed solution is added to obtain a microsphere dispersion system; and carrying out ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and obtaining the fluorescent microsphere quality control product of the flow cytometer after centrifugal washing.
Description
Technical Field
The invention relates to the technical field of fluorescent microspheres for flow cytometry, in particular to a quality control product of fluorescent microspheres for flow cytometry and a preparation method thereof.
Background
The flow cytometer is a cell analyzer for measuring scattered light and marked fluorescence intensity of cells, can analyze tens of thousands of cells at high speed, can simultaneously measure a plurality of cell characteristic parameters from one cell, performs qualitative and quantitative analysis, has the characteristics of high speed, high precision, good accuracy and the like, becomes the most advanced cell quantitative analysis technology of the current generation, and is widely applied to clinical practice. The quality control of the fluorescent microspheres of the current national flow cytometer is all dependent on import and has high price. Although a small part of the fluorescent microsphere standard substances are suitable for the flow cytometry in China, only a single specific fluorescent channel can be met, and the detection of a plurality of channels cannot be met at the same time, so that the operation becomes complicated.
The dyeing of the fluorescent microsphere can be divided into external dyeing and internal dyeing, wherein the external dyeing is obtained by directly coupling the microsphere with a specific group and the fluorescent dye through a chemical bond, and the method is simple and controllable, does not influence the microsphere, but has poor stability, and is not suitable for preparing a quality control product; the internal dyeing comprises the steps of adding dye at the same time when preparing the microsphere, enabling the dye to be wrapped in the microsphere, swelling the finished microsphere, adding the dye, wherein the obtained fluorescent microsphere is good in stability, but harsh in reaction conditions, easy to quench the dye, mild in reaction conditions, high in stability, and important in developing a preparation method of a fluorescent microsphere quality control product suitable for a flow cytometer, besides factors of the dye, conditions such as a swelling agent, a swelling medium and a reaction proportion used for preparing the fluorescent microsphere by a swelling method directly influence the uniformity of the fluorescent intensity of the obtained fluorescent microsphere.
Disclosure of Invention
The invention aims to provide a fluorescent microsphere quality control product of a flow cytometer and a preparation method thereof, which have high uniformity and good stability and can well meet the quality control requirement of the flow cytometer.
In order to achieve the above object, in a first aspect, the present invention provides a method for preparing a fluorescent microsphere quality control product of a flow cytometer, comprising the following steps:
dissolving the weighed blank microspheres in a swelling medium, uniformly mixing by ultrasonic, and simultaneously adding a hydrophobic dye dissolved in a swelling agent into a mixed solution to obtain a microsphere dispersion system;
and carrying out ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and obtaining the fluorescent microsphere quality control product of the flow cytometer after centrifugal washing.
The method comprises the steps of dissolving the weighed blank microspheres in a swelling medium, uniformly mixing by ultrasonic, and simultaneously adding a hydrophobic dye dissolved in a swelling agent into a mixed solution to obtain a microsphere dispersion system, wherein the method comprises the following steps:
weighing 0.3-1 g of blank microspheres with forward scattering light variation coefficient less than 3%, dissolving the blank microspheres in a swelling medium with mass fraction of 1-5%, and carrying out ultrasonic treatment for 1-5 min by using an ultrasonic crusher with ultrasonic intensity of 50% -100% to obtain a mixed solution;
weighing 2-10 mg of hydrophobic dye, dissolving in 20-50 mL of swelling agent, and adding the mixed solution to obtain a microsphere dispersion system.
Wherein, carry on the said microballoon disperse system supersound, shake, rotary evaporation, and get the fluorescent microsphere quality control of the flow cytometer after centrifugal washing, include:
ultrasonic crushing the microsphere dispersion system for 1-5 min by using an ultrasonic crusher with the ultrasonic intensity of 50-100%, and oscillating for 30-90 min on a constant temperature shaking table with the temperature of room temperature and the rotating speed of 100-200 r/min after the ultrasonic treatment is finished;
and performing rotary evaporation and centrifugal washing on the oscillated microsphere dispersion system to obtain the fluorescent microsphere quality control product of the flow cytometer.
Performing rotary evaporation and centrifugal washing on the oscillated microsphere dispersion system to obtain the fluorescent microsphere quality control product of the flow cytometer, wherein the method comprises the following steps:
under the set evaporation condition, the oscillated microsphere dispersion system is subjected to rotary evaporation by a rotary evaporator, and after the evaporation is completed, the microsphere dispersion system is centrifuged for 2 to 5 minutes under a centrifuge with a relative centrifugal force of 2000 to 4000 g;
repeatedly washing with ethanol until the supernatant is colorless, discarding the supernatant, and dispersing and storing the fluorescent microspheres in 0.2% sodium dodecyl sulfate aqueous solution to obtain the quality control product of the fluorescent microspheres of the flow cytometer.
In a second aspect, the invention provides a quality control product of fluorescent microspheres of a flow cytometer, which is suitable for the preparation method of the quality control product of fluorescent microspheres of the flow cytometer in the first aspect,
the quality control product of the fluorescent microsphere of the flow cytometer comprises 0.3 g-1 g of blank microsphere, 100 mL-250 mL of swelling medium, 2 mg-10 mg of hydrophobic dye and 20 mL-50 mL of swelling agent, wherein the mass fraction of the swelling medium is 1-5%.
Wherein the blank microsphere is polystyrene microsphere, the particle size is any one of 3 μm, 7 μm and 10 μm, and the forward scattering light variation coefficient is less than 3%.
Wherein the hydrophobic dye is organic dye nile red.
The invention relates to a fluorescent microsphere quality control product of a flow cytometer and a preparation method thereof, wherein the fluorescent microsphere quality control product comprises 0.3 g-1 g of blank microsphere, 100 mL-250 mL of swelling medium, 2 mg-10 mg of hydrophobic dye and 20 mL-50 mL of swelling agent, the blank microsphere with the forward scattering light variation coefficient smaller than 3% is weighed to be 0.3 g-1 g, the blank microsphere is dissolved in the swelling medium with the mass fraction of 1-5%, the ultrasonic breaking instrument with the ultrasonic intensity of 50-100% is utilized for carrying out ultrasonic treatment for 1 min-5 min, 2 mg-10 mg of hydrophobic dye is weighed, and after the hydrophobic dye is dissolved in the swelling agent with the mass fraction of 20 mL-50 mL, the mixed solution is added to obtain a microsphere dispersion system; and carrying out ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and obtaining the fluorescent microsphere quality control product of the flow cytometer after centrifugal washing.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of steps of a method for preparing a fluorescent microsphere quality control product of a flow cytometer.
FIG. 2 is a histogram of particle size distribution of microspheres before and after staining in example 1 provided by the present invention.
FIG. 3 shows the results of on-line detection of the quality control flow cytometer of example 1 provided by the present invention.
FIG. 4 is a histogram of particle size distribution of microspheres before and after staining in example 2 provided by the present invention.
FIG. 5 shows the results of on-line detection of the quality control flow cytometer obtained in example 2 provided by the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
Referring to fig. 1, the invention provides a preparation method of a fluorescent microsphere quality control product of a flow cytometer, comprising the following steps:
s101, dissolving the weighed blank microspheres in a swelling medium, uniformly mixing by ultrasonic, and simultaneously adding the hydrophobic dye dissolved in the swelling agent into the mixed solution to obtain a microsphere dispersion system.
Specifically, weighing 0.3-1 g of blank microspheres with forward scattering light variation coefficients less than 3%, dissolving the blank microspheres in a swelling medium with mass fraction of 1-5%, and carrying out ultrasonic treatment for 1-5 min by using an ultrasonic crusher with ultrasonic intensity of 50% -100% to obtain a mixed solution; weighing 2-10 mg of hydrophobic dye, dissolving in 20-50 mL of swelling agent, and adding the mixed solution to obtain a microsphere dispersion system, wherein the microspheres are polystyrene microspheres, the particle size is suitable for a flow cytometer, preferably 3-10 mu m, 7 mu m and 10 mu m, and the forward scattered light variation coefficient of the blank microspheres is less than 3%.
S102, carrying out ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and obtaining the fluorescent microsphere quality control product of the flow cytometry after centrifugal washing.
Specifically, an ultrasonic breaker with the ultrasonic intensity of 50-100% is utilized to carry out ultrasonic treatment on the microsphere dispersion system for 1-5 min, and after the ultrasonic treatment is finished, the microsphere dispersion system is oscillated on a constant temperature shaking table with the temperature of room temperature and the rotation speed of 100-200 r/min for 30-90 min; under the set evaporation condition, the oscillated microsphere dispersion system is subjected to rotary evaporation by a rotary evaporator, and after the evaporation is completed, the microsphere dispersion system is centrifuged for 2 to 5 minutes under a centrifuge with a relative centrifugal force of 2000 to 4000 g; repeatedly washing with ethanol until the supernatant is colorless, discarding the supernatant, and dispersing and storing the fluorescent microspheres in 0.2% sodium dodecyl sulfate aqueous solution to obtain the quality control product of the fluorescent microspheres of the cytograph. Wherein the set evaporation condition is that the temperature is set to be 30-37 ℃, the rotating speed is 60-80 r/min, the pressure is 0.05-0.1 MPa, and the time is 10-30 min.
The quality control material of the flow cytometer fluorescent microsphere is polystyrene fluorescent microsphere, the particle size is basically unchanged, and the forward scattered light variation coefficient on the flow cytometer is less than 2%; the fluorescent dye can be excited by 488nm light in the microsphere instead of the surface, and the fluorescence intensity variation coefficient of each channel of fluorescence under the excitation of 488nm light of the flow cytometer is less than 2%.
The invention provides a flow cytometer fluorescent microsphere quality control product, which is suitable for a preparation method of the flow cytometer fluorescent microsphere quality control product, wherein the flow cytometer fluorescent microsphere quality control product comprises 0.3 g-1 g of blank microsphere, 100 mL-250 mL of swelling medium, 2 mg-10 mg of hydrophobic dye and 20 mL-50 mL of swelling agent, and the mass fraction of the swelling medium is 1-5%.
In this embodiment, the blank microspheres are polystyrene microspheres, the particle size is any one of 3 μm, 7 μm and 10 μm, and the forward scattered light variation coefficient is less than 3%; the swelling medium is a high molecular nonionic surfactant aqueous solution, preferably a Pluronic F127 aqueous solution; the hydrophobic dye is organic micromolecular dye nile red; the swelling agent is an organic solvent having a boiling point well below that of water and being miscible with water, preferably tetrahydrofuran.
Example 1:
weighing 0.3g of blank microspheres with the particle size of 3.39 mu m and the forward scattered light variation coefficient of 0.73%, dissolving the blank microspheres in 100mLPluromic F127 aqueous solution (the mass fraction is 1%), and carrying out ultrasonic treatment for 1min (the intensity is set to be 50%) to uniformly disperse the blank microspheres; 2mg of nile red dye is weighed and dissolved in 20mL of tetrahydrofuran, the two are mixed, after ultrasonic treatment is carried out for 2min (the intensity is set to be 100%), the mixture is oscillated on a constant-temperature shaking table for 30min (the room temperature is 21.3 ℃ and the rotating speed is 100 r/min), the mixture is transferred to a rotary evaporation device for rotary evaporation (the temperature is set to be 30 ℃ and the rotating speed is 60r/min, the pressure is 0.05MPa and the time is 10 min), and the product after rotary evaporation is centrifuged (the relative centrifugal force is 3000g and the time is 2 min) and is repeatedly washed by ethanol until the supernatant is colorless. And discarding the supernatant, and dispersing and storing the fluorescent microspheres in 0.2% sodium dodecyl sulfate aqueous solution to obtain the quality control product of the fluorescent microspheres of the cytograph.
As can be seen from FIG. 2, the particle size of the fluorescent microsphere quality control product of the flow cytometer is 3.45 μm, the particle size difference between the microspheres before and after dyeing is not large, and the microspheres still have good dispersibility; as can be seen from FIG. 3, the forward scattered light variation coefficient was 0.78% and the FL1-FL5 fluorescence channel variation coefficients for 488nm light excitation were all less than 2% after on-machine detection using a flow cytometer.
Example 2:
weighing 0.5g of blank microspheres with the particle size of 3.39 mu m and the forward scattered light variation coefficient of 0.73%, dissolving the blank microspheres in 150mLPluromic F127 aqueous solution (the mass fraction is 1%), and carrying out ultrasonic treatment for 2min (the intensity is set to be 50%) to uniformly disperse the blank microspheres; 3.5mg of nile red dye is weighed and dissolved in 35mL of tetrahydrofuran, the two are mixed, the ultrasonic treatment is carried out for 3min (the intensity is set to be 100%), the shaking table is carried out for 40 min (the room temperature is 22.8 ℃ C., the rotating speed is 120 r/min), the shaking table is transferred to a rotary evaporation device for rotary evaporation (the temperature is set to be 37 ℃ C., the rotating speed is 60r/min, the pressure is 0.07MPa, the time is 20 min), the product after rotary evaporation is centrifuged (the relative centrifugal force is 3000g, the time is 3 min), and the product is repeatedly washed by ethanol until the supernatant is colorless. And discarding the supernatant, and dispersing and storing the fluorescent microspheres in 0.2% sodium dodecyl sulfate aqueous solution to obtain the quality control product of the fluorescent microspheres of the cytograph.
As can be seen from FIG. 4, the particle size of the fluorescent microsphere quality control product of the flow cytometer is 3.49 μm, the particle size difference between the microspheres before and after dyeing is not large, and the microspheres still have good dispersibility; as can be seen from FIG. 5, the forward scattered light variation coefficient was 1.36% and the FL1-FL5 fluorescence channel variation coefficients for 488nm light excitation were all less than 2% after on-machine detection using a flow cytometer.
Test example:
and (3) taking part of quality control products obtained in examples 1 and 2 and foreign comparison quality control products of the same type, putting the quality control products into a 37 ℃ environment for light-shielding acceleration, and detecting the stability of the quality control products. The partially accelerated quality control was taken out at regular intervals and examined by flow cytometry, and the fluorescence intensity and the coefficient of variation obtained are shown in Table 1 below.
TABLE 1 flow cytometer fluorescent microsphere quality control acceleration stability detection results
The data in table 1 above shows that with increasing acceleration time, the fluorescence intensity of each channel of examples 1 and 2 and the comparative quality control product is reduced, but the reduction is not large, and at this time, each channel CV is substantially smaller than 2, which indicates that the quality control product obtained according to the present embodiment has excellent stability and fluorescence is not easy to leak.
Advantageous effects
1. The method for dyeing the monodisperse standard particle size microspheres by adopting the swelling method is simple in operation, the microspheres are not greatly influenced before and after swelling, and the dye is coated in the microspheres, so that the stability is good;
2. compared with the traditional surfactants such as sodium dodecyl sulfonate, tween and the like, the novel high-molecular nonionic surfactant, namely the pramipexole, has the functions of emulsification, dispersion and the like of the surfactant, can effectively prevent the mutual adhesion of microspheres in a swelling state through a steric hindrance action mechanism, can uniformly enter the inside of the microspheres, and can also lead the dye to uniformly enter the inside of the microspheres, so that the forward scattered light of the prepared fluorescent microspheres and the variation coefficient of each fluorescent channel are smaller.
3. The nile red is used as the dye adopted by the fluorescent microsphere quality control product of the flow cytometer, and has the advantages that: (1) The dye can be used for preparing the daily quality control of all channels of the flow cytometer, which is applicable to the flow cytometer with blue light (488 nm) of a laser and with the optical filter configuration range of 525 nm-785 nm (for example, the optical filters are configured as 525/45, 575/35, 670/20, 710/50 and 785/62), and the quality control of the flow cytometer is not required to be carried out by using various quality control products with specific single channels, so that the quality control process is simpler and more convenient; (2) Only one dye is used and is an organic micromolecular dye, so that the condition of the swelling process is more controllable, the dyeing of the microspheres is more facilitated, and the operation is simpler and more convenient.
4. Dichloromethane, chloroform and tetrahydrofuran are all common swelling agents, have low boiling points (40 ℃ and 61 ℃ and 66 ℃ in sequence), can effectively dye the microspheres by using a swelling method, adopt tetrahydrofuran which can be mutually dissolved with water (dichloromethane and chloroform are halogenated alkanes and are insoluble in water), are easy to remove by rotary evaporation, can enable the whole reaction end system to be in a dispersing and homogenizing state by simple operation (ultrasonic for 1-5 min), and are more favorable for fluorescent dye to uniformly enter the microspheres, so that the fluorescent intensity of the obtained fluorescent microspheres is highly uniform.
5. The preparation of the quality control product is carried out by adopting the formula and the method, so that the total consumption time is short, and the operation is simple and convenient; the prepared fluorescent microsphere quality control product of the flow cytometer has uniform particle size and high fluorescence intensity, the variation coefficient is less than 2%, and the dye is positioned in the microsphere, and the property of the dye is stable, so that the quality control product has excellent stability, and can meet the daily precision quality control, light path and photomultiplier debugging applicable to the flow cytometer.
The invention relates to a fluorescent microsphere quality control product of a flow cytometer and a preparation method thereof, wherein the fluorescent microsphere quality control product comprises 0.3 g-1 g of blank microsphere, 100 mL-250 mL of swelling medium, 2 mg-10 mg of hydrophobic dye and 20 mL-50 mL of swelling agent, the blank microsphere with the forward scattering light variation coefficient smaller than 3% is weighed to be 0.3 g-1 g, the blank microsphere is dissolved in the swelling medium with the mass fraction of 1-5%, the ultrasonic breaking instrument with the ultrasonic intensity of 50-100% is utilized for carrying out ultrasonic treatment for 1 min-5 min, 2 mg-10 mg of hydrophobic dye is weighed, and after the hydrophobic dye is dissolved in the swelling agent with the mass fraction of 20 mL-50 mL, the mixed solution is added to obtain a microsphere dispersion system; and carrying out ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and obtaining the fluorescent microsphere quality control product of the flow cytometer after centrifugal washing.
The above disclosure is only a preferred embodiment of the present invention, and it should be understood that the scope of the invention is not limited thereto, and those skilled in the art will appreciate that all or part of the procedures described above can be performed according to the equivalent changes of the claims, and still fall within the scope of the present invention.
Claims (3)
1. The preparation method of the fluorescent microsphere quality control product of the flow cytometer is characterized by comprising the following steps of:
dissolving the weighed blank microspheres in a swelling medium, uniformly mixing by ultrasonic, and simultaneously adding a hydrophobic dye dissolved in a swelling agent into a mixed solution to obtain a microsphere dispersion system;
weighing 0.3-1 g of blank microspheres with forward scattering light variation coefficient less than 3%, dissolving the blank microspheres in a swelling medium with mass fraction of 1-5%, and carrying out ultrasonic treatment for 1-5 min by using an ultrasonic crusher with ultrasonic intensity of 50% -100% to obtain a mixed solution;
weighing 2-10 mg of hydrophobic dye, dissolving in 20-50 mL of swelling agent, and adding the mixed solution to obtain a microsphere dispersion system;
the swelling medium is a novel polymer nonionic surfactant pramipexole;
performing ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system, and performing centrifugal washing to obtain a fluorescent microsphere quality control product of the flow cytometer;
the quality control product of the fluorescent microsphere of the flow cytometer comprises 0.3 to 1g of blank microsphere and 2 to 10mg of hydrophobic dye;
the blank microsphere is polystyrene microsphere, the particle size is any one of 3 mu m, 7 mu m and 10 mu m, and the forward scattering light variation coefficient is less than 3%;
the hydrophobic dye is organic dye nile red.
2. The method for preparing the fluorescent microsphere quality control of the flow cytometer according to claim 1, wherein the method for preparing the fluorescent microsphere quality control of the flow cytometer by performing ultrasonic, oscillation and rotary evaporation on the microsphere dispersion system and performing centrifugal washing comprises the following steps:
ultrasonic crushing the microsphere dispersion system for 1-5 min by using an ultrasonic crusher with the ultrasonic intensity of 50-100%, and oscillating for 30-90 min on a constant temperature shaking table with the temperature of room temperature and the rotating speed of 100-200 r/min after the ultrasonic treatment is finished;
and performing rotary evaporation and centrifugal washing on the oscillated microsphere dispersion system to obtain the fluorescent microsphere quality control product of the flow cytometer.
3. The method for preparing the fluorescent microsphere quality control of the flow cytometer according to claim 2, wherein the method for preparing the fluorescent microsphere quality control of the flow cytometer by rotary evaporation and centrifugal washing of the microsphere dispersion system after oscillation comprises the following steps:
under the set evaporation condition, the oscillated microsphere dispersion system is subjected to rotary evaporation by a rotary evaporator, and after the evaporation is completed, the microsphere dispersion system is centrifuged for 2 to 5 minutes under a centrifuge with a relative centrifugal force of 2000 to 4000 g;
repeatedly washing with ethanol until the supernatant is colorless, discarding the supernatant, and dispersing and storing the fluorescent microspheres in 0.2% sodium dodecyl sulfate aqueous solution to obtain the quality control product of the fluorescent microspheres of the cytograph.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8088629B1 (en) * | 2004-10-12 | 2012-01-03 | Luminex Corporation | Methods for forming dyed microspheres and populations of microspheres |
| CN106479483A (en) * | 2016-09-23 | 2017-03-08 | 北京海岸鸿蒙标准物质技术有限责任公司 | A kind of streaming Fluorescent Microsphere Standards matter and preparation method thereof |
| CN107523288A (en) * | 2017-07-24 | 2017-12-29 | 深圳清华大学研究院 | Near-infrared II areas high molecular fluorescent microballoons and preparation method thereof |
| CN110982298A (en) * | 2019-10-31 | 2020-04-10 | 复旦大学 | 1300nm near-infrared fluorescent microsphere with emission peak value ≧ 1300nm, and preparation and application thereof |
| CN110982512A (en) * | 2019-10-31 | 2020-04-10 | 复旦大学 | Multi-near-infrared II-region fluorescent dye co-doped fluorescent microsphere and preparation and application thereof |
| CN111875744A (en) * | 2020-08-17 | 2020-11-03 | 四川迈克生物新材料技术有限公司 | Method for preparing fluorescent microspheres and corresponding fluorescent microspheres |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10392557B2 (en) * | 2014-04-08 | 2019-08-27 | Dna Medicine Institute, Inc. | Multi-dye microparticles |
-
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- 2021-03-11 CN CN202110265274.3A patent/CN113088276B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8088629B1 (en) * | 2004-10-12 | 2012-01-03 | Luminex Corporation | Methods for forming dyed microspheres and populations of microspheres |
| CN106479483A (en) * | 2016-09-23 | 2017-03-08 | 北京海岸鸿蒙标准物质技术有限责任公司 | A kind of streaming Fluorescent Microsphere Standards matter and preparation method thereof |
| CN107523288A (en) * | 2017-07-24 | 2017-12-29 | 深圳清华大学研究院 | Near-infrared II areas high molecular fluorescent microballoons and preparation method thereof |
| CN110982298A (en) * | 2019-10-31 | 2020-04-10 | 复旦大学 | 1300nm near-infrared fluorescent microsphere with emission peak value ≧ 1300nm, and preparation and application thereof |
| CN110982512A (en) * | 2019-10-31 | 2020-04-10 | 复旦大学 | Multi-near-infrared II-region fluorescent dye co-doped fluorescent microsphere and preparation and application thereof |
| CN111875744A (en) * | 2020-08-17 | 2020-11-03 | 四川迈克生物新材料技术有限公司 | Method for preparing fluorescent microspheres and corresponding fluorescent microspheres |
Non-Patent Citations (1)
| Title |
|---|
| Yanxue Cai等.Rapid and Sensitive Detection of Cardiac Troponin I for Point-of-Care Tests Based on Red Fluorescent Microspheres.《Molecules》.2018,第23卷第1102-1-1102-13页. * |
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