A method for rapid measurement of fluorescent microsphere particle size
By testing the signal value decay percentage of fluorescent microspheres to determine the particle size, the problem of complex particle size measurement results in the prior art is solved, and a fast, accurate and economical particle size measurement of fluorescent microspheres is achieved.
Patent Information
- Application Number
- CN202410159226.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-04
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2044-02-04
AI Technical Summary
Existing methods for testing fluorescent microsphere particle size, such as laser particle size distribution meter and dynamic light scattering, have complex problems of result shift and preprocessing, and scanning electron microscopy is accurate, time-consuming and expensive.
By continuously testing the signal value decay percentage of fluorescent microspheres of different particle sizes, fitting a linear relationship, determining a standard curve, and using self-made fluorescent microspheres and independently developed fluorescent signal value testing machine with fixed light intensity fixed wavelengths, simplifying pre-processing And measure particle size quickly.
Accurate measurement consistent with the scanning electron microscope results is achieved, with simple pre-processing, short time-consuming, low-price, and no change in sample status, ensuring the accuracy and speed of measurement.
Smart Images
Figure CN118168993B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microspheres, and in particular to a method for rapidly measuring the particle diameter of fluorescent microspheres. Background Art
[0002] At present, the main methods for testing the particle size of polymer microspheres are: laser particle size distribution analyzer, dynamic light scattering (DLS), scanning electron microscopy (SEM), etc.
[0003] The main detection process of the laser particle size distribution method is to prepare the sample into an aqueous solution of a certain concentration and add it to the sample pool of the laser particle size distribution instrument. When the shading rate reaches the minimum concentration requirement of the instrument, the detection begins to obtain the particle size and distribution of the polymer microspheres. For the same sample, the laser particle size distribution instrument has a variety of peak states after detection, such as double peaks, multiple peaks, broad peaks, etc., and the detection results vary greatly. In addition, the pretreatment changes the pH of the real sample, which causes trouble in judging the real particle size of the polymer microspheres.
[0004] When light propagates, its alternating electromagnetic field causes the electrons of the molecules in the medium to produce forced vibrations. This vibration becomes a secondary wave source that radiates electromagnetic waves in all directions, which is the cause of light scattering. When the polarizability of the microparticles or molecules is different from that of the surrounding medium, scattered light can be observed. If there is no energy transfer in the scattering, the frequency of the scattered light should be the same as the frequency of the incident light, that is, the so-called static light scattering occurs. But in fact, the particles or molecules in the medium are constantly in Brownian motion, and the incident light quasi-elasticly collides with the molecules or particles in thermal motion, causing them to undergo tiny energy changes. As a result, the scattered light field is broadened with the frequency of the incident light as the center, and the so-called dynamic light scattering occurs at this time. However, because the scattering intensity of the particles is related to the laser intensity in addition to the particle size, the scattering intensity of the ultrafine (less than tens of nanometers) particles is often mainly related to the laser light intensity. Therefore, the selection of the laser light source is particularly important, which directly affects the resolution of the instrument. In addition, different laser particle size analyzers are equipped with different data processing systems, which is also the main reason for the deviation between the measured particle size and the actual particle size.
[0005] SEM imaging is to obtain the signals of secondary electrons and backscattered electrons through the detector. If the sample is not conductive or has poor conductivity, it will cause the accumulation of excess electrons or free particles on the sample surface and cannot be conducted away in time. After a certain degree, charging and discharging will occur repeatedly, which will eventually affect the transmission of electronic signals and cause image distortion, deformation, shaking, etc. Therefore, polymer microspheres need to be treated with surface gold spraying. The size of the gold spraying particles and the target material, the height of the sample placement, time, current, and the presence or absence of protective gas all have an impact, which is time-consuming and the SEM is expensive.
[0006] Existing methods for testing the particle size of fluorescent microspheres, such as laser particle size distribution analyzer and dynamic light scattering (DLS), require the sample to be prepared into an aqueous solution of a certain concentration. The test results are often offset from the actual particle size and are usually larger than the scanning electron microscope (SEM) test results. The scanning electron microscope (SEM) can clearly observe the morphology of the microspheres and the test results are relatively accurate, but the SEM requires the sample to be gold-plated. The size of the gold-sprayed particles and the target material, the height of the sample placement, time, current, and the presence or absence of protective gas all have an impact, and consistency cannot be guaranteed. It is time-consuming and expensive. Summary of the invention
[0007] The present invention firstly provides a method for rapidly measuring the particle size of fluorescent microspheres, which comprises the step of determining the particle size of the fluorescent microspheres by continuously testing the attenuation percentage of the signal value of fluorescent microspheres with different particle sizes.
[0008] Optionally, the method further comprises the step of fitting a linear relationship between different particle sizes and corresponding signal value attenuation percentages to determine a standard curve.
[0009] The present invention also provides a method for quickly measuring the particle size of fluorescent microspheres, comprising the following steps:
[0010] (1) Fluorescent microspheres of different particle sizes are diluted with purified water to a value of about 10,000 MV, and then the fluorescent microspheres of different particle sizes are sprayed on PVC plates with nitrocellulose membranes, placed in an oven for baking, and the baked PVC plates are cut and placed in the card slots to form reagent cards for later use;
[0011] (2) Testing the signal value of fluorescent microspheres of different particle sizes; statistically analyzing the signal value attenuation percentages corresponding to the fluorescent microspheres of different particle sizes; fitting a linear relationship between the different particle sizes and the corresponding signal value attenuation percentages; determining a standard curve; and determining the particle size of the microspheres based on the signal value attenuation percentages of the fluorescent microspheres.
[0012] Optionally, the oven baking condition is 37° C. for 30 min.
[0013] Optionally, the fluorescence signal value test uses a fluorescence signal value testing machine PMDT9000.
[0014] Optionally, the parameters of the fluorescence signal value testing machine PMDT9000 are an excitation wavelength of 365 nm and an emission wavelength of 610 nm.
[0015] The present invention also provides a method for preparing fluorescent microspheres for testing, which comprises the following steps:
[0016] (1) Preparation of carboxyl microspheres: adjust the alcohol-water ratio of the reaction system, add corresponding proportions of anhydrous ethanol and purified water, emulsifier solution and purified styrene monomer into a three-necked flask, and stir; then introduce nitrogen to remove oxygen, absorb the initiator solution, keep the device in a closed reaction state, continue stirring, then inject the carboxyl monomer, and absorb the initiator solution again; continue stirring, seal the reaction; stop heating, add hydrochloric acid, continue stirring, and acid hydrolyze overnight; centrifuge and wash with ethanol solution and purified water, and store at 4°C for later use;
[0017] (2) Preparation of fluorescent microspheres: Weigh the europium complex into a transparent glass bottle, add a swelling agent, then add an emulsifier aqueous solution, ultrasonicate to fully emulsify it, then add the carboxyl microspheres and purified water described in step (1), then put the glass bottle into a water bath and stir overnight; adjust the temperature of the rotary evaporator, perform vacuum distillation, and centrifuge and wash the fluorescent microspheres after vacuum distillation with an ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume as the carboxyl microspheres added during swelling;
[0018] (3) Carboxyl microspheres of different sizes can be prepared by adjusting the alcohol-water ratio of the reaction system;
[0019] Optionally, the different particle sizes include 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm.
[0020] Optionally, the preparation of carboxyl microspheres (1): adjust the alcohol-water ratio of the reaction system, set the oil bath temperature to 80°C, add corresponding proportions of anhydrous ethanol and purified water, 0.4% styrene by volume emulsifier solution and purified styrene monomer into a three-necked flask, and start stirring at 400 r / min; then introduce nitrogen to remove oxygen, use a pointed needle glass injector to draw 1% styrene by volume initiator solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device sealed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 3% styrene by volume carboxyl monomer, and at the same time use a pointed needle glass injector to draw 1% styrene by volume initiator solution; continue stirring at 400 r / min, seal and react for 8h; stop heating, add 90ml of 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight; centrifuge and wash with ethanol solution and purified water, and store at 4°C for later use.
[0021] Optionally, the preparation of the fluorescent microspheres (2) is as follows: 0.5% carboxyl microsphere volume of europium complex is weighed into a 20 ml transparent glass bottle, half the volume of carboxyl microsphere volume of swelling agent is added, then 0.4% carboxyl microsphere volume of emulsifier aqueous solution is added, and the solution is placed in an ultrasonic machine for full emulsification, followed by the addition of the carboxyl microspheres in the first step and 4 times the amount of purified water as carboxyl microspheres, followed by the placement of the glass bottle in a water bath, the temperature of the water bath is adjusted to 35°C, and the glass bottle is stirred overnight. The temperature of the rotary evaporator is adjusted to 40°C, and the vacuum pump is turned on at the same time. The vacuum pump is in a negative pressure state, and the vacuum distillation is performed under reduced pressure for 2 hours. The fluorescent microspheres after reduced pressure distillation are centrifugally washed with ethanol solution and purified water; after washing, the supernatant is removed, and the lower layer of fluorescent microspheres is redissolved with pure water to the same volume as the carboxyl microspheres added during swelling.
[0022] Finally, the present invention provides a product for rapidly measuring the particle size of fluorescent microspheres, which comprises reagents and equipment for implementing the above-mentioned method for rapidly measuring the particle size of fluorescent microspheres. Optionally, the reagents comprise fluorescent microspheres for testing, and the equipment comprises a fluorescence signal value testing machine.
[0023] Compared with the prior art, the present invention has at least the following beneficial effects:
[0024] The present invention provides a method for determining the particle size of fluorescent microspheres by testing the attenuation values of fluorescent microspheres with different particle sizes. The test result of this method is consistent with that of a scanning electron microscope (SEM), but the pretreatment is simple and the original state of the test sample is not changed.
[0025] The present invention can determine the particle size of fluorescent microspheres by testing the attenuation value of fluorescent microspheres with different particle sizes. The fluorescent microspheres with different particle sizes are made by our company, and the testing machine is a corresponding machine with fixed light intensity and fixed wavelength independently developed by our company; the pretreatment operation of the invention is simple, time-saving, low-cost and highly accurate. The specific operation method is to spray the fluorescent microspheres with different particle sizes diluted to 10,000 MV values on a PVC plate with a nitrocellulose membrane at the same spraying rate, which greatly shortens the pretreatment time and does not change the authenticity of the fluorescent microspheres; at the same time, the company's fixed light source and fixed wavelength instrument is used to detect the fluorescence signal value, and the signal value is tested continuously for multiple times. The particle size is determined by the percentage of signal value attenuation. The present invention can quickly and accurately determine the particle size of the fluorescent microspheres while ensuring the authenticity of the sample. The fluorescence intensity is related to the concentration of the microspheres and the particle size, and the particle size cannot be judged only by the fluorescence intensity. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 For the standard curve. DETAILED DESCRIPTION
[0027] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.
[0028] Example 1 Principle of the method for rapid measurement of fluorescent microsphere particle size
[0029] The invention determines the particle size of fluorescent microspheres by continuously testing the signal value attenuation percentage of fluorescent microspheres with different particle sizes; the fluorescent microspheres used for testing are made by the company; the testing machine is a PMDT9000 fluorescence tester with fixed light intensity and fixed wavelength independently developed by the company; the pretreatment operation of the invention is simple, time-saving, low-cost and highly accurate.
[0030] The specific experimental plan is as follows:
[0031] Step 1: Stick the nitrocellulose membrane to the PVC board, turn on the film sprayer, set the rate to 0.06μl / mm, dilute the fluorescent microspheres of different particle sizes with purified water to around 10,000 MV value, and then spray the fluorescent microspheres of different particle sizes on the PVC board with nitrocellulose membrane, put it in an oven at 37℃ for 30min, cut the baked PVC board into 400mm with a strip cutter and put it into the card slot to buckle it into a reagent card for use.
[0032] Step 2: Use the fluorescence signal value test machine PMDT9000 (excitation wavelength 365nm, emission wavelength 610nm) independently developed by our company to conduct three groups of signal value tests on fluorescent microspheres of different particle sizes, and test each group 20 times in succession; statistical data is collected to obtain the signal value attenuation percentage corresponding to fluorescent microspheres of different particle sizes, and a linear relationship is fitted from different particle sizes and the corresponding signal value attenuation percentages, and a standard curve is determined, so as to determine the particle size of the microspheres according to the signal value attenuation percentage of the fluorescent microspheres.
[0033] When testing, you can first dilute it to 10,000 MV and then measure the attenuation, or you can directly measure the attenuation.
[0034] Example 2 Preparation method of fluorescent microspheres for testing
[0035] Step 1: Preparation of carboxyl microspheres: Adjust the alcohol-water ratio of the reaction system, set the oil bath temperature to 80°C, add the corresponding proportion of anhydrous ethanol and purified water, 0.4% styrene volume emulsifier solution and refined styrene monomer to the three-necked flask, and start stirring at 400r / min; then introduce nitrogen to remove oxygen, use a pointed needle glass injector to absorb 1% styrene volume initiator solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 3% styrene volume carboxyl monomer, and use a pointed needle glass injector to absorb 1% styrene volume initiator solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml of 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, and store at 4°C for use.
[0036] Step 2: Preparation of fluorescent microspheres: Weigh 0.5% carboxyl microsphere volume of europium complex into a 20ml transparent glass bottle, add half the volume of carboxyl microsphere volume of swelling agent, then add 0.4% carboxyl microsphere volume of emulsifier aqueous solution, put it into an ultrasonic machine to fully emulsify it, then add the carboxyl microspheres in the first step and 4 times the purified water of carboxyl microspheres, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume as the carboxyl microspheres added during swelling.
[0037] By adjusting the alcohol-water ratio of the reaction system, carboxyl microspheres of different particle sizes can be prepared, thereby preparing fluorescent microspheres of 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm.
[0038] Example 3 Preparation of 100 nm fluorescent microspheres
[0039] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 72ml of purified water and 18ml of anhydrous ethanol (ethanol-water ratio 2:8), 0.3ml of 10% SDS and 7ml of refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml of methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml of 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 100nm.
[0040] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0041] Example 4 Preparation of 150 nm fluorescent microspheres
[0042] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 63ml purified water and 27ml anhydrous ethanol (ethanol-water ratio 3:7), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 150nm.
[0043] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0044] Example 5 Preparation of 200nm fluorescent microspheres
[0045] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 54ml purified water and 36ml anhydrous ethanol (ethanol-water ratio 4:6), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 200nm.
[0046] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0047] Example 6 Preparation of 250 nm fluorescent microspheres
[0048] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 45ml purified water and 45ml anhydrous ethanol (ethanol-water ratio 5:5), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 250nm.
[0049] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0050] Example 7 Preparation of 300nm fluorescent microspheres
[0051] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 36ml purified water and 54ml anhydrous ethanol (ethanol-water ratio 6:4), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 300nm.
[0052] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0053] Example 8 Preparation of 350nm fluorescent microspheres
[0054] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 27ml purified water and 63ml anhydrous ethanol (ethanol-water ratio 7:3), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and start stirring at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 350nm.
[0055] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0056] Example 9 Preparation of 400nm fluorescent microspheres
[0057] Step 1: Preparation of carboxyl microspheres: Set the oil bath temperature to 80°C, add 18ml purified water and 72ml anhydrous ethanol (ethanol-water ratio 8:2), 0.3ml 10% SDS and 7ml refined styrene monomer to a three-necked flask and stir at 400r / min; then introduce nitrogen to remove oxygen, use a sharp needle glass injector to absorb 700μl of 10% KPS solution (the injector needs to be preheated in warm water to prevent KPS from crystallizing in the injector), keep the device closed and react, continue stirring for 0.5h, then pierce the rubber stopper with a needle and inject 2ml methyl methacrylate, and use a sharp needle glass injector to absorb 700μl of 10% KPS solution again; continue stirring at 400r / min, seal and react for 8h; stop heating, add 90ml 5% hydrochloric acid, continue stirring, and acid hydrolyze overnight. Centrifuge and wash with ethanol solution and purified water, store at 4°C for use, and the prepared carboxyl microspheres are 400nm.
[0058] Step 2: Preparation of fluorescent microspheres: Weigh 0.02g ((1,10-phenanthroline) tri[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III)) into a 20ml transparent glass bottle, add 2ml dichloromethane, then add 0.15ml of 10% SDS aqueous solution, put it into an ultrasonic machine for ultrasonication to fully emulsify it, then add 4ml of carboxyl microspheres in step 1 and 16ml of purified water, then put the glass bottle into a water bath, adjust the temperature of the water bath to 35℃, and stir overnight. Adjust the temperature of the rotary evaporator to 40℃, turn on the vacuum pump at the same time, and the vacuum pump is in a negative pressure state. Reduced pressure distillation for 2h, and the fluorescent microspheres after reduced pressure distillation are centrifuged and washed with ethanol solution and purified water; after washing, remove the supernatant, and redissolve the lower layer of fluorescent microspheres with pure water to the same volume of 4ml as the carboxyl microspheres added during swelling, and store at 4℃ for later use.
[0059] Example 10 Method for Rapidly Measuring the Particle Size of Fluorescent Microspheres
[0060] Step 1: Stick the nitrocellulose membrane to the PVC board, turn on the film sprayer, set the rate to 0.06μl / mm, dilute the fluorescent microspheres of different particle sizes with purified water to around 10,000 MV value, and then spray the fluorescent microspheres of 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm on the stuck PVC board respectively, put it in an oven at 37℃ for 30min, cut the baked PVC board into 400mm with a strip cutting machine and put it into the card slot to buckle it into a reagent card for standby use.
[0061] Step 2: Use the PMDT9000 (excitation wavelength 365nm, emission wavelength 610nm) independently developed by our company to test the signal values of three groups of fluorescent microspheres of 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, and 400nm, and test each group 20 times in a row; statistical data is obtained to obtain the signal value attenuation percentage corresponding to fluorescent microspheres of different particle sizes. The linear relationship between different particle sizes and the corresponding signal value attenuation percentage is fitted to determine the standard curve, so as to determine the particle size of the microsphere according to the attenuation percentage of the fluorescent microsphere signal value. The results are shown in Table 1, Table 2, Table 3, and Table 4.
[0062] Table 1 Test results of 100nm and 150nm
[0063]
[0064] Table 2 Different test results of 200nm and 250nm
[0065]
[0066] Table 3 Test results of 300nm, 350nm and 400nm
[0067]
[0068] Table 4 Signal attenuation results of different fluorescent microsphere particle sizes
[0069]
[0070] Example 11 Preparation and verification of standard curve
[0071] Step 1: Dilute the homemade fluorescent microspheres of different particle sizes with purified water to about 10,000 MV value, stick the nitrocellulose membrane to the PVC board, turn on the film sprayer, set the rate to 0.06μl / mm, spray the fluorescent microspheres of 120nm, 150nm, 180nm, 230nm, 300nm, 330nm, and 400nm on the stuck PVC board respectively, put it in an oven at 37℃ for 30min, cut the baked PVC board into 400mm with a strip cutting machine and put it into the card slot to buckle it into a reagent card for standby use.
[0072] Step 2: Use the PMDT9000 (excitation wavelength 365nm, emission wavelength 610nm) independently developed by our company to test the signal values of five groups of fluorescent microspheres of 120nm, 150nm, 180nm, 230nm, 300nm, 330nm, and 400nm, and test each group 20 times in a row; collect statistics, calculate the attenuation percentage of the signal values of different fluorescent microspheres, and bring the attenuation percentage into the standard curve to calculate the average value of the five groups of data particle size, and find the deviation between the theoretical value and the calculated value, and the deviation is within 3%. When testing, you can dilute to this MV value and then measure the attenuation, or you can directly measure the attenuation.
[0073]
[0074]
[0075]
[0076]
[0077] The linear relationship between different particle sizes and the corresponding signal value attenuation percentages was fitted to determine the standard curve, so that the particle size of the microspheres was determined according to the signal value attenuation percentage of the fluorescent microspheres. The results show that the present invention determines the particle size of the fluorescent microspheres by continuously testing the signal value attenuation percentages of fluorescent microspheres with different particle sizes.
[0078] In summary, the present invention can determine the particle size of fluorescent microspheres by testing the attenuation value of fluorescent microspheres of different particle sizes. The fluorescent microspheres of different particle sizes are made by our company, and the testing machine is a corresponding machine with fixed light intensity and fixed wavelength independently developed by our company; the pretreatment operation of the invention is simple, time-saving, low-cost and highly accurate. The pretreatment time is greatly shortened and the fluorescent microspheres are not processed to change their authenticity; at the same time, the instrument with fixed light source and fixed wavelength of our company is used to detect the fluorescence signal value, and the signal value is tested continuously for multiple times. The particle size is determined by the percentage of signal value attenuation. While ensuring the authenticity of the sample, the present invention can quickly and accurately determine the particle size of the fluorescent microspheres.
[0079] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for rapidly measuring the particle size of fluorescent microspheres, characterized in that: The following steps are involved: (1) Fluorescent microspheres of different particle sizes were diluted with purified water to a value of about 10,000 mV, and then sprayed on PVC plates with nitrocellulose membranes, placed in an oven for baking, and the baked PVC plates were cut and placed in the card slots to form reagent cards for later use; (2) Using a fluorescence signal value testing machine PMDT9000, three groups of signal value tests were performed on the fluorescent microspheres of each particle size, and each group was tested 20 times in succession. The parameters of the fluorescence signal value testing machine PMDT9000 were as follows: an excitation wavelength of 365 nm and an emission wavelength of 610 nm. Statistical data were obtained to obtain the attenuation percentage of the signal value of the fluorescent microspheres with different particle sizes in the 20th test. The attenuation percentage of the signal value of the 20th test was the attenuation percentage of the signal value of the 20th test relative to the signal value of the 1st test. A linear relationship was fitted from the attenuation percentages of different particle sizes and corresponding signal values, and a standard curve was determined. Thus, the particle size of the fluorescent microspheres to be tested was determined according to the attenuation percentage of the signal value of the fluorescent microspheres to be tested.
2. The method for rapidly measuring the particle size of fluorescent microspheres according to claim 1, characterized in that: The oven baking condition is 37° C. for 30 min.