A method for preparing time-resolved fluorescent PS / cPGMA nanoparticles

Abstract

The application belongs to the technical field of polymer microsphere preparation, and discloses a preparation method of time-resolved fluorescence PS / cPGMA nanoparticles, wherein a chelate ligand of Eu is dissolved in anhydrous ethanol and stirred and dissolved; the pH value of a mixed solution is adjusted to 6.0-7.0 by using ammonia water, and an anhydrous ethanol solution containing EuCl3·6H2O is added dropwise to carry out a dark reaction; standing, distillation, and Eu chelate are obtained; PS / cPGMA core-shell type nanoparticles are dispersed in deionized water to prepare a 10% PS microsphere aqueous solution, and SDS is added to ultrasonic dispersion; the Eu chelate is dissolved in dichloromethane, the swelling liquid of the dissolved Eu chelate is added dropwise into the PS / cPGMA core-shell type nanoparticles, and stirring is carried out and then condensation is carried out in a refrigerator; after the reaction is completed, water is added for dilution, centrifugation is carried out, and deionized water is added for redissolution. The application uses a PGMA shell type material to improve the stability and sensitivity of the time-resolved fluorescence nanoparticles in an aqueous solution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polymer microsphere preparation, and particularly relates to a preparation method of time-resolved fluorescent PS / cPGMA nanospheres. BACKGROUND

[0002] Currently, time-resolved fluorescence immunochromatography (TRFIA) is a non-radioactive labeling immunological analysis technology developed on the basis of traditional fluorescent immunological analysis technologies such as radioimmunoassay technology, enzyme-linked immunological analysis technology and luminescent immunological analysis technology. TRFIA uses time-resolved fluorescent nanospheres (TRFN) as a labeling material and combines the high specificity of antigen-antibody and high sensitivity in the field of ultra-micro detection. TRFIA is a new detection technology, and its principle is to use rare earth ions with long fluorescent half-life as a label. Since the label has a large Stokes shift (150 nm) and a fluorescent lifetime that is 5-6 orders of magnitude higher than the fluorescent lifetime of background substances, the signal of the label can be effectively eliminated by delaying the measurement time and measuring the signal of the label after the fluorescence of the background substances is fully attenuated, thereby obtaining high sensitivity.

[0003] As a special functional microparticle, TRFN is commonly used to coat lanthanide chelates with polystyrene (PS) nanospheres. A plurality of fluorescent molecules can be coated in each nanosphere, which greatly improves the labeling efficiency of fluorescence and effectively improves the analysis sensitivity. Meanwhile, the surface of the fluorescent nanosphere is modified with a suitable density of carboxyl or other functional groups, which is used for covalent coupling with proteins or antibodies, thereby improving the stability of the label. More importantly, since the rare earth ions embedded in the microspheres have been chelated, there is no need for dissociation enhancement. Therefore, the problem that traditional dissociation enhanced lanthanide fluorescent immunoassay (DELFIA) can only be used in liquid phase and cannot be used in solid phase interface reaction is fundamentally solved, thereby solving the technical bottleneck of applying time-resolved fluorescence to the immunochromatography platform. On this basis, a quantitative detection technology with a sensitivity that is 1-3 orders of magnitude higher than that of ordinary colloidal gold or red latex immunochromatography can be developed.

[0004] The most common method for preparing time-resolved nanoparticles is to add europium (Eu) and its ligand to the carboxylated polystyrene emulsion, for example, Hutinen P et al. prepared polystyrene nanoparticles with time-resolved fluorescence by adding europium (Eu) and its ligand to the carboxylated polystyrene, and chelating on the surface of carboxylated polystyrene, and adjusting the carboxyl content on the surface of carboxylated polystyrene, it is found that with the increase of carboxyl content on the surface of carboxylated polystyrene, the fluorescence decay time is slightly prolonged, but the Eu chelate mainly exists in the form of physical adsorption on the surface of carboxylated polystyrene, which is easy to fall off or cause fluorescence quenching when in contact with water. For example, Desbiens J et al. combined Eu chelate with polystyrene by miniemulsion polymerization method to prepare polystyrene nanoparticles loaded with Eu(tta)3Phen, and the maximum average loading capacity of the final particles is about 2% (w / w). The instability of the miniemulsion at a higher loading level can lead to a decrease in particle conversion and Eu content. For example, Tao Dongliang et al. dispersed rare earth complex Eu(tta)3Phen in isopropyl alcohol, and prepared a new type of fluorescent material SiO2 / Eu(tta)3Phen by hydrolyzing tetraethyl orthosilicate (TEOS). The fluorescence intensity of the rare earth complex is doubled after being wrapped, but the fluorescence lifetime is also reduced. For example, Peng Chao et al. prepared polystyrene microspheres containing europium complex by swelling method, and investigated the effects of different swelling agents, swelling agent dosage, swelling time and fluorescence stability, etc. The fluorescence intensity of the fluorescent nanoparticles can be adjusted by adjusting the amount of europium complex added.

[0005] However, the above-mentioned methods prepare polystyrene nanoparticles with time-resolved fluorescence by emulsion polymerization, swelling and other methods, but the nanoparticles have certain uniformity, but the fluorescence intensity is often low and the stability is poor, which leads to problems such as leakage of fluorescent dyes and fluorescence quenching during long-term storage.

[0006] Through the above analysis, the problems and defects of the prior art are that the existing commercial time-resolved fluorescent microspheres have the problems of poor stability of nanoparticles, insufficient fluorescence intensity, leakage of fluorescent dyes from polystyrene nanospheres during long-term storage, or fluorescence quenching, which affects the stability and accuracy of the detection results of the time-resolved immunochromatographic test strip. SUMMARY

[0007] In view of the problems existing in the prior art, the present application provides a preparation method of time-resolved fluorescent PS / cPGMAo nanoparticles, and particularly relates to a time-resolved fluorescent PS / cPGMA core-shell nanoparticle and a preparation method thereof.

[0008] The present application is achieved by a preparation method of time-resolved fluorescent PS / cPGMA nanoparticles, which comprises the following steps: dissolving a chelate ligand of Eu in anhydrous ethanol and stirring the solution at room temperature; adjusting the pH value of the mixed solution to 6.0-7.0 by using ammonia water, adding an anhydrous ethanol solution containing EuCl3·6H2O dropwise, and stirring the solution at room temperature in the dark; standing overnight, removing ethanol by distillation, and obtaining a Eu chelate; dispersing PS / cPGMA core-shell nanoparticles in deionized water, preparing a 10% PS microsphere aqueous solution, and adding SDS for ultrasonic dispersion; dissolving the Eu chelate in dichloromethane, adding the swollen solution of the dissolved Eu chelate to the PS / cPGMA core-shell nanoparticles, stirring the solution, and then placing the solution in a refrigerator for condensation reaction; after the reaction is completed, diluting the solution with water, centrifuging the solution, and adding deionized water for redissolution.

[0009] Further, the preparation method of time-resolved fluorescent PS / cPGMA nanoparticles comprises the following steps:

[0010] Step one, forming a Eu chelate by chelation of Eu and a ligand of Eu;

[0011] Step two, swelling PS / cPGMA core-shell nanoparticles;

[0012] Step three, adding the swollen PS / cPGMA core-shell nanoparticles to the Eu chelate;

[0013] Step four, condensing and washing the swollen and dyed PS / cPGMA core-shell nanoparticles.

[0014] Further, the ligand of the Eu chelate in step one comprises 2-thiophenecarbonyl trifluoroacetone, 4,4,4-trifluoro-1,2-naphthalene-1,3-butanedione, dibenzoylmethane, 1,10-phenanthroline, and n-trioctylphosphine oxide.

[0015] Further, in step one, any one or two of the ligands of the Eu chelate form a ligand or a bidentate ligand, the pH value is adjusted to 6.0-7.0 by using ammonia water, and a Eu chelate is obtained.

[0016] Further, in step two, the amount of europium chelate added during swelling is 1-50% of the mass of the carboxyl nanoparticles, the proportion of the aqueous solution in the swelling is 1-90%, and the proportion of the swelling agent is 1-90%.

[0017] Further, in step two, the swelling step of the carboxyl nanoparticles is: dispersing the carboxyl nanoparticles in deionized water containing 0.1-5% sodium dodecyl benzene sulfonate.

[0018] Further, in step two, the swelling agent is any one or several of dichloromethane, anhydrous ethanol, isopropanol, butanol, ethylene glycol, benzyl alcohol, tetrahydrofuran, chloroform, acetone or N,N-dimethylformamide.

[0019] Further, in step two, the swelling temperature is 25-150°C, the swelling time is 0.5-10h, and the stirring speed is 100-1000rpm.

[0020] Further, in step three, the PS / cPGMA nanoparticles used have a particle size of 40-600nm, a shell thickness of 2-50nm, a cross-linking degree of 0.5-10%, and a carboxyl density of 0.1-1mmol / g.

[0021] Further, the condensation process in step four includes: reducing the temperature to 4-37°C and diluting the swelling agent concentration by 1-10 times, the condensation time is 3-48h, and the condensation speed is 50-200rpm; washing with deionized water or anhydrous ethanol 1-5 times, the centrifugal force is 4000-15000rpm, and the centrifugal time is 10-60min.

[0022] In combination with the above technical solutions and the technical problems solved, the technical solutions to be protected by the present application have the following advantages and positive effects:

[0023] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty in solving the problems, the technical solutions to be protected by the present application are closely combined with the results and data obtained during the research and development process, and the technical problems solved by the technical solutions are analyzed in detail and deeply. Some creative technical effects brought about after solving the problems are described as follows:

[0024] (1) The preparation method of the time-resolved fluorescent PS / cPGMA core-shell nanoparticles provided by the present application uses PGMA shell material to improve the stability of the time-resolved fluorescent nanoparticles in aqueous solution.

[0025] (2) The preparation method of the time-resolved fluorescence PS / cPGMA core-shell type nanoparticle provided by the present application can increase the content of europium and its complex loaded on the carboxyl polystyrene nanoparticle by swelling method, and the particle uniformity and stability are both good, the fluorescence intensity of the prepared time-resolved fluorescence nanoparticle is improved, and thus the sensitivity of the time-resolved immunochromatography technology is improved.

[0026] (3) The preparation method of the time-resolved fluorescence PS / cPGMA core-shell type nanoparticle provided by the present application wraps the shell material of PGMA on the carboxyl nanoparticle with time resolution, thereby improving the stability of europium and its complex on the nanoparticle, improving the fluorescence intensity of the time-resolved fluorescence nanoparticle, reducing fluorescence leakage and fluorescence quenching, and improving the stability of the time-resolved fluorescence nanoparticle in long-term storage.

[0027] Secondly, the technical effect and advantages of the technical solution to be protected by the present application are described as follows from the perspective of the product as a whole or from the perspective of the product:

[0028] The present application provides a preparation method of time-resolved fluorescence PS / cPGMA core-shell type nanoparticle, which solves the problems of the existing time-resolved nanoparticle, such as the aggregation of Eu and its complex loaded on the polystyrene (PS) nanoparticle in the form of copolymerization or physical adsorption, insufficient fluorescence intensity, fluorescence quenching, etc. The time-resolved fluorescence nanoparticle PS / cPGMA core-shell type nanoparticle prepared by the present application has better uniformity; the time-resolved fluorescence nanoparticle PS / cPGMA core-shell type nanoparticle prepared by the present application has higher fluorescence intensity under excitation of the same wavelength light path; and the time-resolved fluorescence nanoparticle PS / cPGMA core-shell type nanoparticle prepared by the present application has less fluorescence leakage and quenching in long-term storage, and has better nanoparticle stability.

[0029] Thirdly, the creativity of the claims of the present application is also embodied in the following important aspects:

[0030] (1) The technical solution of the present application fills the technical gap in the industry at home and abroad: the PS / cPGMA core-shell type nanoparticle is used to prepare time-resolved fluorescence nanoparticle, and no one has used the PS / cPGMA core-shell type nanoparticle to prepare time-resolved fluorescence nanoparticle.

[0031] (2) The technical solution of the present application solves the technical problems that people have been eager to solve but have failed to succeed: the technical problems of insufficient stability of time-resolved nanoparticle, insufficient fluorescence intensity, fluorescence leakage, and fluorescence quenching on time-resolved fluorescence microspheres are solved by the core-shell type result of the PS / cPGMA core-shell type nanoparticle. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly introduced as follows. Obviously, the drawings described below only illustrate some of the embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0033] Figure 1 is a flow chart of a preparation method of time-resolved fluorescent PS / cPGMA nanoparticles provided by the embodiments of the present application;

[0034] Figure 2 is a particle size test result graph of time-resolved fluorescent PS / cPGMA provided by the embodiments of the present application;

[0035] Figure 3 is a comparison graph of emission wavelengths (450-700 nm) of time-resolved fluorescent PS / cPGMA nanoparticles and time-resolved fluorescent PS nanoparticles under an excitation wavelength of 365 nm provided by the embodiments of the present application;

[0036] Figure 4A is a comparison graph of fluorescence decay degrees of time-resolved fluorescent PS / cPGMA nanoparticles at 37℃ for 30 days provided by the embodiment 12 of the present application;

[0037] Figure 4B is a comparison graph of fluorescence decay degrees of time-resolved fluorescent PS nanoparticles at 37℃ for 30 days provided by the embodiment 13 of the present application;

[0038] Figure 5 is a test strip schematic diagram of time-resolved fluorescent PS / cPGMA nanoparticles provided by the embodiments of the present application;

[0039] Figure 6 is a standard curve drawing result schematic diagram provided by the embodiments of the present application;

[0040] Figure 7 is an IgET value, C value and T / C value stability detection result schematic diagram provided by the embodiments of the present application. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0042] In view of the problems in the prior art, the present application provides a preparation method of time-resolved fluorescent PS / cPGMA nanoparticles, which will be described in detail below in combination with drawings.

[0043] As Figure 1As shown, the preparation method of the time-resolved fluorescence PS / cPGMA nanoparticle provided by the embodiment of the present application comprises the following steps:

[0044] S101, forming an Eu chelate by chelation of europium and a ligand of europium;

[0045] S102, swelling the PS / cPGMA core-shell nanoparticle;

[0046] S103, adding the swollen PS / cPGMA core-shell nanoparticle into the Eu chelate;

[0047] S104, condensing and washing the swollen and dyed PS / cPGMA core-shell nanoparticle.

[0048] The ligand of the Eu chelate in step S101 provided by the embodiment of the present application comprises 2-thiophene formyl trifluoroacetone (TTA), 4,4,4-trifluoro-1,2-naphthalene-1,3-butanedione (NTA), dibenzoylmethane (DBM), 1,10-phenanthroline (Phen) and n-trioctyl phosphine oxide (TOPO), and one or two of the ligands of the Eu chelate form a mono-ligand or a di-ligand, the pH value is adjusted to 6.0-7.0 by ammonia water, and the Eu chelate is obtained.

[0049] In step S102 provided by the embodiment of the present application, the amount of the Eu chelate added in the swelling process is 1-50% of the mass of the carboxyl nanoparticle, the proportion of the aqueous solution in the swelling process is 1-90%, and the proportion of the swelling agent is 1-90%.

[0050] In step S102 provided by the embodiment of the present application, the swelling step of the carboxyl nanoparticle is: dispersing the carboxyl nanoparticle in deionized water containing 0.1-5% of sodium dodecyl benzene sulfonate.

[0051] The swelling agent provided by the embodiment of the present application is any one or several of dichloromethane, anhydrous ethanol, isopropyl alcohol, butanol, ethylene glycol, benzyl alcohol, tetrahydrofuran, chloroform, acetone or N,N-dimethylformamide.

[0052] The swelling temperature in step S102 provided by the embodiment of the present application is 25-150℃, the swelling time is 0.5-10h, and the stirring speed is 100-1000rpm.

[0053] The PS / cPGMA nanoparticle in step S103 provided by the embodiment of the present application has a particle size of 40-600nm, a shell thickness of 2-50nm, a cross-linking degree of 0.5-10%, and a carboxyl density of 0.1-1mmol / g on the surface.

[0054] The condensation process in step S104 provided by the embodiment of the present application comprises: reducing the temperature to 4-37°C and diluting the concentration of the swelling agent by 1-10 times, the condensation time is 3-48h, the condensation rotation speed is 50-200rpm; washing 1-5 times with deionized water or anhydrous ethanol, the centrifugal force is 4000-15000rpm, and the centrifugal time is 10-60min.

[0055] Figure 4 shows the stability of the time-resolved fluorescent PS / cPGMA nanoparticles prepared in Example 2 and the time-resolved fluorescent PS nanoparticles under light-off 37°C Figure 2

[0056] Instrument model:

[0057] 1) Take 1ml of time-resolved fluorescent PS / cPGMA nanoparticles with a solid content of 0.01% to a dish for detection

[0058] 2) The detection results are shown in Table 1 Figure 2

[0059] Figure 4 shows the stability of the time-resolved fluorescent PS / cPGMA nanoparticles prepared in Example 2 and the time-resolved fluorescent PS nanoparticles under light-off 37°C Figure 3 Instrument model: BioTek synergy H1

[0060] 1) Take 100μL of time-resolved fluorescent nanoparticles with a solid content of 0.01% and add them to a 96-well test plate

[0061] 2) Detection method setting:

[0062] Excitation wavelength: 365nm Emission wavelength: 450-700nm

[0063] Step: 1nm Gain: 50

[0064] 3) The detection results are shown in Table 2

[0065] Figure 3 F7 is the result of time-resolved fluorescent PS / cPGMA core-shell nanoparticles, and E7 is the result of time-resolved fluorescent PS nanoparticles

[0066] Figure 4 shows the stability of the time-resolved fluorescent PS / cPGMA nanoparticles prepared in Example 2 and the time-resolved fluorescent PS nanoparticles under light-off 37°C

[0067] Instrument equipment: SAN TN constant temperature oven

[0068] BioTek synergy H1

[0069] 1) Put 1ml of time-resolved fluorescent nanoparticles with a solid content of 1% into a 37°C constant temperature oven for light-off storage​​​

[0070] 2) Take out 10 μL per day to dilute to a solid content of 0.01%, take 100 μL to add to a 96-well test plate for detection

[0071] 3) Detection method settings:

[0072] Excitation wavelength: 365 nm Emission wavelength: 615 nm

[0073] Step: 1 nm Gain: 50

[0074] 4) The detection results are shown in Figure 4, Figure 4A which are the results of time-resolved fluorescence PS / cPGMA nanoparticles stored in the dark at 37°C for 30 days, Figure 4B which are the results of time-resolved fluorescence PS nanoparticles stored in the dark at 37°C for 30 days

[0075] In order to prove the creativity and technical value of the technical solutions of the present application, this part is an application example of the technical solutions of the claims on specific products or related technologies.

[0076] Application Example

[0077] Assembly of time-resolved fluorescence PS / cPGMA nanoparticle test strips

[0078] Take out the PVC base plate, remove all adhesive strips, first paste the NC film, paste the water absorption paper above the NC film, use the membrane drawing and gold spraying instrument to draw the T line (IgE monoclonal antibody, 0.8 mg / mL, spraying volume is 1 μL / cm) and C line (SA, 0.8 mg / mL, spraying volume is 1 μL / cm) on the NC film, the distance between the two lines is 4 mm, and then dry in a 37°C oven for 12 h, then paste the binding pad (add 10 μL 0.1% time-resolved fluorescence labeled IgE antibody + 10 μL 0.1% time-resolved fluorescence labeled Biotin + diluent 5% sucrose, 0.1% PVPK30, 10 μL / cm) under the NC film, and then paste the sample pad under the binding pad, so that the sample pad, binding pad, NC film, and water absorption paper overlap by about 1 mm, after assembly, cut it into 4.0 mm wide test strips with a cutting machine, put it into a card shell, and store it in a tin foil bag with a desiccant at 2-8°C for standby.

[0079] Detection of time-resolved fluorescence PS / cPGMA nanoparticle test strips (as shown in Figure 5 )

[0080] Take 100 μL of the sample to be tested prepared with the sample diluent, drop it into the sample hole of the prepared immunochromatography test strip, place it on a horizontal table for 20 min, then place it in a fluorescence immunochromatography instrument for detection, and record the fluorescence intensity of the C and T lines. The structure of the test strip is shown in Figure 5 .

[0081] Preparation of time-resolved fluorescent PS / cPGMA nanoparticle-labeled antibody

[0082] Activation of time-resolved fluorescent PS / cPGMA nanoparticle

[0083] (1) Take 50 μL of time-resolved fluorescent nanoparticles with a solid content of 1%, add 450 μL of MES (0.01 mol / L pH 5.5), mix with a shaking mixer, centrifuge at 14000 rpm at 4°C for 15 min, remove the supernatant, resuspend the precipitate with 500 μL of MES (0.01 mol / L pH 5.5), then add 132 μL of 1 mg / mL NHS solution and 114 μL of 1 mg / mL EDC solution in turn, shake and mix after shaking, and activate at room temperature for 15 min at low speed;

[0084] (2) Centrifuge at 14000 rpm at 4°C for 15 min, remove the supernatant, add 500 μL of BB (0.05 mol / L pH 8.0), resuspend the microspheres with ultrasonic, and mix thoroughly.

[0085] Preparation of time-resolved fluorescent PS / cPGMA nanoparticle-labeled IgE antibody

[0086] (1) Add 50 μg of IgE antibody to the above time-resolved fluorescent nanoparticle solution, mix thoroughly, and react at 37°C for 2 h;

[0087] (2) Centrifuge at 14000 rpm at 4°C for 15 min, remove the supernatant, resuspend with 500 μL of Tris-HCl (0.05 mol / L pH 8.0, 2% BSA), and block at 37°C for 1 h;

[0088] (3) Centrifuge at 14000 rpm at 4°C for 15 min, remove the supernatant, wash twice with 500 μL of BB (0.05 mol / L pH 8.0), resuspend with 250 μL of Tris-HCl (0.05 mol / L pH 8.0, 0.1% Tween 20), mix thoroughly, and store at 2-8°C for standby;

[0089] Preparation of time-resolved fluorescent PS / cPGMA nanoparticle-labeled Biotin

[0090] (1) After activating the time-resolved fluorescent nanoparticles as described above, add 100 μg of IgG antibody, mix thoroughly, and react at 37°C for 2 h;

[0091] (2) Centrifuge at 14000 rpm at 4°C for 15 min, remove the supernatant, resuspend with 500 μL of Tris-HCl (0.05 mol / L pH 8.0, 2% BSA), and block at 37°C for 1 h;

[0092] (3) 14000 rpm 4℃ centrifugation for 15 min, remove supernatant, resuspended with 500 μL BB (0.05 mol / L pH 8.0) ultrasonic, fully mixed after adding 15 μL 1 mg / mL Sulfo-NHS Biotin solution, 37℃ incubation for 30 min;

[0093] (4) 14000 rpm 4℃ centrifugation for 15 min, remove supernatant, washed with 500 μL BB (0.05 mol / L pH 8.0) twice, resuspended with 250 μL Tris-HCl (0.05 mol / L pH 8.0, 0.1% Tween 20) ultrasonic, fully mixed after 2-8℃ storage for standby;

[0094] Time-resolved fluorescence PS / cPGMA nanoparticle test strip standard curve and sensitivity

[0095] IgE high value linear standard 1024 ng / mL was diluted by multiple dilution, so that the standard concentration was 8, 16, 32, 64, 128, 256, 512, 1024 ng / mL respectively. The developed test strip was used for repeated detection of each concentration point of the standard for 10 times. After dilution according to the volume ratio of sample diluent PBS (0.05 mol / L pH 7.4, 1% BSA, 0.1% PEG4000, 0.05% PC300) to standard was 3:1, 100 μL was taken and dropped into the sample well, and after 20 min, it was placed in the fluorescence quantitative analyzer for detection, and the detection was repeated for 3 times. The standard curve was drawn with the logarithmic value of each concentration value of the standard as the abscissa and the logarithmic value of the average value of T / C as the ordinate.

[0096] The standard curve drawing result is shown in Figure 6 , and the measured IgE is linear in the range of 8-1024 ng / mL, y=0.7062x+1.6697, R 2 =0.990;

[0097] Precision

[0098] Three different batches of test strips were randomly selected respectively, and the IgE standard with a concentration of 8, 64, 512 ng / mL was detected, and each concentration was determined in parallel for 10 times, and the precision (CV) of the test strip within and between batches was calculated, and the calculation formula was as follows:

[0099]

[0100] The batch and batch precision test results are shown in the table, and according to the table, the batch variation coefficient of the IgE quality control with a concentration of 8, 64, 512 ng / mL is less than 10%, and the batch variation coefficient is also less than 15%, which meets the precision requirement

[0101]

[0102] Accuracy of time-resolved fluorescence PS / cPGMA nanoparticle test strip

[0103] The test strip was detected by adding IgE quality control with known concentrations (8, 64, 512 ng / mL) in negative samples, respectively, and each concentration was repeated 10 times. The average value and variation of each concentration recovery rate were calculated, and the calculation formula was as follows:

[0104] Recovery rate = measured concentration / true concentration x 100%

[0105] The test strip accuracy detection results are shown in Tables 3.3 and 3.4. The recovery rates of IgE samples with concentrations of 8, 64, 512 ng / mL were 100.88%, 98.05%, and 99.22%, respectively; which met the requirements of 85%-115% recovery rate. The above results showed that the test strip had good accuracy.

[0106]

[0107]

[0108] Stability of time-resolved fluorescence PS / cPGMA nanoparticle test strip

[0109] The prepared test strip was dried and packaged, and placed at 37°C for accelerated testing. IgE standard with a concentration of 64 ng / mL was used to detect at 0, 5, 10, 15, 20, 25, and 30 days. According to the changes of T value, C value, and T / C value during the accelerated test, the stability of the test strip was analyzed.

[0110] The stability detection results are shown in the figure. The T value, C value, and T / C value of the test strip were Figure 7 It can be seen that the T value, C value, and T / C value of the two test strips had no significant change during the 37°C accelerated test, indicating that the test strip had good stability.

[0111] Example 1

[0112] TTA 1.3 g was dissolved in 20 mL of anhydrous ethanol in a beaker and stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted with ammonia water to 6.0-7.0. 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.74 g was added dropwise to the mixed solution under magnetic stirring. After the addition was completed, the solution was irradiated with a UV lamp and turned pink. The beaker was wrapped with tin paper and the reaction was continued under stirring for 12 h at room temperature in the dark. After standing overnight, the ethanol was removed by distillation, and Eu(TTA)3chelate was obtained and stored in a desiccator in the dark for later use.

[0113] The PS / cPGMA core-shell nanoparticles (Zhejiang Ipsylon Biotechnology Co., Ltd.) were dispersed in deionized water to prepare a 10% (w / w) PS microsphere aqueous solution 10 mL in a flat flask. The average particle size of the PS / cPGMA core-shell nanoparticles was 40.2 nm, the PDI was 0.003, the shell thickness was 10 nm, and the crosslinking degree was 4%. 0.1 g of SDS was added and ultrasonically dispersed. 0.01 g of Eu(TTA)3was dissolved in 0.1 mL of dichloromethane, and the swelling solution containing the dissolved Eu chelate was added to the PS / cPGMA core-shell nanoparticles. The reaction was carried out at a temperature of 25°C with a stirring speed of 1000 rpm for 10 h. After the reaction was completed, it was placed in a refrigerator at 4°C with a stirring speed of 50 rpm for 10 h. After dilution with water, it was centrifuged once to remove the Eu chelate that did not enter the PS / cPGMA core-shell nanoparticles. The centrifugal speed was 15000 rpm and the time was 60 min. 100 mL of deionized water was added to redissolve it to 1% (w / w). The average particle size of the time-resolved fluorescent PS / cPGMA core-shell nanoparticles was 43.3 nm, and the PDI was 0.013. The particle size was slightly larger than that of the original PS / cPGMA core-shell nanoparticles, which was caused by the addition of europium chelate in the PS / cPGMA core-shell structure. The surface still maintained good uniformity (generally considered that PDI < 0.05 is good uniformity).

[0114] Example 2

[0115] TTA 1.33 g and Phen 0.36 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and magnetically stirred at room temperature for 10 min to make them fully dissolved; the pH value of the mixed solution in the flask was adjusted with ammonia water, so that the solution pH was 6.0-7.0; under the condition of magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.74 g was added dropwise into the mixed solution, and after the addition was completed, the precipitate was separated out in the solution, and the solution was red under ultraviolet lamp irradiation. The flask was wrapped with tin paper and continued to stir the reaction under light shielding at room temperature for 12 h; after standing overnight, the solution was filtered with 50% ethanol / water for 2-3 times, and then the powder was placed at 80°C for drying, to obtain Eu(TTA)3Phen chelate, which was stored in a desiccator under light shielding for later use.

[0116] The PS / cPGMA core-shell nanoparticles (Zhejiang Ipsylon Biotechnology Co., Ltd.) were dispersed in deionized water to prepare a 10% (w / w) PS microsphere aqueous solution 10 mL in a flat flask, the average particle size of the PS / cPGMA core-shell nanoparticles was 151.7 nm, PDI was 0.006, the shell thickness was 2 nm, and the crosslinking degree was 2%; 0.25 g of SDS was added and ultrasonically dispersed, 1 mL of deionized water was added, 0.23 g of Eu(TTA)3Phen was dissolved in 9 mL of tetrahydrofuran, and the swelling solution of the dissolved Eu chelate was added dropwise into the PS / cPGMA core-shell nanoparticles; the reaction was carried out at a temperature of 67°C and a stirring speed of 100 rpm for 5 h; after the reaction was completed, it was placed in a refrigerator at 4°C and condensed at a stirring speed of 100 rpm for 1 h; after dilution with water by 5 times, the Eu chelate not entering into the PS / cPGMA core-shell nanoparticles was washed away by ethanol centrifugation for 5 times, and the speed was 8000 rpm and the centrifugation time was 30 min; 100 mL of deionized water was added to redissolve to 1% (w / w), and the average particle size of the time-resolved fluorescence PS / cPGMA core-shell nanoparticles was 153.0 nm, and PDI was 0.003.

[0117] Example 3

[0118] TTA 0.1667 g and TOPO 0.0967 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and magnetically stirred at room temperature for 10 min to make them fully dissolved; the pH value of the mixed solution in the flask was adjusted with ammonia water, so that the solution pH was 6.0-7.0; under the condition of magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise into the mixed solution, and after the addition was completed, the solution was red under ultraviolet lamp irradiation. The flask was wrapped with tin paper and continued to stir the reaction under light shielding at room temperature for 12 h; after standing overnight, the ethanol in the solution was removed by distillation, and finally Eu(TTA)3TOPO chelate was obtained, which was stored in a desiccator under light shielding for later use.

[0119] Preparation of Eu(TTA)3(TOPO)2 chelate complex: TTA 0.1667 g and TOPO 0.193 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution in the beaker was adjusted with ammonia water to 6.0-7.0. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped with tin paper and the reaction was continued under stirring for 12 h at room temperature in the dark. After standing overnight, the ethanol was removed by distillation, and the final Eu(TTA)3(TOPO)2 chelate complex was obtained and stored in a desiccator in the dark for later use.

[0120] Example 4

[0121] Preparation of Eu(TTA)3(TOPO)2 chelate complex: TTA 0.1667 g and TOPO 0.193 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution in the beaker was adjusted with ammonia water to 6.0-7.0. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped with tin paper and the reaction was continued under stirring for 12 h at room temperature in the dark. After standing overnight, the ethanol was removed by distillation, and the final Eu(TTA)3(TOPO)2 chelate complex was obtained and stored in a desiccator in the dark for later use.

[0122] Preparation of Eu(NTA)3 chelate: NTA 0.1997 g was dissolved in 20 mL of absolute ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 by adding ammonia water. Under magnetic stirring, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was completed, the solution was irradiated with a UV lamp and turned pink. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. The solution was left to stand overnight, and the ethanol was removed by distillation. The final Eu(NTA)3 chelate was obtained and stored in a desiccator in the dark until use.

[0123] Example 5

[0124] Preparation of Eu(NTA)3 chelate: NTA 0.1997 g was dissolved in 20 mL of absolute ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 by adding ammonia water. Under magnetic stirring, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was completed, the solution was irradiated with a UV lamp and turned pink. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. The solution was left to stand overnight, and the ethanol was removed by distillation. The final Eu(NTA)3 chelate was obtained and stored in a desiccator in the dark until use.

[0125] Preparation of Eu(TTA)3Phen chelate complex: NTA 0.1997 g and Phen 0.0496 g were dissolved in 20 mL absolute ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was completed, precipitates were separated out, and the solution was red under UV light. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. After standing overnight, the powder was filtered with 50% ethanol / water for 2-3 times, and then dried at 80°C. The final Eu(TTA)3Phen chelate complex was obtained and stored in a desiccator in the dark.

[0126] Example 6

[0127] Preparation of Eu(TTA)3Phen chelate complex: NTA 0.1997 g and Phen 0.0496 g were dissolved in 20 mL absolute ethanol in a beaker, and stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was completed, precipitates were separated out, and the solution was red under UV light. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. After standing overnight, the powder was filtered with 50% ethanol / water for 2-3 times, and then dried at 80°C. The final Eu(TTA)3Phen chelate complex was obtained and stored in a desiccator in the dark.

[0128] Preparation of Eu(NTA)3TOPO chelate: NTA 0.1667 g and TOPO 0.0967 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped in tin paper and the reaction was continued under stirring in the dark at room temperature for 12 h. After standing overnight, the ethanol was removed by distillation, and the final Eu(NTA)3TOPO chelate was obtained and stored in a desiccator in the dark for later use.

[0129] Example 7

[0130] Preparation of Eu(NTA)3TOPO chelate: NTA 0.1667 g and TOPO 0.0967 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped in tin paper and the reaction was continued under stirring in the dark at room temperature for 12 h. After standing overnight, the ethanol was removed by distillation, and the final Eu(NTA)3TOPO chelate was obtained and stored in a desiccator in the dark for later use.

[0131] Preparation of Eu(NTA)3(TOPO)2 chelate complex: NTA 0.1667 g and TOPO 0.193 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped in tin paper and the reaction was continued under stirring in the dark at room temperature for 12 h. After standing overnight, the ethanol was removed by distillation, and the final Eu(NTA)3(TOPO)2 chelate complex was obtained and stored in a desiccator in the dark for later use.

[0132] Example 8

[0133] Preparation of Eu(NTA)3(TOPO)2 chelate complex: NTA 0.1667 g and TOPO 0.193 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, the solution was red under ultraviolet light. The beaker was wrapped in tin paper and the reaction was continued under stirring in the dark at room temperature for 12 h. After standing overnight, the ethanol was removed by distillation, and the final Eu(NTA)3(TOPO)2 chelate complex was obtained and stored in a desiccator in the dark for later use.

[0134] Preparation of Eu(DEM)3 chelate: DEM 0.168 g was dissolved in 20 mL of absolute ethanol in a beaker, and was stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 by adding ammonia water. Then, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution under magnetic stirring. The solution was pink under UV irradiation. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. The solution was distilled to remove ethanol, and Eu(DEM)3 chelate was obtained and stored in a desiccator in the dark.

[0135] Example 9

[0136] Preparation of Eu(DEM)3 chelate: DEM 0.168 g was dissolved in 20 mL of absolute ethanol in a beaker, and was stirred magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 by adding ammonia water. Then, 20 mL of absolute ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution under magnetic stirring. The solution was pink under UV irradiation. The beaker was wrapped with tin foil and the reaction was continued under stirring for 12 h at room temperature in the dark. The solution was distilled to remove ethanol, and Eu(DEM)3 chelate was obtained and stored in a desiccator in the dark.

[0137] Preparation of Eu(DEM)3Phen chelate: Eu(DEM)3Phen chelate was prepared by dissolving DEM 0.168 g and Phen 0.0496 g in 20 mL anhydrous ethanol in a beaker, and stirring magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 using ammonia water. While stirring magnetically, 20 mL of anhydrous ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, precipitates were separated out of the solution, and the solution was red under UV light. The beaker was wrapped in tin foil and the reaction was continued to stir in the dark at room temperature for 12 h. After standing overnight, the powder was filtered 2-3 times with 50% ethanol / water, and then dried at 80°C. The final Eu(DEM)3Phen chelate was stored in a desiccator in the dark until use.

[0138] Example 10

[0139] Preparation of Eu(DEM)3Phen chelate: Eu(DEM)3Phen chelate was prepared by dissolving DEM 0.168 g and Phen 0.0496 g in 20 mL anhydrous ethanol in a beaker, and stirring magnetically for 10 min at room temperature to ensure complete dissolution. The pH of the solution was adjusted to 6.0-7.0 using ammonia water. While stirring magnetically, 20 mL of anhydrous ethanol solution containing EuCl3-6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was complete, precipitates were separated out of the solution, and the solution was red under UV light. The beaker was wrapped in tin foil and the reaction was continued to stir in the dark at room temperature for 12 h. After standing overnight, the powder was filtered 2-3 times with 50% ethanol / water, and then dried at 80°C. The final Eu(DEM)3Phen chelate was stored in a desiccator in the dark until use.

[0140] Preparation of Eu(NTA)3 chelate doped PS / cPGMA core-shell nanoparticles: PS / cPGMA core-shell nanoparticles (Zhejiang Ipsylon Biotechnology Co., Ltd.) were dispersed in deionized water to prepare a 10% (w / w) aqueous solution of PS microspheres 10 mL in a flat flask. The average particle size of the PS / cPGMA core-shell nanoparticles was 150.8 nm, the PDI was 0.003, the shell thickness was 10 nm, and the cross-linking degree was 4%. 0.01 g of SDS was added and ultrasonically dispersed. 1 mL of deionized water was added, and 0.5 g of Eu(NTA)3 was dissolved in 9 mL of a swelling agent (swelling agent: ethylene glycol: benzyl alcohol = 8:1). The swelling solution containing the dissolved Eu chelate was added dropwise to the PS / cPGMA core-shell nanoparticles. The reaction was carried out at a temperature of 25°C with a stirring speed of 500 rpm for 10 h. After the reaction was completed, it was placed at a temperature of 15°C with a stirring speed of 200 rpm for 5 h. After dilution with water, ethanol was added and centrifuged once to remove the Eu chelate that did not enter the PS / cPGMA core-shell nanoparticles. The centrifugal speed was 8000 rpm and the time was 30 min. 100 mL of deionized water was added to redissolve the time-resolved fluorescent PS / cPGMA core-shell nanoparticles to a concentration of 1% (w / w). The average particle size of the time-resolved fluorescent PS / cPGMA core-shell nanoparticles was 152.3 nm, and the PDI was 0.009.

[0141] Example 11

[0142] Preparation of Eu(DEM)3TOPO chelate: DEM 0.168 g and TOPO 0.0967 g were dissolved in 20 mL of anhydrous ethanol in a beaker, and the solution was stirred magnetically at room temperature for 10 min to ensure complete dissolution. The pH of the solution in the beaker was adjusted to 6.0-7.0 using ammonia water. Under magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g was added dropwise to the mixed solution. After the addition was completed, the solution was red under ultraviolet light. The beaker was wrapped with tin paper and the reaction was continued under stirring in the dark at room temperature for 12 h. After standing overnight, the ethanol was removed by distillation, and Eu(DEM)3TOPO chelate was obtained. The product was stored in a desiccator in the dark for later use.

[0143] Preparation of Eu(NTA)3 chelate doped PS / cPGMA core-shell nanoparticles: PS / cPGMA core-shell nanoparticles (Zhejiang Ipsylon Biotechnology Co., Ltd.) were dispersed in deionized water to prepare a 10% (w / w) aqueous solution of PS microspheres 10 mL in a flat flask. The average particle size of the PS / cPGMA core-shell nanoparticles was 601.5 nm, the PDI was 0.011, the shell thickness was 2 nm, and the cross-linking degree was 10%. 0.5 g of SDS was added and ultrasonically dispersed. 0.5 g of Eu(NTA)3 was dissolved in 90 mL of a swelling agent (swelling agent: ethanol: tetrahydrofuran = 8:1). The swelling solution containing the dissolved Eu chelate was added dropwise to the PS / cPGMA core-shell nanoparticles. The reaction was carried out at a temperature of 25°C with a stirring speed of 1000 rpm for 10 h. After the reaction was completed, it was placed at 4°C with a stirring speed of 100 rpm for condensation for 1 h. After dilution with water by a factor of 10, ethanol was centrifuged once to remove the Eu chelate that did not enter the PS / cPGMA core-shell nanoparticles. The centrifugal speed was 4000 rpm and the time was 60 min. 100 mL of deionized water was added to redissolve the time-resolved fluorescent PS / cPGMA core-shell nanoparticles to 1% (w / w). The average particle size of the time-resolved fluorescent PS / cPGMA core-shell nanoparticles was 607.2 nm, and the PDI was 0.009.

[0144] Example 12

[0145] Dissolution of DEM 0.168 g and TOPO 0.193 g in 20 mL of anhydrous ethanol in a beaker, magnetic stirring at room temperature for 10 min, so that it is fully dissolved; adjust the pH value of the mixed solution in the flask with ammonia water, so that the pH of the solution is 6.0-7.0; under the condition of magnetic stirring, 20 mL of anhydrous ethanol solution containing EuCl3·6H2O 0.0916 g is added dropwise to the mixed solution, and the solution is red under ultraviolet lamp irradiation. The flask is wrapped with tin paper at room temperature and continues to stir in the dark for 12 h; stand overnight, distill the ethanol from the solution, and finally obtain Eu(DEM)3(TOPO)2 chelate, which is stored in a desiccator in the dark for use.

[0146] Preparation of time-resolved fluorescent PS / cPGMA nanoparticles: 10% (w / w) of PS / cPGMA core-shell nanoparticles (Zhejiang Ipsylon Biotechnology Co., Ltd.) was prepared by dispersing the nanoparticles in deionized water. 10 mL of the solution was placed in a flat flask. The average particle size of the PS / cPGMA core-shell nanoparticles was 152.3 nm with a PDI of 0.002, a shell thickness of 50 nm, and a cross-linking degree of 10%. 0.5 g of SDS was added and ultrasonically dispersed. 0.5 g of Eu(NTA)3 was dissolved in 90 mL of a swelling agent (a mixture of ethylene glycol and tetrahydrofuran in a ratio of 8:1). The swelling solution containing the dissolved Eu chelate was added dropwise to the PS / cPGMA core-shell nanoparticles. The reaction was carried out at a temperature of 150°C with a stirring speed of 100 rpm for 5 h. After the reaction was completed, the mixture was placed in a 37°C water bath with a stirring speed of 50 rpm for 5 h. After dilution with water, ethanol was added and centrifuged for 5 times to remove the Eu chelate that did not enter the PS / cPGMA core-shell nanoparticles. The centrifugal speed was 15000 rpm and the time was 10 min. 100 mL of deionized water was added to dissolve the time-resolved fluorescent PS / cPGMA core-shell nanoparticles to a concentration of 1% (w / w). The average particle size of the time-resolved fluorescent PS / cPGMA core-shell nanoparticles was 154.5 nm with a PDI of 0.009.

[0147] Example 13

[0148] A comparative experiment was carried out using carboxyl polystyrene microspheres (Zhejiang Ipsylon Biotechnology Co., Ltd.).

[0149] The time-resolved fluorescent PS nanoparticles were prepared according to a literature method (Peng Chao, Chen Lei, Wan Qianhong. Preparation of polystyrene fluorescent microspheres containing europium complexes by swelling method [J].

[0150] The particle size test results of the time-resolved fluorescent PS / cPGMA are shown in Figure 2 The particle size was 153.0 nm with a PDI of 0.003, indicating that the prepared time-resolved fluorescent PS / cPGMA microspheres had very good uniformity.

[0151] The comparison chart of the emission wavelength (450-700 nm) of the time-resolved fluorescent PS / cPGMA nanoparticles and the time-resolved fluorescent PS nanoparticles at an excitation wavelength of 365 nm is shown in Figure 3 As can be seen from Figure 3 the comparison chart, the highest value of the time-resolved fluorescent PS / cPGMA is about 1 times higher than that of the time-resolved fluorescent PS nanoparticles.

[0152] The comparison chart of the fluorescence decay degree of the time-resolved fluorescent PS / cPGMA nanoparticles at 37°C for 30 days is shown in Figure 4A The comparison chart of the fluorescence decay degree of the time-resolved fluorescent PS nanoparticles at 37°C for 30 days is shown in Figure 4B

[0153] ​Compared with the figure, the PS / cPGMA nanoparticles attenuated only about 0.2% under the condition of 30 days at 37°C in the dark, while the PS nanoparticles attenuated about 12% under the condition of 30 days at 37°C in the dark, which indicated that the core-shell type of PS / cPGMA nanoparticles was beneficial to reduce the problem of fluorescence value reduction caused by fluorescence leakage.

[0154] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any modification, equivalent replacement and improvement within the technical range disclosed by the present application and in the spirit and principle of the present application should be covered within the protection scope of the present application.

Claims

1. A method of preparing time-resolved fluorescent PS / cPGMA nanoparticles, characterized in that, The preparation method of the time-resolved fluorescent PS / cPGMA nanoparticles comprises the following steps: dissolving a chelate ligand of Eu in anhydrous ethanol and stirring the solution at room temperature; adjusting the pH value of the mixed solution to 6.0-7.0 by using ammonia water, adding an anhydrous ethanol solution containing EuCl3·6H2O dropwise, and stirring the solution at room temperature in the dark; standing overnight, removing ethanol by distillation, and obtaining Eu chelate; dispersing the PS / cPGMA core-shell nanoparticles in deionized water, preparing a 10% PS microsphere aqueous solution, and adding SDS for ultrasonic dispersion; dissolving the Eu chelate in dichloromethane, adding the swollen solution of the Eu chelate to the PS / cPGMA core-shell nanoparticles, stirring the solution, and then placing the solution in a refrigerator for condensation reaction; after the reaction is completed, diluting the solution with water, centrifuging the solution, and adding deionized water for redissolution. The PS / cPGMA nanoparticles used have a particle size of 40-600 nm, a shell thickness of 2-50 nm, a crosslinking degree of 0.5-10%, and a carboxyl density of 0.1-1 mmol / g. The condensation process comprises the following steps: reducing the temperature to 4-37℃ and diluting the swelling agent by 1-10 times, condensing for 3-48 h at a rotation speed of 50-200 rpm, and washing the solution with deionized water or anhydrous ethanol for 1-5 times at a centrifugal force of 4000-15000 rpm and a centrifugal time of 10-60 min.

2. The method of claim 1, wherein the time-resolved fluorescent PS / cPGMA nanoparticles are prepared by the method comprising the steps of: The preparation method of the time-resolved fluorescent PS / cPGMA nanoparticles comprises the following steps: Step one: forming an Eu chelate by chelation of Eu and a ligand of Eu; Step two: swelling the PS / cPGMA core-shell nanoparticles; Step three: adding the swollen PS / cPGMA core-shell nanoparticles to the Eu chelate; Step four: condensing and washing the swollen and dyed PS / cPGMA core-shell nanoparticles.

3. The method of claim 2, wherein the time-resolved fluorescent PS / cPGMA nanoparticles are prepared by the method comprising the steps of: The ligand of the Eu chelate in step one comprises 2-thiophenecarbonyl trifluoroacetone, 4,4,4-trifluoro-1,2-naphthalene-1,3-butanedione, dibenzoylmethane, 1,10-phenanthroline, and n-trioctyl phosphine oxide.

4. The method of claim 2, wherein the time-resolved fluorescent PS / cPGMA nanoparticles are prepared by the method comprising the steps of: In step one, any one or two of the ligands of the Eu chelate forms a mono-ligand or a di-ligand, the pH value is adjusted to 6.0-7.0 by using ammonia water, and the Eu chelate is obtained.

5. The method of claim 2, wherein the time-resolved fluorescent PS / cPGMA nanoparticles are prepared by the method comprising the steps of: In step two, the amount of the Eu chelate added in the swelling process is 1-50% of the mass of the carboxyl nanoparticles, the proportion of the aqueous solution in the swelling process is 1-90%, and the proportion of the swelling agent is 1-90%.

6. The method of claim 2, wherein the time-resolved fluorescent PS / cPGMA nanoparticles are prepared by the method comprising the steps of: In step two, the swelling step of the carboxyl nanoparticles is: dispersing the carboxyl nanoparticles in deionized water containing 0.1-5% sodium dodecyl benzene sulfonate.

7. The method for preparing time-resolved fluorescent PS / cPGMA nanoparticles as described in claim 2, characterized in that, In step two, the swelling agent is any one or several of dichloromethane, anhydrous ethanol, isopropyl alcohol, butanol, ethylene glycol, benzyl alcohol, tetrahydrofuran, chloroform, acetone, or N,N-dimethylformamide.

8. The method for preparing time-resolved fluorescent PS / cPGMA nanoparticles as described in claim 2, characterized in that, In step two, the swelling temperature is 25-150℃, the swelling time is 0.5-10 h, and the stirring rotation speed is 100-1000 rpm.

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