Preparation method and application of high-sensitivity and high-stability time-resolved fluorescent microspheres

Abstract

This invention discloses a method for preparing and applying highly sensitive and stable time-resolved fluorescent microspheres, relating to the field of immunochromatography. The preparation method of this invention includes the following steps: (1) preparation of highly stable polystyrene microspheres with high carboxyl density; (2) preparation of highly stable europium ion chelates; and (3) preparation of highly stable fluorescent microspheres. This invention constructs a highly stable fluorescent microsphere with high carboxyl density, linked by strong covalent bonds from the inside out, through a strategy combining "functional monomer copolymerization" and "surface covalent labeling." This microsphere exhibits significant advantages and innovation in chemical stability, anti-leakage capability of the fluorescent label, and surface functional group density. The time-resolved fluorescent microsphere test strip developed based on this microsphere has advantages such as good stability and high detection sensitivity, enabling rapid detection and analysis of analytes.

Description

Technical Field

[0001] This invention relates to the field of immunochromatography technology, specifically to a method for preparing and applying highly sensitive and stable time-resolved fluorescent microspheres. Background Technology

[0002] Point-of-Care Testing (POCT) technologies mainly include dry chemistry, immunochromatography, chemiluminescence, microfluidics, biosensors, and enzyme-linked immunosorbent assays (ELISA). Among these, immunochromatography has become the best choice for on-site testing due to its advantages of simple operation, rapid testing, and high sensitivity and specificity. Immunochromatography is a detection technique that combines chromatography with antigen-antibody specific immune reactions. It mainly includes chromatographic techniques such as colloidal gold, fluorescence, chemiluminescence, and magnetic nanomaterials. Time-resolved fluorescent microsphere technology is one type of fluorescent chromatography, initially used to study the rapid domain movement of macromolecules and the interaction of fluorescent dyes. With technological advancements, its application in the detection field has gradually expanded, especially in immunoassays, where highly sensitive detection of target substances is achieved by labeling antibodies or antigens.

[0003] Time-resolved fluorescent microspheres are fluorescent microspheres encapsulated with thousands of coordinating rare earth ions (Eu, Tβ, Sm, Dy). These coordinating rare earth metal ions exhibit fluorescence and have a long fluorescence lifetime (10-1000 microseconds), far exceeding the fluorescence lifetime of background substances (1-10 nanoseconds). This allows for improved detection sensitivity by collecting fluorescence at different time points. Furthermore, encapsulating the coordinating rare earth ions within the microspheres enhances their stability in solution, making the fluorescence intensity less susceptible to environmental influences. Secondly, the large Stokes shift (large difference between excitation and emission wavelengths) of the fluorescence emitted by the coordinating rare earth ions, typically excited at 340 nm and received at 610 nm, ensures complete separation of the emission and excitation spectra. This reduces the risk of internal quenching of fluorescence between microspheres due to excessively high microsphere concentrations. Therefore, by delaying the measurement time and allowing the background fluorescence to completely decay before detecting the specific fluorescence signal, the sensitivity and linear range of the test strip can be improved. In traditional emulsion copolymerization, hydrophilic carboxyl-containing monomers (such as acrylic acid) tend to be distributed at the interface (surface) between the aqueous phase and the microspheres. This results in carboxyl groups being mainly present on the surface of the microspheres, with limited internal and overall density. This restricts the capacity for subsequent biomolecule bonding and makes the microspheres susceptible to external environmental interference. This can lead to low labeling efficiency, poor batch-to-batch repeatability, low fluorescence intensity, and reduced detection sensitivity in the fabricated test strips. Therefore, it is essential to optimize the existing fluorescent microsphere preparation process to improve microsphere performance and ensure good stability, repeatability, sensitivity, and specificity of the test strips. Summary of the Invention

[0004] Therefore, one of the objectives of this invention is to provide a method for preparing highly sensitive and stable time-resolved fluorescent microspheres.

[0005] Another object of the present invention is to provide the application of the above-mentioned time-resolved fluorescent microspheres in immunochromatographic test strips.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing highly sensitive and stable time-resolved fluorescent microspheres, comprising the following steps:

[0008] (1) Preparation of high-stability, high-carboxyl-density polystyrene microspheres: Styrene monomer and glycolic acid monomer were dissolved in acetonitrile solvent, deionized water and trifluoroacetic acid were added, and the reaction was carried out in a water bath under nitrogen protection for 3 h. After cooling to room temperature, ethyl acetate was extracted, the mixture was evaporated, and the solid was pulped with n-heptane. After filtration and vacuum drying, the mixture was dissolved in deionized water containing dextran and stirred magnetically until homogeneous. After nitrogen purging three times, the mixture was sealed and heated to 60 °C. An initiator was added, and the reaction was stirred under nitrogen for 12 h. After cooling to room temperature, the mixture was filtered to obtain a polymer nanosphere solution. After dialyzing in a dialysis bag for 72 h, a preservative solution was added and the mixture was stored at 4 °C to obtain polystyrene microspheres with high carboxyl density.

[0009] (2) Preparation of high-stability europium ion chelate: europium ion standard acetonitrile solution, 1,4,7,10-tetramonoheterocyclic dodecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester, nitrogen protection at low temperature 0℃, sealed and stirred evenly, and stirred overnight at low speed.

[0010] (3) Preparation of high-stability fluorescent microspheres: The high-stability high-carboxyl density microspheres prepared in step 1 were immersed in a swelling agent, acetonitrile and deionized water were added, and the mixture was stirred evenly. The europium ion chelate prepared in step 2 was added, and the mixture was stirred at 0°C for 12 hours. The organic solvent in the solution was removed by vacuum low-temperature rotary evaporation, and the eluent was collected by passing it through a molecular sieve column. The eluent was then freeze-dried using a freeze dryer, diluted to the standard mass volume with a preservative solution, and stored at 4°C.

[0011] Preferably, in step (1), the styrene monomer is 50 mm and the glycolic acid monomer is 3.0 mm;

[0012] Preferably, in step (1), the mass ratio of acetonitrile solvent, deionized water and trifluoroacetic acid is 1:0.6-0.7:0.16-0.17;

[0013] Preferably, in step (1), the initiator is 5 mL of 0.2 mM potassium persulfate;

[0014] Preferably, in step (2), the europium ion standard acetonitrile solution can be replaced with any one of europium nitrate hydrate, europium fluoride, or europium nitride;

[0015] Preferably, in step (3), the mass ratio of acetonitrile to deionized water is 2:1;

[0016] Preferably, in steps (1) and (3), the preservative solution is 0.01% sodium azide.

[0017] The application of the time-resolved fluorescent microspheres described in this invention in immunochromatographic test strips.

[0018] The present invention has the following advantages:

[0019] This invention utilizes a strategy combining "functional monomer copolymerization" and "surface covalent labeling" to construct highly stable fluorescent microspheres with high carboxyl group density, linked by strong covalent bonds from the inside out. These microspheres exhibit significant advantages and innovation in chemical stability, anti-leakage capability of the fluorescent label, and surface functional group density. Compared to aqueous methods (such as CN202211445051A), this invention abandons the traditional aqueous copolymerization approach and innovatively adopts a route of "homogeneous synthesis of functionalized precursors followed by molding." In an anhydrous organic solvent (acetonitrile), styrene and glycolic acid undergo a controlled polymerization reaction catalyzed by acid to obtain carboxyl-containing oligomers. The oligomers are subjected to soap-free emulsion polymerization in the presence of dextran to form polymer microspheres with carboxyl groups distributed throughout the microsphere backbone and a total carboxyl group content of ≥0.45 mmol / g on the surface and inside. This method ensures that all reactant molecules are in the same phase and have equal probability of molecular collisions. It can produce copolymers with more uniform carboxyl functional groups distributed on the polymer backbone and more defined chemical composition, and also ensures the chemical purity of the microsphere material from the source.

[0020] Furthermore, this invention constructs a stable system of "fully covalent bonding": the microsphere framework, carboxyl groups, chelating agents, and europium ions are all connected by strong chemical bonds. Unlike core-shell structures (such as CN202211157658), this system does not have physical barriers, but extends the covalent bonding from "connecting biomolecules" to "fixing fluorescent groups," achieving a fully covalently bonded stable structure from the inside out, thus exhibiting superior chemical stability. Compared to molecular anchoring methods (such as CN201911392474), covalent bonds are stronger than coordination / hydrogen bonds. Compared to physical embedding methods (such as CN201810217199), the preparation method of this invention fundamentally solves the problem of fluorescent dye leakage, resulting in extremely high stability.

[0021] This invention pre-synthesizes and purifies a well-defined europium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate under optimal conditions. Functionalized microspheres are moderately swollen in an organic solvent to expand their internal network. Subsequently, the purified DOTA-Eu chelate (in its active ester form) is introduced, undergoing a highly efficient amidation reaction with the activated carboxyl groups on the surface / interior of the microspheres under mild conditions, forming strong covalent bonds. The binding energy of the covalent bonds is much higher than that of physical interactions, fundamentally preventing the leakage of fluorescent groups. The extremely high stability of the DOTA-Eu chelate itself constitutes a "double insurance," ensuring that the fluorescent microspheres maintain stable signals even under harsh environments. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0023] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0024] Figure 1 This is a scanning electron microscope (SEM) image of the microspheres.

[0025] Figure 2 This is a particle size distribution diagram of the microspheres;

[0026] Figure 3 This is a schematic diagram of the test strip assembly. Detailed Implementation

[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Preparation method of high-sensitivity and high-stability time-resolved fluorescent microspheres

[0029] 1. Preparation of high-stability, high-carboxyl-density polystyrene microspheres

[0030] (1) Dissolve 50 mm styrene monomer and 3.0 mm glycolic acid monomer in 30 mL acetonitrile solvent, add 20 mL deionized water, add to a round bottom flask, add 5 mL trifluoroacetic acid, add and stir in a water bath under nitrogen protection for 180 min, cool to room temperature, extract with ethyl acetate, evaporate to dryness, slurry with n-heptane, filter, vacuum dry, and set aside for use.

[0031] (2) Take the solid obtained in step (1), dissolve it in deionized water containing dextran, add it to a round-bottom flask, stir it evenly with magnetic force, replace it with nitrogen three times, seal it and add it to 60°C, add 5 mL of 0.2 mM potassium persulfate, stir the reaction under nitrogen for 12 h, cool it to room temperature, filter it to obtain a polymer nanosphere solution, dialyze it with a 1000 Da dialysis bag for 72 h, replace the dialysate once every 12 h to obtain a purified dialysate, add 0.01% sodium azide and store it at 4°C to obtain polystyrene microspheres with carboxyl group density.

[0032] 2. Preparation of highly stable ionic chelates

[0033] Take europium ion standard acetonitrile solution and 1,4,7,10-tetraheterocyclic dodecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester, and under nitrogen protection at 0°C, seal and stir until homogeneous, then stir overnight at low speed. The europium ion standard acetonitrile solution can be replaced with other europium ion standard solutions, europium nitrate hydrate, europium fluoride, europium nitride, etc.

[0034] 3. Preparation of highly stable fluorescent microspheres

[0035] The highly stable, high-carboxyl-density microspheres prepared in step 1 were immersed in a swelling agent, acetonitrile and deionized water were added, and the mixture was stirred until homogeneous. The europium ion chelate prepared in step 2 was added, and the mixture was stirred at 0°C for 12 hours. The organic solvent in the solution was removed by vacuum low-temperature rotary evaporation, and the eluent was collected by passing it through a molecular sieve column. The eluent was then freeze-dried using an LGJ-S50 standard freeze dryer, diluted to a standard mass volume with 0.01% sodium azide solution, and stored at 4°C.

[0036] Example 2: Performance comparison of the present invention with commercially available time-resolved fluorescent microspheres

[0037] (1) Characterization and detection

[0038] The time-resolved fluorescent microspheres prepared above and samples from commercial manufacturers A and B were characterized by scanning electron microscopy (SEM) to obtain the SEM test results, as follows: Figure 1As shown, the time-resolved fluorescent microspheres prepared according to Example 1 and the time-resolved fluorescent microspheres from manufacturers A and B are regular spheres with high uniformity between particles.

[0039] (2) Particle size detection

[0040] The time-resolved fluorescent microspheres prepared above, along with samples from commercially available manufacturers A and B, were tested using a Malvern Zetasizer Nano-S90 laser particle size analyzer. The results are as follows: Figure 2 As shown, the time-resolved fluorescent microspheres prepared according to Example 1 and the time-resolved fluorescent microspheres from manufacturers A and B all exhibit a typical narrow single-peak morphology in particle size distribution, and the PDI is all <0.05, indicating that they have excellent monodispersity and can all be used to prepare lateral chromatography rapid test strips.

[0041] Example 3: Preparation of the time-resolved fluorescent microsphere immunochromatographic test strip of the present invention

[0042] 1. Preparation of fluorescent microsphere pads

[0043] (1) Cleaning of microspheres: Take the time-resolved fluorescent microspheres prepared according to Example 1, sonicate for 1 min, take 100 μL of microspheres and add 900 μL MES (10 mmol / L, pH 6.0). Centrifuge at 16000 r / min for 10 min, remove the supernatant, add 1 ml MES to resuspend the microspheres, and sonicate for 2-5 min to mix well;

[0044] (2) Activation of microspheres: Weigh 20 mg NHS and EDC, dissolve them in MES, and prepare fresh each time, i.e., 20 mg / mL NHS and EDC; take 30 μL NHS and add it to the cleaned microspheres, mix quickly; then take 5 μL EDC and add it to the microspheres, mix quickly, and rotate at room temperature for 20 min.

[0045] (3) Cleaning to remove residual activator: Centrifuge the activated microspheres at 16000 r / min for 10 min, remove the supernatant, and add 1 ml MES to resuspend the microspheres; Centrifuge at 16000 r / min for 10 min, discard the supernatant, and add 1 ml MES to resuspend the microspheres for later use;

[0046] (4) Coupling of microspheres with antibodies: Add the optimal concentration of feline panleukopenia virus monoclonal antibody to the activated and cleaned microspheres, mix quickly, and then rotate at room temperature for 2 hours.

[0047] (5) Blocking: Add 100 μL of blocking solution (containing 10% BSA) and mix by rotating at room temperature for 1 h;

[0048] (6) Remove unbound antibodies: Centrifuge at 16000 r / min for 10 min, discard the supernatant, add 1 ml MES to resuspend the microspheres, centrifuge at 16000 r / min for 10 min, discard the supernatant, and remove unbound antibodies;

[0049] (7) Resuspension and reconstitution: Finally, resuspend the microspheres in 1 ml of reconstitution solution (0.02 M Tris-HCl + 10% sucrose) to obtain the antibody-microsphere labeled conjugate. Store at 4 °C for later use.

[0050] The composite was uniformly sprayed onto the glass fiber at a rate of 3 μL / cm and dried in an oven for 5-7 hours.

[0051] 2. Preparation of coating membrane

[0052] The glass cellulose membrane was adhered to a PVC substrate and equilibrated at 40% humidity for 30 minutes. Then, the prepared quality control line and test line coating solution was evenly applied to the glass cellulose membrane using a membrane drawing instrument. The membrane was then placed in an oven to dry for 16 hours.

[0053] 3. Assembly of fluorescent microsphere test strips

[0054] The prepared fluorescent microsphere pads, NC membranes (coated with control lines and test lines), sample pads, and absorbent filter paper were then placed together. Figure 3 Paste the strips sequentially onto a PVC base plate, overlapping each layer by 1-2 mm. Cut the strips into 4.0 mm wide strips and insert them into a cartridge. This is the feline panleukopenia virus antigen fluorescent microsphere test strip.

[0055] Example 4: Application of the time-resolved fluorescent microsphere immunochromatographic test strip of the present invention

[0056] The feline panleukopenia virus antigen fluorescent microsphere test strip prepared by this invention is mainly used to detect feline panleukopenia virus antigen in feline anal swab samples, and can also be used to assess the level of feline panleukopenia virus in laboratory culture.

[0057] 1. How to use the test strips

[0058] 1.1 Testing of Anal Swab Samples: Insert a disposable sampling swab into the cat's anus and rotate it 3 times. Unscrew the cap of the sample diluent tube, fully immerse the swab in the sample diluent, and hold the swab handle to squeeze the tube wall back and forth 5 times before discarding it. Tighten the cap, mix thoroughly, and the clear upper layer is the test solution. Use a pipette to draw 100 μl of the test solution and add it dropwise vertically into the sample well of the test strip.

[0059] 1.2 Test of virus samples: Dilute feline panleukopenia virus with serial dilution buffer. Use a pipette to draw 100 μl of the test solution and add it dropwise vertically to the sample well of the test strip.

[0060] Immediately after adding the sample, insert the test strip into the detection well of the fluorescence immunoassay analyzer and press "Standard Test". The instrument will automatically read the value after a 10-minute countdown. Alternatively, after adding the sample, allow it to react at room temperature (25±2℃) for 10 minutes, then insert the test strip into the detection well of the fluorescence immunoassay analyzer and immediately press "Rapid Test" to read the value automatically.

[0061] 2. Result Determination

[0062] After the test is completed, the test results can be read on the display screen. If the test result C value is <6000, it indicates that the test is invalid and the instrument screen will display the result as "invalid". The test strip needs to be replaced and the test repeated, or other possible causes of test failure should be investigated.

[0063] A sample test result with a T / C value ≥ 0.25 is considered positive, a T / C value < 0.19 is considered negative, and a T / C value ≤ 0.19 < 0.25 is considered suspicious and should be repeated. If the repeated test result has a T / C value ≥ 0.22, it is considered positive; if the T / C value < 0.22, it is considered negative.

[0064] Example 5: Performance comparison of immunochromatographic test strips prepared from the time-resolved fluorescent microspheres of the present invention and those prepared from commercially available microspheres.

[0065] The time-resolved fluorescent microspheres prepared according to Example 1 and commercially available microspheres from manufacturers A and B were conjugated with feline panleukopenia virus antibodies (the optimal labeling concentration for each was determined). Another feline panleukopenia virus antibody was coated onto the NC membrane, and feline panleukopenia virus antigen fluorescent microsphere test strips were prepared according to Example 3. These were test strip 1 (prepared using time-resolved fluorescent microspheres prepared according to the present invention), test strip 2 (prepared using time-resolved fluorescent microspheres from manufacturer A), and test strip 3 (prepared using time-resolved fluorescent microspheres from manufacturer B).

[0066] (1) Sensitivity detection of three feline panleukopenia virus antigen fluorescent microsphere test strips

[0067] The assembled three test strips were used to detect serially diluted feline panleukopenia virus, following the method described in Example 3. The results are shown in Table 1. The lowest detection concentrations of the three test strips were 6 × 10⁻⁶. 2 TCID 50 / 0.1ml, 1.2×10 3 TCID 50 / 0.1ml and 6×10 2 TCID 50 / 0.1ml, the sensitivity of test strip 1 is the same as that of test strip 3, both of which are higher than that of test strip 2.

[0068] Table 1. Sensitivity test results of three test strips

[0069]

[0070] (2) Specific detection of three feline panleukopenia virus antigen fluorescent microsphere test strips

[0071] Following the detection method described in Example 3, three test strips were used to detect feline herpesvirus, feline calicivirus, and feline coronavirus. The results are shown in Table 2. All three test strips made of time-resolved fluorescent microspheres showed no reaction, indicating good specificity.

[0072] Table 2. Specificity test results of the three test strips

[0073]

[0074] (3) Stability test of feline panleukopenia virus antigen fluorescent microsphere test strip

[0075] The three test strips were placed at 40°C for one month, and 1.2 × 10⁻⁶ was detected according to the method described in Example 3. 3 TCID 50 / 0.1ml of feline panleukopenia virus, the results showed that the CV of test strip 1 and test strip 2 was <10%, and the two test strips were basically stable (Table 3).

[0076] Table 3. Stability test results of three types of test strips

[0077]

[0078] In summary, the feline panleukopenia virus test strip prepared by the microspheres of this invention exhibits superior overall fluorescence intensity, sensitivity, and stability compared to manufacturers A and B, providing a high-performance labeled probe for highly sensitive fluorescence immunoassay.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing high-sensitivity and high-stability time-resolved fluorescent microspheres, characterized in that, Includes the following steps: (1) Preparation of high-stability, high-carboxyl-density polystyrene microspheres: Styrene monomer and glycolic acid monomer were dissolved in acetonitrile solvent, deionized water and trifluoroacetic acid were added, and the reaction was carried out in a water bath under nitrogen protection for 3 h. After cooling to room temperature, ethyl acetate was extracted, the mixture was dried by rotary evaporation, and the solid was pulped with n-heptane. After filtration and vacuum drying, the mixture was dissolved in deionized water containing dextran and stirred magnetically until homogeneous. After nitrogen purging three times, the mixture was sealed and heated to 60°C. An initiator was added, and the reaction was stirred under nitrogen for 12 h. After cooling to room temperature, the mixture was filtered to obtain a polymer nanosphere solution. After dialyzing in a dialysis bag for 72 h, a preservative solution was added and the mixture was stored at 4°C to obtain polystyrene microspheres with carboxyl density. (2) Preparation of high-stability europium ion chelate: europium ion standard acetonitrile solution, 1,4,7,10-tetramonoheterocyclic dodecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester, nitrogen protection at low temperature 0℃, sealed and stirred evenly, and stirred overnight at low speed. (3) Preparation of high-stability fluorescent microspheres: The high-stability high-carboxyl density microspheres prepared in step 1 were immersed in a swelling agent, acetonitrile and deionized water were added, and the mixture was stirred evenly. The europium ion chelate prepared in step 2 was added, and the mixture was stirred at 0°C for 12 hours. The organic solvent in the solution was removed by vacuum low-temperature rotary evaporation, and the eluent was collected by passing it through a molecular sieve column. The eluent was then freeze-dried using a freeze dryer, diluted to the standard mass volume with a preservative solution, and stored at 4°C.

2. The method for preparing high-sensitivity and high-stability time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (1), the styrene monomer is 50 mm and the glycolic acid monomer is 3.0 mm; the mass ratio of acetonitrile solvent, deionized water and trifluoroacetic acid is 1:0.6-0.7:0.16-0.17; the initiator is 5 mL of 0.2 mM potassium persulfate; and the preservative solution is 0.01% sodium azide.

3. The method for preparing high-sensitivity and high-stability time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (2), the europium ion standard acetonitrile solution can be replaced with any one of europium nitrate hydrate, europium fluoride, or europium nitride.

4. The method for preparing high-sensitivity and high-stability time-resolved fluorescent microspheres according to claim 1, characterized in that: In step (3), the mass ratio of acetonitrile to deionized water is 2:1; the preservative solution is 0.01% sodium azide.

5. Highly sensitive and highly stable time-resolved fluorescent microspheres prepared by the preparation method according to any one of claims 1-4.

6. The application of the high-sensitivity, high-stability time-resolved fluorescent microspheres according to claim 5 in the preparation of immunochromatographic test strips.

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