Carboxyl polystyrene microsphere preserving fluid and application

Abstract

The invention relates to the technical field of latex microspheres, in particular to a carboxyl polystyrene microsphere preserving fluid and application thereof, and the carboxyl polystyrene microsphere preserving fluid comprises a buffer solution, a surfactant, sodium 2, 4-dichlorophenoxyacetate and a preservative. The buffer solution is any one of a boric acid buffer solution, a tris buffer solution and a carbonate buffer solution, the interfacial agent is a twen series nonionic interfacial agent, and the preservative is NaN3 or Proclin 300. Sodium 2, 4-dichlorphenoxyacetate in the carboxyl polystyrene microsphere preserving fluid is negatively charged, chlorine atoms on a benzene ring are electron withdrawing groups, and oxygen atoms connected with the benzene ring and surrounding SP2 hybridized carbon can form a p-pi conjugation effect, so that the electronegativity of the benzene ring is jointly improved, the sodium 2, 4-dichlorphenoxyacetate is more easily adsorbed to the surface of the microsphere, and the preservation effect of the carboxyl polystyrene microsphere is improved. The stability and surface potential of surface charges of the microspheres are maintained, so that the microspheres are not easy to agglutinate in the antibody binding process.

Description

Technical Field

[0001] The present invention relates to the technical field of latex microspheres, and specifically relates to a carboxyl polystyrene microsphere preservation solution and its application. Background Art

[0002] Latex-enhanced immunoturbidimetry is a relatively stable and accurate immunoturbidimetric technique. Its principle is to coat the antibody corresponding to the substance to be tested on 10nm-1μm latex microspheres, which increases the volume of the antigen-antibody complex. After light passes through, the intensity changes of transmitted light and scattered light are more significant, thereby improving the sensitivity of the test.

[0003] Patent CN116087502A discloses a microsphere preservation solution to improve the dispersibility of microspheres, including polyols, buffers, protein protectants, polymers, non-ionic surfactants and preservatives. The microsphere preservation solution suspends and disperses the microspheres through the synergistic effect of polyols, polymers and non-ionic surfactants, and cooperates with the buffers and protein protectants to maintain the long-term stability of the microspheres, thereby improving the accuracy of the test results. This patent is specifically for fluorescently encoded microspheres with covalently cross-linked antibodies or antigens on the surface.

[0004] Patent CN109370568B discloses a preservation solution for fluorescent microspheres, including ethanol, buffer, inorganic sodium salt, sugar, organic dispersant and water. The ethanol, sugar and organic dispersant work synergistically to enhance the protection of the surface active groups of the fluorescent microsphere particles. At the same time, the inorganic sodium salt adjusts the system ion concentration and the buffering effect of the buffer, thereby achieving a better protection effect on the fluorescent microspheres and improving the stability of the fluorescence signal and the stability of the biological activity.

[0005] Patent CN115786323A discloses a magnetic microsphere preservation solution, which includes acidic substances, metal salts and buffer solutions. The metal ions and hydrogen ions not only effectively increase the magnetic responsiveness of the magnetic beads, but also improve their extraction performance in rapid extraction procedures (especially in RNA extraction, such as RNA extraction of the new coronavirus).

[0006] Commonly used latex microspheres with surface carboxyl groups can bind antigens and antibodies through covalent coupling. However, the microspheres themselves interact with each other, and prolonged storage often leads to self-aggregation. This aggregation problem can be addressed through ultrasonic cleaning and ultrasonic disruption. However, during EDC / NHS activation and antibody binding, the charge on the microsphere surface changes, leading to agglutination. This agglutination problem is difficult to resolve through ultrasonic disruption, thus affecting the accuracy of experimental results. Therefore, it is extremely important to ensure the monodispersity of the microspheres during long-term storage and after antibody binding.

[0007] However, there are few reports on carboxyl polystyrene microsphere storage solutions. Therefore, there is an urgent need to provide a carboxyl polystyrene latex microsphere storage solution to improve the monodispersity of the microspheres during long-term storage and the monodispersity after binding to antibodies. Summary of the Invention

[0008] To address the problems of poor long-term storage stability and easy aggregation of carboxylated polystyrene microspheres, as well as the easy aggregation and difficulty in dispersion of the microspheres during EDC / NHS activation and antibody binding, the present invention provides a carboxylated polystyrene microsphere storage solution and its application.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A carboxyl polystyrene microsphere preservation solution comprises a buffer, a surfactant, sodium 2,4-dichlorophenoxyacetate and a preservative.

[0011] The buffer is any one of borate buffer, tris buffer and carbonate buffer to maintain the pH stability of the preservation solution.

[0012] The working concentration of the buffer is 0.5 mM to 50 mM. Further, the working concentration of the buffer is 1 mM to 25 mM. Further, the working concentration of the buffer is 10 mM to 20 mM.

[0013] Wherein, the surfactant is a tween series non-ionic surfactant, preferably one of tween 20 and tween 80.

[0014] The mass fraction of the surfactant is 0.01%-0.5%. Further, the mass fraction of the surfactant is 0.05%-0.3%. Furthermore, the mass fraction of the surfactant is 0.1%-0.2%.

[0015] The mass fraction of the sodium 2,4-dichlorophenoxyacetic acid is 0.01%-0.05%. Furthermore, the mass fraction of the sodium 2,4-dichlorophenoxyacetic acid is 0.01%-0.02%.

[0016] Wherein, the preservative is NaN3 or Proclin 300. Preferably, it is Proclin 300.

[0017] Wherein, the mass fraction of the preservative is 0.04%-0.1%.

[0018] The carboxyl polystyrene microsphere storage solution of the present invention can be used for microsphere storage and antibody coupling, thereby improving the monodispersity and stability of the microspheres during long-term storage and antibody coupling.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The sodium 2,4-dichlorophenoxyacetic acid in the carboxyl polystyrene microsphere preservation solution of the present invention is negatively charged, the chlorine atom on the benzene ring is an electron-withdrawing group, and the oxygen atom connected to the benzene ring is negatively charged with the surrounding SP 2 The hybridized carbon will form a p-π conjugation effect, increase the electronegativity of the benzene ring, and be more easily adsorbed to the surface of the microspheres, thereby maintaining the stability of the surface charge and surface potential of the microspheres, making it difficult for the microspheres to aggregate during the antibody binding process.

[0021] (2) The surfactant in the carboxyl polystyrene microsphere preservation solution of the present invention contains hydrophilic groups and lipophilic groups. The lipophilic groups interact with the microspheres, and the hydrophilic groups are dispersed in water, which effectively improves the monodispersity of the microspheres, ensures the long-term storage stability of the microspheres, and makes the microspheres less likely to agglomerate during the EDC / NHS activation process, thereby improving their monodispersity.

[0022] (3) The buffer solution in the carboxyl polystyrene microsphere storage solution of the present invention can provide a stable pH range for the microspheres, making the microspheres less susceptible to the effects of the storage environment and improving the stability of the microspheres. For example, CO2 in the air can interfere with pH and affect their performance.

[0023] (4) The buffer, surfactant and sodium 2,4-dichlorophenoxyacetic acid work synergistically to improve the monodispersity and stability of the microspheres during long-term storage and antibody coupling. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0025] Example 1 Preparation of Carboxyl Polystyrene Microsphere Preservative Solution

[0026] Prepare carboxylated polystyrene microsphere storage solutions according to the following six recipes. Prepare the buffer solution first, then add the other reagents to the buffer solution in the appropriate proportions.

[0027] Microsphere storage solution blank control (B-0): pure water.

[0028] Microsphere storage solution 1 (B-1): 10 mM sodium bicarbonate-sodium carbonate buffer (pH 8.5), Tween 20 0.1% (w / w), sodium salicylate 0.02% (w / w), Proclin 300 0.05% (w / w).

[0029] Microsphere storage solution 2 (B-2): 10 mM sodium bicarbonate-sodium carbonate buffer (pH 8.5), Tween 20 0.1% (w / w), sodium 4-chlorophenoxyacetate 0.02% (w / w), Proclin 300 0.05% (w / w).

[0030] Microsphere storage solution 3 (B-3): 10 mM sodium bicarbonate-sodium carbonate buffer (pH 8.5), Tween 20 0.1% (w / w), sodium phenoxyacetate 0.02% (w / w), Proclin 300 0.05% (w / w).

[0031] Microsphere storage solution 4 (B-4): 10 mM sodium bicarbonate-sodium carbonate buffer (pH 8.5), Tween 20 0.2

[0032] % (w / w), sodium 2,4-dichlorophenoxyacetate 0.01% (w / w), Proclin 300 0.04% (w / w).

[0033] Microsphere storage solution 5 (B-5): 20 mM tris buffer (pH 8.0), Tween 20 0.2% (w / w), sodium 2,4-dichlorophenoxyacetate 0.01% (w / w), Proclin 300 0.04% (w / w).

[0034] Microsphere storage solution 6 (B-6): 20 mM borate buffer (pH 8.5), Tween 20 0.2% (w / w), sodium 2,4-dichlorophenoxyacetate 0.015% (w / w), Proclin 300 0.04% (w / w).

[0035] Example 2 Microsphere Treatment

[0036] Take 3.5 mL of carboxyl polystyrene microspheres (5% solids content, product number LD102028, provided by Nanjing Liding Medical Biotechnology Co., Ltd.) and divide them into 7 equal portions of 0.5 mL each. Transfer the mixture to a centrifuge tube, centrifuge, remove the supernatant, and add 0.5 mL of microsphere preservation solution B-0, B-1, B-2, B-3, B-4, B-5, and B-6 prepared in Example 1, respectively. Mix by ultrasonication and set aside.

[0037] Example 3 Labeling Antibodies (Taking RBP Antibody as an Example)

[0038] (1) Activation: The microspheres treated in Example 2 were mixed with an appropriate amount of 25 mM MES buffer, and then an appropriate amount of 100 mg / mL EDC solution (prepared immediately before use) was added and activated at room temperature for 15 min (the latexes after EDC activation were recorded as E-0, E-1, E-2, E-3, E-4, E-5, and E-6, respectively).

[0039] (2) Coupling: Add appropriate amount of RBP antibody (Nanjing Liding Medical Biotechnology Co., Ltd.) and couple at room temperature for 1 h.

[0040] (3) Blocking: Add an appropriate amount of blocking solution (containing 1% BSA and 0.2% glycine) and react at room temperature for 1 hour.

[0041] (4) Removal of unbound antibodies: Centrifuge at 8000 rpm for 10 min and resuspend in 2 mL of PBS buffer.

[0042] (5) Dispersion: Ultrasonicate for 10 min to disperse the latex.

[0043] (6) Dilution: Use PBS buffer to dilute the latex at a volume ratio of 3:1 (the latex after antibody coupling is recorded as L-0, L-1, L-2, L-3, L-4, L-5, and L-6).

[0044] Example 4 Performance Test

[0045] (1) Calibrator testing

[0046] The above 7 latex reagents were tested using a Hitachi AU7180 fully automatic biochemical analyzer with a test wavelength of 570 nm. The volumes of the calibrator, quality control product R1 (Nanjing Liding Medical Biotechnology Co., Ltd.), and R2 (RBP-binding antibody latex prepared in Example 3) were 2 μL, 100 μL, and 100 μL, respectively, and the absorbance of the 4 calibrators was tested.

[0047] (2) Particle size test

[0048] The Otsuka nanoSAQLA particle size analyzer was used for the test. Each sample was tested three times and the average value was taken.

[0049] The microspheres were processed and labeled using the same process. The labeled antibody was a monoclonal antibody against retinol-binding protein (RBP) from Nanjing Liding Medical Biotechnology Co., Ltd. The labeled antibody was diluted to the same working concentration using the same solution for calibration. Table 1 shows the absorbance test results of the calibrators on the day of preparation (tested on the same day of preparation). Table 2 shows the average particle size and dispersion index (PDI) test results of the microspheres dispersed in different storage solutions (B-0, B-1, B-2, B-3, B-4, B-5, B-6), activated with EDC (E-0, E-1, E-2, E-3, E-4, E-5, E-6), and bound to the antibody (L-0, L-1, L-2, L-3, L-4, L-5, L-6).

[0050] Table 1 △Abs test results of calibrators

[0051]

[0052] Table 2 Latex average particle size and PDI test results

[0053]

[0054]

[0055] It can be seen from Table 1 that the absorbance and background value of L-0 after binding to the antibody are the highest, and the correlation with the calibration material is poor. Combined with the particle size analysis in Table 2, it can be seen that the latex is severely agglutinated; L-3 has a lower background value and reactivity than L-0, but the correlation is poor. Combined with the particle size analysis in Table 2, it can be seen that the latex is agglutinated at this time and the monodispersity is poor; L-1 / L-2 has a lower background value and reactivity than L-3, but the correlation is good. Combined with the particle size analysis, the monodispersity of the latex is slightly improved. This is because the hydroxyl and chlorine on the benzene ring in sodium salicylate and sodium 4-chlorophenoxyacetate in B-1 and B-2 are electron-withdrawing groups, which are easier to bind to the microspheres than sodium phenoxyacetate, thereby improving the stability and monodispersity of the microspheres; L-4 / L-5 / L-6 have the lowest background value and the correlation R 2 >0.98, combined with the particle size analysis, the latex PDI <0.05, indicating good monodispersity. This is because the benzene ring of sodium 2,4-dichlorophenoxyacetate in L-4 / L-5 / L-6 has two chlorine atoms, which are electron-withdrawing groups. The oxygen atom connected to the benzene ring is closely connected to the surrounding SP 2The hybridized carbon will form a p-π conjugation effect, which will jointly increase the electronegativity of the benzene ring, making it easier to adsorb to the surface of the microspheres, resulting in better monodispersity and stability. Combined with the experimental data in Table 2, before the microspheres were activated, the particle size and dispersibility were not much different. However, after activation and binding to antibodies, the particle size and dispersion index of E-0 / E-1 / E-2 / E-3 / L-0 / L-1 / L-2 / L-3 and E-4 / E-5 / E-6 / L-4 / L-5 / L-6 were quite different. The main manifestation was that the former had a large particle size and poor dispersibility, mainly due to severe agglomeration. This is also consistent with the result of high absorbance of L-0 / L-1 / L-2 / L-3 in Table 1. However, the absorbance value of L-4 / L-5 / L-6 was low, and the correlation R 2 >0.98, and the dispersion index of the latex after EDC activation and antibody binding was small, indicating that the microspheres stored in B-4 / B-5 / B-6 still showed good monodispersity despite the process of EDC activation and antibody binding.

[0056] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A carboxyl polystyrene microsphere storage solution, characterized in that: Contains buffer, surfactant, sodium 2,4-dichlorophenoxyacetate and preservative.

2. The carboxyl polystyrene microsphere storage solution according to claim 1, characterized in that: The buffer is any one of borate buffer, tris buffer and carbonate buffer.

3. The carboxyl polystyrene microsphere storage solution according to claim 2, characterized in that: The working concentration of the buffer is 0.5 mM-50 mM.

4. The carboxyl polystyrene microsphere storage solution according to claim 1, characterized in that: The surfactant is a tween series non-ionic surfactant.

5. The carboxyl polystyrene microsphere storage solution according to claim 4, characterized in that: The mass fraction of the surfactant is 0.01%-0.5%.

6. The carboxyl polystyrene microsphere storage solution according to claim 1, characterized in that: The mass fraction of the sodium 2,4-dichlorophenoxyacetic acid is 0.01%-0.05%.

7. The carboxyl polystyrene microsphere storage solution according to claim 1, characterized in that: The preservative is NaN3 or Proclin 300.

8. The carboxyl polystyrene microsphere storage solution according to claim 7, characterized in that: The mass fraction of the preservative is 0.04%-0.1%.

9. Use of the carboxyl polystyrene microsphere storage solution according to any one of claims 1 to 8 in microsphere storage and antibody coupling.

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