Carboxyl polystyrene microsphere preservative solution and application

Abstract

The present application relates to latex microsphere technical field, especially a kind of carboxyl polystyrene microsphere preservative and application, including buffer, interface active agent, 2,4-dichlorophenoxy sodium acetate and preservative.The buffer is any one in boric acid buffer, tris buffer and carbonate buffer, interface active agent is tween series non-ionic interface active agent, and preservative is NaN3Or Proclin 300.The 2,4-dichlorophenoxy sodium acetate in the carboxyl polystyrene microsphere preservative of the present application is negatively charged, and the chlorine atom on benzene ring is electron-withdrawing group, and the oxygen atom connected with benzene ring is connected with the surrounding SP 2 Hybrid carbon will form p-pi conjugation effect, jointly improve the electrongativity of benzene ring, more easily adsorbed to microsphere surface, keep the stability of microsphere surface charge and surface potential, so that microsphere is not easy to aggregate in the process of combining antibody.

Description

Technical Field

[0001] This invention relates to the field of latex microsphere technology, specifically to a carboxylated polystyrene microsphere preservation solution and its applications. Background Technology

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

[0003] Patent CN116087502A discloses a microsphere preservation solution to improve the dispersibility of microspheres. The solution includes a polyol, a buffer, a protein protectant, a polymer, a nonionic surfactant, and a preservative. The microsphere preservation solution suspends and disperses the microspheres through the synergistic effect of the polyol, polymer, and nonionic surfactant, and works synergistically with the buffer and protein protectant to maintain the long-term stability of the microspheres, thereby improving the accuracy of the detection results. Specifically, this patent targets fluorescently encoded microspheres with surface covalently cross-linked antibodies or antigens.

[0004] Patent CN109370568B discloses a preservation solution for fluorescent microspheres, comprising ethanol, buffer solution, inorganic sodium salt, sugar, organic dispersant, and water. The ethanol, sugar, and organic dispersant work synergistically to enhance the protection of the active groups on the surface of the fluorescent microspheres. At the same time, the inorganic sodium salt regulates the ion concentration of the system, and the buffer solution provides buffering effect, thereby achieving a better protection effect for the fluorescent microspheres and improving the stability of the fluorescence signal and the stability of 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 enhance their extraction performance in rapid extraction processes (especially in RNA extraction, such as RNA extraction from the novel coronavirus).

[0006] Commonly used latex microspheres with carboxyl-modified surfaces can bind antigens and antibodies via covalent coupling. However, interactions exist between the microspheres themselves, and prolonged storage often leads to microsphere aggregation. This aggregation problem can be solved by ultrasonic cleaning or ultrasonic disruption. However, during EDC / NHS activation and antibody binding, changes in the surface charge of the microspheres cause aggregation, which is difficult to resolve by ultrasonic disruption, thus affecting the accuracy of experimental results. Therefore, addressing the monodispersity of microspheres during long-term storage and after antibody binding is extremely important.

[0007] However, there are few reports on the preservation solution of carboxylated polystyrene microspheres. Therefore, there is an urgent need to provide a preservation solution for carboxylated polystyrene latex microspheres to improve the monodispersity of microspheres during long-term preservation and after binding with 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 tendency of microspheres to aggregate and be difficult to disperse after EDC / NHS activation and antibody binding processes, this invention provides a carboxylated polystyrene microsphere preservation solution and its application.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

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

[0011] The buffer solution 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 solution is 0.5 mM-50 mM. Further, the working concentration of the buffer solution is 1 mM-25 mM. Even further, the working concentration of the buffer solution is 10 mM-20 mM.

[0013] The surfactant is a nonionic surfactant from the Tween series, preferably one of Tween20 and Tween80.

[0014] The surfactant has a mass fraction of 0.01%-0.5%. Further, the surfactant has a mass fraction of 0.05%-0.3%. Even further, the surfactant has a mass fraction of 0.1%-0.2%.

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

[0016] The preservative is either NaN3 or Proclin 300, preferably Proclin 300.

[0017] The preservative has a mass fraction of 0.04%-0.1%.

[0018] The carboxylated polystyrene microsphere preservation solution described in this invention can be used for microsphere preservation and antibody conjugation, improving the monodispersity and stability of microspheres during long-term preservation and antibody conjugation.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) The sodium 2,4-dichlorophenoxyacetic acid in the carboxylated polystyrene microsphere preservation solution of the present invention carries a negative charge, the chlorine atom on the benzene ring is an electron-withdrawing group, and the oxygen atom connected to the benzene ring is connected to the surrounding SP 2 Hybridized carbon forms a p-π conjugation effect, which increases the electronegativity of the benzene ring, making it easier to adsorb onto the surface of the microspheres. This maintains the stability of the surface charge and surface potential of the microspheres, making them less prone to aggregation during antibody binding.

[0021] (2) The surfactant in the carboxylated 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 stability of the microspheres for long-term preservation, and makes the microspheres less likely to aggregate during the EDC / NHS activation process, thus improving their monodispersity.

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

[0023] (4) The synergistic effect of buffer, surfactant and sodium 2,4-dichlorophenoxyacetate improves the monodispersity and stability of microspheres during long-term preservation and antibody conjugation. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0025] Example 1: Preparation of Carboxylated Polystyrene Microsphere Preservation Solution

[0026] Prepare carboxylated polystyrene microsphere preservation solutions according to the following 6 formulations. When preparing the solutions, first prepare the buffer solution, and then add the other reagents to the buffer solution in the appropriate proportions.

[0027] Blank control for microsphere preservation solution (B-0): pure water.

[0028] Microsphere preservation 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 preservation 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 preservation 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 preservation 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 preservation 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 preservation 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 carboxylated polystyrene microspheres (5% solids content, catalog number LD102028, provided by Nanjing Liding Medical Biotechnology Co., Ltd.), divide them into 7 equal portions of 0.5 mL each. Transfer them to centrifuge tubes, centrifuge, remove the supernatant, and add 0.5 mL of the microsphere preservation solutions B-0, B-1, B-2, B-3, B-4, B-5, and B-6 prepared in Example 1 respectively. Mix well by sonication and set aside.

[0037] Example 3: Labeled Antibody (using RBP antibody as an example)

[0038] (1) Activation: Mix the microspheres treated in Example 2 with an appropriate amount of 25mM MES buffer, and then add an appropriate amount of 100mg / mL EDC solution (prepared and used immediately). Activate at room temperature for 15min (the latexes activated by EDC are respectively labeled as E-0, E-1, E-2, E-3, E-4, E-5, E-6).

[0039] (2) Conjugation: Add an appropriate amount of RBP antibody (Nanjing Liding Medical Biotechnology Co., Ltd.) and conjugate at room temperature for 1 hour.

[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) Remove unbound antibodies: Centrifuge at 8000 rpm for 10 min, then resuspend in 2 mL PBS buffer.

[0042] (5) Dispersion: Disperse the latex by ultrasonically breaking it up for 10 minutes.

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

[0044] Example 4 Performance Test

[0045] (1) Calibration test

[0046] The above seven latex reagents were tested using a Hitachi AU7180 fully automated biochemical analyzer at a wavelength of 570 nm. The volumes of the calibrators, quality control samples R1 (Nanjing Liding Medical Biotechnology Co., Ltd.), and R2 (RBP antibody-bound latex prepared in Example 3) were 2 μL, 100 μL, and 100 μL, respectively. The absorbance of the four calibrators was also tested.

[0047] (2) Particle size test

[0048] The samples were tested using an Otsuka nanoSAQLA nanoparticle size analyzer. Each sample was tested three times, and the average value was taken.

[0049] The same process was used to process and label the microspheres. The labeling antibody was a retinol-binding protein (RBP) monoclonal antibody from Nanjing Liding Medical Biotechnology Co., Ltd. The labeled antibody was diluted to the same working concentration using the same solution for calibrator testing. Table 1 shows the absorbance test results of the calibrators on the day of preparation (tested on the same day as preparation). Table 2 shows the average particle size and dispersion index (PDI) test results of the microspheres dispersed in different preservation solutions (B-0, B-1, B-2, B-3, B-4, B-5, B-6), after EDC activation (E-0, E-1, E-2, E-3, E-4, E-5, E-6), and after binding antibodies (L-0, L-1, L-2, L-3, L-4, L-5, L-6).

[0050] Table 1. ΔAbs test results of the calibrators

[0051]

[0052] Table 2. Results of latex average particle size and PDI test.

[0053]

[0054]

[0055] Table 1 shows that L-0 has the highest absorbance and background value after antibody binding, and poor correlation with the calibrator. Particle size analysis in Table 2 indicates severe latex aggregation. L-3 has lower background and reactivity than L-0, but poor correlation. Particle size analysis in Table 2 shows latex aggregation and poor monodispersity at this stage. L-1 / L-2 has lower background and reactivity than L-3, but good correlation. Particle size analysis shows slightly improved latex monodispersity. This is because the hydroxyl and chlorine groups on the benzene rings of sodium salicylate and sodium 4-chlorophenoxyacetate in B-1 and B-2 are electron-withdrawing groups, making them more readily bound to the microspheres than sodium phenoxyacetate, thus improving the stability and monodispersity of the microspheres. L-4 / L-5 / L-6 have the lowest background value and the highest correlation (R). 2 >0.98, combined with particle size analysis showing a latex PDI <0.05, indicates good monodispersity. This is because the sodium 2,4-dichlorophenoxyacetate in L-4 / L-5 / L-6 has two chlorine atoms in its benzene ring, which are electron-withdrawing groups. The oxygen atom connected to the benzene ring interacts with the surrounding SP... 2Hybridized carbon atoms form a p-π conjugation effect, collectively increasing the electronegativity of the benzene ring, making it easier to adsorb onto the microsphere surface, resulting in better monodispersity and stability. Based on the experimental data in Table 2, before microsphere activation, the particle size and dispersibility were not significantly different. However, after activation and antibody binding, the particle size and dispersibility of E-0 / E-1 / E-2 / E-3 / L-0 / L-1 / L-2 / L-3 differed significantly from those of E-4 / E-5 / E-6 / L-4 / L-5 / L-6, mainly due to the former's larger particle size and poorer dispersibility, primarily indicating more severe aggregation. This is consistent with the high absorbance of L-0 / L-1 / L-2 / L-3 in Table 1. However, the absorbance values ​​of L-4 / L-5 / L-6 were lower, with a correlation coefficient R... 2 The latex dispersion index was >0.98, and the latex dispersion index after EDC activation and antibody binding was relatively small, indicating that the microspheres stored in B-4 / B-5 / B-6 still exhibited good monodispersity despite undergoing EDC activation and antibody binding.

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

Claims

1. A carboxyl polystyrene microsphere preservation solution, characterized in that: The product comprises a buffer solution, a surfactant, sodium 2,4-dichlorophenoxyacetate, and a preservative. The buffer solution is any one of borate buffer, Tris buffer, and carbonate buffer, with a working concentration of 10 mM-20 mM. The surfactant is Tween 20 with a mass fraction of 0.2%. The sodium 2,4-dichlorophenoxyacetate has a mass fraction of 0.01%-0.015%.

2. The carboxylated polystyrene microsphere preservation solution according to claim 1, characterized in that: The preservative is NaN3 or Proclin 300.

3. The carboxylated polystyrene microsphere preservation solution according to claim 2, characterized in that: The preservative has a mass fraction of 0.04%.

4. The use of the carboxylated polystyrene microsphere preservation solution according to any one of claims 1-3 in microsphere preservation and antibody conjugation.

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