Magnetic nanometer microsphere storage buffer and preparation process

Abstract

This invention discloses a magnetic nanosphere storage buffer and its preparation process, belonging to the field of chemiluminescence reagent technology. The magnetic nanosphere storage buffer includes: a buffer solution, a microsphere suspending agent, a microsphere charge protectant, a protein protectant, an enzyme inhibitor, and a preservative; wherein the microsphere suspending agent is hydroxypropyl methylcellulose (HPMC) type II. The buffer solution provided in this application has the following advantages: (1) The microsphere storage buffer solution needs to have good suspension properties for the magnetic beads to avoid large-particle-size magnetic beads with high Fe3O4 content settling too quickly, affecting the accuracy and precision of the measurement. (2) The microsphere storage buffer solution can maintain the performance stability of the microspheres in the range of -20 to 45℃. It can maintain its good dispersibility and not agglomerate even after repeated freeze-thaw cycles at -20℃ and accelerated high-temperature treatment at 45℃.

Description

Technical Field

[0001] This invention belongs to the field of chemiluminescence reagent technology, specifically relating to a magnetic nanosphere storage buffer and its preparation process. Background Technology

[0002] In the field of IVD (in vitro diagnostics), magnetic nanospheres have become a key raw material in various detection technologies such as electrochemiluminescence, direct chemiluminescence, and enzyme immunoassay chemiluminescence due to their unique advantages. As a solid-phase carrier for magnetic separation, these microspheres can efficiently couple streptavidin, antigens, or antibodies to form immune complexes, providing a solid foundation for subsequent separation and detection. In practical applications, magnetic nanospheres are easily affected by factors such as particle size, Fe3O4 content, thermal stability, and freeze-thaw stability, resulting in different suspension properties or states in different storage buffers. However, magnetic beads with poor suspension properties may settle or aggregate during instrument loading (20-30 days on the instrument), leading to a decrease in the solid content of magnetic microspheres during the immune reaction, resulting in poor precision or accuracy of instrument measurements, and even off-target effects in quality control samples.

[0003] To address the aforementioned issues, patent CN116087504B discloses an immunomagnetic bead protective solution, its preparation method, and its application. The main components of the immunomagnetic bead protective solution include an amphiphilic polymer, a HOVE-based block fluorinated copolymer, a protective protein, a protease inhibitor, glycerol and / or a polymer, a complexing agent, an amphiphilic surfactant, and a preservative. The HOVE block fluorinated copolymer is selected from poly(HOVE-b-HFBOVE), poly(HOVE-b-TFEOVE), poly(HOVE-b-PFPOVE), or combinations thereof; the amphiphilic polymer is selected from poly(methacryloyloxyethylphosphorylcholine), polysulfobetaine methacrylate, polycarboxybetaine methacrylate, or combinations thereof; the protective protein includes BSA and L-lysine; the polymer is polyethylene glycol 200; the complexing agent is EDTA; and the amphiphilic surfactant is Tween-20.

[0004] While the above solutions can address the stability and aggregation issues during the preservation of immunomagnetic beads, they do not help with the suspension of immunomagnetic beads with high Fe3O4 content or large particle size (above 1 μm). Summary of the Invention

[0005] This invention discloses a magnetic nanosphere storage buffer and its preparation process to solve the problems of low stability and aggregation in the preservation process of immunomagnetic beads in the prior art.

[0006] A first aspect of this application provides a magnetic nanosphere storage buffer, comprising: a buffer solution, a microsphere suspending agent, a microsphere charge protectant, a protein protectant, an enzyme inhibitor, and a preservative; wherein the microsphere suspending agent is hydroxypropyl methylcellulose (HPMC) type II.

[0007] Optionally, the buffer solution is selected from citrate-sodium citrate or MES-NaOH buffer solution, with a concentration of 10-50 mmol / L and a pH of 6.0-7.0.

[0008] Optionally, the viscosity of the hydroxypropyl methylcellulose is 15000 mPa·s-100000 mPa·s, and the addition amount is 0.5-1.5 g / L.

[0009] Optionally, the microsphere charge protection agent is one or both of polyoxyethylene (2,10) soybean amine and alkyl glycoside 600, with an addition amount of 0.05-0.2 v / v.

[0010] Optionally, the enzyme inhibitor is benzoamidine hydrochloride, and the amount added is 0.2-0.5 g / L.

[0011] Optionally, the protein protectant is selected from one of BSA, newborn calf serum, and gamma globulin, wherein the amount of BSA or gamma globulin added is 5-20 g / L, and the amount of newborn calf serum added is 4-5 v / v.

[0012] Optionally, the preservative is selected from Proclin 300, Proclin 950, and diazolidinyl urea, wherein the amount of Proclin 300 or Proclin 950 added is 0.05-0.2 v / v, and the amount of diazolidinyl urea added is 0.5-2 g / L.

[0013] A second aspect of this application provides a process for preparing a magnetic nanosphere storage buffer solution, comprising:

[0014] Add appropriate amounts of buffer solution, microsphere charge protectant, enzyme inhibitor and preservative to purified water, stir well, filter, and obtain buffered protein solution;

[0015] Heat purified water to 70-90℃, and add hydroxypropyl methylcellulose while stirring until the hydroxypropyl methylcellulose is completely suspended in the water to obtain a hydroxypropyl methylcellulose solution.

[0016] The hydroxypropyl methylcellulose solution and the buffer protein solution were combined and stirred until homogeneous to obtain the magnetic nanosphere storage buffer solution.

[0017] The magnetic nanosphere storage buffer provided in this application needs to have the following advantages: (1) The microsphere storage buffer needs to have good suspension properties for the magnetic beads to avoid large-diameter magnetic beads with high Fe3O4 content settling too quickly, which would affect the accuracy and precision of the measurement. (2) The microsphere storage buffer can maintain the performance stability of the microspheres in the range of -20 to 45℃. It can maintain its good dispersibility and not agglomerate even after repeated freeze-thaw cycles at -20℃ and accelerated high-temperature treatment at 45℃. Detailed Implementation

[0018] 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.

[0019] This invention provides a magnetic nanosphere storage buffer, comprising a buffer solution, a microsphere suspending agent, a microsphere charge protectant, a protein protectant, an enzyme inhibitor, and a preservative.

[0020] The buffer solution is a citrate-sodium citrate or MES-NaOH buffer solution with a concentration of 10-50 mmol / L and a pH of 6.0-7.0. Low-concentration MES-NaOH or citrate-sodium citrate buffer systems maintain the stability of the magnetic beads; high-concentration MES-NaOH or citrate-sodium citrate buffer systems can easily cause antigen-antibody detachment or affect the spatial conformation of the antigen-antibody, thus weakening their activity. Therefore, the preferred concentration in this invention is 10-50 mmol / L.

[0021] The microsphere suspending agent is hydroxypropyl methylcellulose (HPMC) type II, with a viscosity of 15000 mPa·s-100000 mPa·s and an addition amount of 0.5-1.5 g / L. Hydroxypropyl methylcellulose is a nonionic cellulose mixed ether compound. It is a semi-synthetic, inactive, viscoelastic polymer and a hydrophilic surfactant, which can effectively improve the suspension properties of magnetic beads, especially large-particle-size beads with high Fe3O4 content; at the same time, it can protect the magnetic beads from aggregation at high and low temperatures.

[0022] The microsphere charge protectant is one or two of polyoxyethylene (2,10) soybean amine and alkyl glycoside 600, with an addition amount of 0.05-0.2 v / v%. The microsphere charge protectant can be effectively adsorbed onto the surface of the magnetic beads, protecting the electric double layer of the magnetic beads and enhancing the dispersibility of the magnetic beads.

[0023] The enzyme inhibitor is benzoamidine hydrochloride, added at a concentration of 0.2-0.5 g / L. Benzamide hydrochloride can effectively inhibit proteases, trypsin-like enzymes, and serine proteases, reducing the degradation of antigens and antibodies by enzymes introduced by inert proteins.

[0024] The protein protectant is selected from inert proteins, specifically one of BSA, newborn calf serum, and gamma globulin. The addition amount of BSA and gamma globulin is 5-20 g / L, and the addition amount of newborn calf serum is 4-5 v / v%. As a protective protein for antigens, antibodies, or streptavidin, it does not participate in the reaction and enhances the stability of the magnetic beads.

[0025] The preservative is one of Proclin 300, Proclin 950, and diazolidinyl urea, wherein the addition amount of Proclin 300 or Proclin 950 is 0.05-0.2 v / v, and the addition amount of diazolidinyl urea is 0.5-2 g / L.

[0026] To better describe the technical problems, technical solutions, and advantages of the present invention, the following description will be combined with implementation examples.

[0027] I. Preparation process of immunomagnetic bead preservation solution

[0028] Example 1

[0029] (1) Prepare 2-morpholinoethanesulfonic acid (MES) buffer protein solution

[0030] Take 1.95g of 2-morpholinoethanesulfonic acid (MES), 1mL of alkyl glycoside 600, 0.2g of benzoamide hydrochloride and 2mL of Proclin 300 in 700mL of purified water. Stir at 200r / min and add 20g of BSA while stirring. Then adjust the pH to 7.0±0.05 with 1mol / L NaOH solution, bring the volume to 800mL, and filter through a 0.22μm filter membrane.

[0031] (2) Preparation of hydroxypropyl methylcellulose solution

[0032] Heat 200 mL of purified water to 70-90 °C, then add 0.5 g of hydroxypropyl methylcellulose HPMC-II type while stirring at 15000 mPa·s until the hydroxypropyl methylcellulose is completely suspended in the water, about 5-10 min.

[0033] (3) Merging

[0034] Combine the hydroxypropyl methylcellulose solution with the 2-morpholinoethanesulfonic acid (MES) buffer protein solution and stir for 10 min to obtain the magnetic nanosphere storage buffer.

[0035] In this embodiment, the final concentration of MES in the buffer solution = 2-morpholinoethanesulfonic acid (MES) / 195 g·mol -1 *1000=10mmol / L.

[0036] Comparative Example 1

[0037] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain microsphere suspending agent.

[0038] Table 1. Formulations of Immunomagnetic Bead Preservation Solution for Example 1 and Comparative Example 1

[0039]

[0040] Example 2

[0041] (1) Prepare 2-morpholinoethanesulfonic acid (MES) buffer-protein solution

[0042] Take 9.75 g of 2-morpholinoethanesulfonic acid (MES), 0.5 mL of polyoxyethylene (2,10) soybean amine, 0.5 g of benzalkonium hydrochloride and 0.5 mL of Proclin 950 and add them to 700 mL of purified water. Stir at 200 r / min and add 5 g of γ-globulin while stirring. Then adjust the pH to 6.00 ± 0.05 with 1 mol / L NaOH solution, bring the volume to 800 mL and filter through a 0.22 μm filter membrane.

[0043] (2) Preparation of hydroxypropyl methylcellulose solution

[0044] Heat 200 mL of purified water to 70-90 °C, then add 1.5 g of hydroxypropyl methylcellulose HPMC-II type while stirring at 15000 mPa·s until the hydroxypropyl methylcellulose is completely suspended in the water, about 5-10 min.

[0045] (3) Merging

[0046] The hydroxypropyl methylcellulose solution and the 2-morpholinoethanesulfonic acid (MES) buffer protein solution were combined and stirred at 200 rpm for 10 min until completely dissolved, which is the magnetic nanosphere storage buffer.

[0047] In this embodiment, the final concentration of MES in the buffer solution = 2-morpholinoethanesulfonic acid / 195 g·mol -1 *1000=10mmol / L.

[0048] Comparative Example 2

[0049] The only difference between Comparative Example 2 and Example 2 is that Comparative Example 2 does not add microsphere suspending agent, but adds 2g of sucrose instead of microsphere suspending agent.

[0050] Table 2. Formulations of Immunomagnetic Bead Preservation Solution for Example 2 and Comparative Example 2

[0051]

[0052]

[0053] Example 3

[0054] (1) Preparation of citric acid-sodium citrate buffer-protein solution

[0055] Take 0.275g of citric acid monohydrate, 2.242g of trisodium citrate, 1.5mL of alkyl glycoside 600, 0.2g of benzoamide hydrochloride, and 2.0g of diazolidinyl urea in 700mL of purified water. Stir at 100r / min while adding 40mL of newborn calf serum. Then adjust the pH to 6.0±0.05 with 1mol / L NaOH solution, bring the volume to 800mL, and filter through a 0.22μm filter membrane.

[0056] (2) Preparation of hydroxypropyl methylcellulose solution

[0057] Heat 200 mL of purified water to 80 °C, then add 0.5 g of hydroxypropyl methylcellulose HPMC-II type while stirring at 15000 mPa·s until the hydroxypropyl methylcellulose is completely suspended in the water, about 5-10 min.

[0058] (3) Merging

[0059] Combine the hydroxypropyl methylcellulose solution with the citric acid-sodium citrate buffer-protein solution and stir for 10 minutes to obtain the magnetic nanosphere storage buffer.

[0060] In this embodiment, the final concentration of the citrate-sodium citrate buffer solution = sodium citrate monohydrate / 210 g·mol -1 *1000+ trisodium citrate / 258.1g·mol -1 *1000=0.275 / 210*1000+2.242 / 258.1*1000=10mmol / L.

[0061] Comparative Example 3

[0062] The only difference between Comparative Example 3 and Example 3 is that Comparative Example 3 does not add microsphere suspending agent, but adds 2g of glycerol instead of microsphere suspending agent.

[0063] Table 3. Formulations of Immunomagnetic Bead Preservation Solution for Example 3 and Comparative Example 3

[0064]

[0065]

[0066] Example 4

[0067] (1) Preparation of citric acid-sodium citrate buffer-protein solution

[0068] Take 1.375g of citric acid monohydrate, 11.21g of trisodium citrate, 2.0mL of polyoxyethylene (2,10) soybean amine, 0.2g of benzoamide hydrochloride and 0.5g of diazolidinyl urea in 700mL of purified water. Stir at 100r / min and add 50mL of newborn calf serum while stirring. Then adjust the pH to 7.0±0.05 with 1mol / L NaOH solution, bring the volume to 800mL, and filter through a 0.22μm filter membrane.

[0069] (2) Preparation of hydroxypropyl methylcellulose solution

[0070] Heat 200 mL of purified water to 70-80 °C, then add 1.5 g of hydroxypropyl methylcellulose HPMC-II type at 100000 mPa·s while stirring until the hydroxypropyl methylcellulose is completely suspended in the water, about 5-10 min.

[0071] (3) Merging

[0072] Combine the hydroxypropyl methylcellulose solution with the citric acid-sodium citrate buffer-protein solution and stir for 10 minutes to obtain the magnetic nanosphere storage buffer.

[0073] In this embodiment, the final concentration of the citrate-sodium citrate buffer solution = sodium citrate monohydrate / 210 g·mol -1 *1000+ trisodium citrate / 258.1g·mol -1 *1000=1.375 / 210*1000+11.21 / 258.1*1000=50mmol / L.

[0074] Comparative Example 4

[0075] The only difference between Comparative Example 4 and Example 4 is that Comparative Example 4 does not add microsphere suspending agent, but adds 2g of PEG10000 instead of microsphere suspending agent.

[0076] Table 4. Formulations of Immunomagnetic Bead Preservation Solution for Example 4 and Comparative Example 4

[0077]

[0078] II. Preparation of Immunomagnetic Beads

[0079] Streptavidin was coated using three different sizes of carboxylated magnetic beads (600-700 nm, 1000-1200 nm, and 2500-2800 nm). The prepared streptavidin magnetic beads were dispersed in different immunomagnetic bead preservation solutions to verify the suspension, precision, signal value, stability, and aggregation under extreme conditions of the magnetic beads.

[0080] cTnI protein I antibody was coated using three different sizes of carboxyl magnetic beads (600-700 nm, 1000-1200 nm, and 2500-2800 nm). The prepared cTnI protein I magnetic beads were dispersed in different immunomagnetic bead preservation solutions, and the suspension, precision, signal value, stability, and aggregation under extreme conditions of the magnetic beads were validated. 1. Method for preparing streptavidin magnetic beads.

[0081] (1) 5 mL of carboxyl magnetic beads (50 mg / mL) were placed in a 250 mL PP bottle. The carboxyl magnetic bead storage solution was removed, and 25 mL of 10 mmol / L MES (pH = 6.0) was added to redisperse the mixture. The mixture was sonicated for 1 min, magnetically separated, and the liquid was discarded. 25 mL of 10 mmol / L MES (pH = 6.0) was added again.

[0082] (2) Weigh EDC and NHS, and prepare 50 mg / mL EDC and NHS solutions respectively using 10 mmol / L MES (pH = 6.0). Take 0.2 mL of each solution and quickly add them to the magnetic bead suspension. Then, place the solution at 37°C and react at 220 rpm for 30 min.

[0083] (3) Clean the EDC thoroughly, wash it twice, remove the suspension, and add 5 mL of 10 mmol / L MES (pH = 6.0);

[0084] (4) Prepare 50 mg / mL streptavidin, add 100 μL (10.0 mg) to the magnetic bead suspension, and place it at 37 °C and react at 220 rpm for 12 h;

[0085] (5) Transfer to a 500mL PP bottle, magnetically separate, discard the supernatant, add 250mL of 20Mm Tris-HCl buffer (containing 0.9% sodium chloride + 0.15% Tween-20 + 0.1% P300), and sonicate five times. After each 5min interval, discard the 20Mm Tris-HCl buffer (containing 0.9% sodium chloride + 0.15% Tween-20 + 0.1% P300), add magnetic bead preservation solution, and obtain magnetic beads with a concentration of 1mg / mL streptavidin.

[0086] 2. Preparation method of cTnI protein I magnetic beads

[0087] (1) 5 mL of carboxyl magnetic beads (50 mg / mL) were placed in a 250 mL PP bottle. The carboxyl magnetic bead storage solution was removed, and 25 mL of 10 mmol / L MES (pH = 6.0) was added to redisperse the mixture. The mixture was sonicated for 1 min, magnetically separated, and the liquid was discarded. 25 mL of 10 mmol / L MES (pH = 6.0) was added again.

[0088] (2) Weigh EDC and NHS, and prepare 50 mg / mL EDC and NHS solutions respectively using 10 mmol / L MES (pH = 6.0). Take 0.2 mL of each solution and quickly add them to the magnetic bead suspension. Then, place the solution at 37°C and react at 220 rpm for 30 min.

[0089] (3) Magnetic separation, wash twice, remove suspension, and add 25 mL of 10 mmol / L MES (pH = 6.0);

[0090] (4) Add 4 mg of cTnI protein I antibody to the magnetic bead suspension and place it at 37°C and 220 rpm for 12 h.

[0091] (5) Transfer to a 500mL PP bottle, magnetically separate, discard the supernatant, add 250mL of 20Mm Tris-HCl buffer (containing 0.9% sodium chloride + 0.15% Tween-20 + 0.1% P300), and sonicate five times. After each 5min interval, discard the 20Mm Tris-HCl buffer (containing 0.9% sodium chloride + 0.15% Tween-20 + 0.1% P300), add magnetic bead preservation solution, and obtain magnetic beads with a concentration of 1mg / mL cTnI protein I.

[0092] III. Evaluation Results of Magnetic Bead Preservation Solution

[0093] (1) Comparison of the suspendability of streptavidin magnetic beads and troponin I magnetic beads after the addition of hydroxypropyl methylcellulose.

[0094] Table 5. Effects of different immunomagnetic bead preservation solutions on the suspension properties of streptavidin and troponin I magnetic beads.

[0095]

[0096]

[0097] Table 5 shows that, without the addition of hydroxypropyl methylcellulose, the settling velocity of streptavidin magnetic beads and troponin I magnetic beads with a particle size of 600-700 nm is approximately 2.1-2.5 cm / h, the settling velocity of streptavidin magnetic beads and troponin I magnetic beads with a particle size of 1000-1200 nm is approximately 4.2-4.8 cm / h, and the settling velocity of streptavidin magnetic beads and troponin I magnetic beads with a particle size of 2500-2800 nm is approximately 7.2-7.9 cm / h. With the addition of hydroxypropyl methylcellulose, the settling velocity of streptavidin magnetic beads and troponin I magnetic beads with a particle size of 600-700 nm is approximately 2.1-2.5 cm / h, the settling velocity of tropavidin magnetic beads and troponin I magnetic beads with a particle size of 1000-1200 nm is approximately 4.2-4.8 cm / h, and the settling velocity of tropavidin magnetic beads and troponin I magnetic beads with a particle size of 2500-2800 nm is approximately 7.2-7.9 cm / h. The sedimentation rate of troponin I magnetic beads decreased to 0.6-1.5 cm / h, while that of streptavidin and troponin I magnetic beads with a particle size of 1000-1200 nm decreased to 1.8-2.9 cm / h, and that of streptavidin and troponin I magnetic beads with a particle size of 2500-2800 nm decreased to 2.3-4.3 cm / h. In particular, the suspension properties of streptavidin and troponin I magnetic beads were significantly improved by adding hydroxypropyl methylcellulose to the immunomagnetic bead preservation solution. The application of this buffer reduces the sedimentation rate of the magnetic beads in the chemiluminescence kit, resulting in more consistent immunoreactions at different time points.

[0098] (2) Comparison of the precision of streptavidin magnetic beads and troponin I magnetic beads after the addition of hydroxypropyl methylcellulose.

[0099] To compare the precision of streptavidin magnetic beads and troponin I magnetic beads after the addition of hydroxypropyl methylcellulose, two high-sensitivity troponin I detection kits with different components were prepared for precision testing.

[0100] A. High-sensitivity troponin I assay kit 1 (3 components): streptavidin magnetic beads; biotin antibody Ab1-Bio component; acridine ester labeled antibody Ab2-AE component.

[0101] B. High-sensitivity troponin I assay kit 2 (2 components): troponin I-Ab1, composed of carboxyl magnetic beads of different particle sizes coated with different sizes; and acridine ester-labeled antibody Ab2-AE component.

[0102] C. The precision testing procedure was as follows: quality control samples L and H were measured twice daily for a total of 20 days. The results are shown in Tables 6-1 and 6-2. In this embodiment, quality control samples L and H were sourced from Getein Biotechnology Co., Ltd., model SCI24002Z.

[0103] Table 6-1 Effects of different immunomagnetic bead preservation solutions on the precision of streptavidin magnetic beads and troponin I magnetic bead carrier assay for troponin I quality control product L.

[0104]

[0105]

[0106] As shown in Table 6-1, the high-sensitivity troponin I assay kit 1 (3 components) used the magnetic bead buffer (without hydroxypropyl methylcellulose) of comparative examples 1-4 to determine the troponin I quality control L. The average precision values ​​of carboxyl magnetic beads of different particle sizes were 4.03%, 5.3%, and 5.78%, respectively, while the average precision values ​​of examples 1-4 (with hydroxypropyl methylcellulose) were 3.13%, 3.38%, and 3.45%, respectively.

[0107] The high-sensitivity troponin I assay kit 2 (two components) used magnetic bead buffer (without hydroxypropyl methylcellulose) from Comparative Examples 1-4 to determine the troponin I quality control L. The mean precision values ​​for different particle sizes of carboxyl magnetic beads were 5.50%, 5.35%, and 5.28%, respectively, while the mean precision values ​​for Examples 1-4 (with hydroxypropyl methylcellulose) were 3.90%, 3.63%, and 3.53%, respectively. Overall, the mean precision values ​​of Examples 1-4 were 1-2% better than those of Comparative Examples 1-4.

[0108] Table 6-2 Effects of different immunomagnetic bead preservation solutions on the precision of streptavidin magnetic beads and troponin I magnetic bead carrier assay for troponin I quality control product H.

[0109]

[0110]

[0111] As shown in Table 6-2, regardless of whether it is the high-sensitivity troponin I assay kit 1 (3 components) or the high-sensitivity troponin I assay kit 2 (2 components) for measuring troponin I quality control H, the average precision of Examples 1-4 is 1-2% better than that of Comparative Examples 1-4.

[0112] (3) Comparison of the signal values ​​of samples measured by the hs-cTnI detection kit after the addition of hydroxypropyl methylcellulose.

[0113] Table 7. Formulations of Immunomagnetic Bead Preservation Solution for Comparative Example 1 and Example 5

[0114]

[0115] As shown in Table 8, after adding 0.1% hydroxypropyl methylcellulose to the preservation solution of streptavidin magnetic beads and troponin I magnetic beads, the luminescence value deviation of the four cTnI concentration samples was within 5%, and the low value gradient was consistent, indicating that the addition of 0.1% hydroxypropyl methylcellulose had no effect on the signal value and gradient of the reagents.

[0116] Table 8. Effect of hydroxypropyl methylcellulose on the signal value of the hs-cTnI detection kit.

[0117]

[0118] (4) The effect of microsphere charge protection agent on magnetic bead aggregation or agglomeration under extreme environments

[0119] Immunomagnetic beads are subject to high temperatures or repeated freeze-thaw cycles during long-distance transportation, limiting their application. To address this issue, this patent compares the effects of different charge-protecting agents on the aggregation or agglomeration of magnetic beads under extreme conditions, based on the formulations in Table 1. The control group consisted of no additives, Tween-20, and PEG10000; the experimental group consisted of polyoxyethylene (2,10) soybean amine and alkyl glycoside 600. The experimental results are shown in Tables 10 and 11.

[0120] Table 9. Formulations of Immunomagnetic Bead Preservation Solution for Example 1 and Comparative Examples 6-9

[0121]

[0122]

[0123] Table 10. Effect of charge protection agent on signal value of hs-cTnI detection kit

[0124]

[0125] Table 11 Effects of charge protection agents on the stability of immunomagnetic beads

[0126]

[0127]

[0128] Table 10 shows that under extreme conditions of accelerated freezing at 45℃ for 3 days or freeze-thaw cycles at -20℃ for 3 days, the control group did not add charge protection agents or added existing charge protection agents such as Tween-20 and PEG10000. In the experimental group of this invention, the storage solution containing polyoxyethylene (2,10) soybean amine and alkyl glycoside 600 did not cause agglomeration of streptavidin magnetic beads or troponin I magnetic beads. Table 11 shows that under extreme conditions of accelerated freezing at 45℃ for 53 days or freeze-thaw cycles at -20℃ for 3 days, the storage solution containing polyoxyethylene (2,10) soybean amine and alkyl glycoside 600 in the experimental group showed a magnetic bead shrinkage rate of less than 10% during accelerated freezing and thawing, which was superior to that of existing technologies such as Tween-20 and PEG10000.

[0129] Compared with existing technologies, the immunomicrosphere storage solution provided by this invention has the following advantages: ① Good dispersibility, improving the suspension of magnetic microspheres with particle sizes of 600-700nm, 1000-1200nm, and 2500-2800nm ​​by 2 times; ② Good reagent carrier precision (1%); ③ Added charge protection agents alkyl glycoside 600 and polyoxyethylene (2,10) soybean amine, preventing agglomeration; ④ Heat resistance at 45℃ and no agglomeration after repeated freeze-thaw cycles at -20℃.

[0130] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A magnetic nanomicrospheres storage buffer, characterized in that, include: The microspheres contain a buffer solution, a microsphere suspending agent, a microsphere charge protectant, a protein protectant, an enzyme inhibitor, and a preservative; wherein the microsphere suspending agent is hydroxypropyl methylcellulose (HPMC) type II.

2. The magnetic nanomicrospheres storage buffer according to claim 1, wherein, The buffer solution is selected from citrate-sodium citrate or MES-NaOH buffer solution, with a concentration of 10-50 mmol / L and a pH of 6.0-7.

0.

3. The magnetic nanomicrospheres storage buffer according to claim 1, wherein, The hydroxypropyl methylcellulose has a viscosity of 15000 mPa·s-100000 mPa·s and is added at a rate of 0.5-1.5 g / L.

4. The magnetic nanomicrospheres storage buffer of claim 1, wherein, The microsphere charge protection agent is one or both of polyoxyethylene (2,10) soybean amine and alkyl glycoside 600, with an addition amount of 0.05-0.2 v / v.

5. The magnetic nanomicrospheres storage buffer of claim 1, wherein, The enzyme inhibitor is benzoamidine hydrochloride, and the addition amount is 0.2-0.5 g / L.

6. The magnetic nanomicrospheres storage buffer of claim 1, wherein, The protein protectant is selected from one of BSA, newborn calf serum, and gamma globulin, wherein the amount of BSA or gamma globulin added is 5-20 g / L, and the amount of newborn calf serum added is 4-5 v / v.

7. The magnetic nanomicrospheres storage buffer of claim 1, wherein, The preservative is selected from Proclin 300, Proclin 950, and diazolidinyl urea, wherein the amount of Proclin 300 or Proclin 950 added is 0.05-0.2 v / v, and the amount of diazolidinyl urea added is 0.5-2 g / L.

8. A preparation process for a magnetic nanosphere storage buffer solution, characterized in that, include: Add appropriate amounts of buffer solution, microsphere charge protectant, enzyme inhibitor and preservative to purified water, stir well, filter, and obtain buffered protein solution; Heat purified water to 70-90℃, and add hydroxypropyl methylcellulose while stirring until the hydroxypropyl methylcellulose is completely suspended in the water to obtain a hydroxypropyl methylcellulose solution. The hydroxypropyl methylcellulose solution and the buffer protein solution were combined and stirred until homogeneous to obtain the magnetic nanosphere storage buffer solution.

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