Multilayer streptavidin-coated magnetic microparticles, method for preparing the same and use thereof

Abstract

The present application relates to the field of biotechnology, especially to a multi-layer streptavidin coated magnetic microparticle, a preparation method and application thereof. The present application uses a self-assembly method, and uses NHS-PEG(n)-NHS as a connecting arm between streptavidin layers. When the streptavidin molecular layer is increased, the amount of biotin combined with the magnetic microparticle is significantly improved. The PEG connecting arm between the streptavidin molecular layers increases the spatial structure, effectively reduces the spatial steric hindrance between the streptavidin molecules, and thus significantly reduces the difficulty of capturing target macromolecules. At the same time, the magnetic microparticle coated by the process has obvious advantages in dispersibility. The streptavidin magnetic microparticle coated by the method can be applied to a magnetic microparticle chemiluminescence detection platform and used in an immunological detection kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a multi-layer streptavidin coated magnetic micro-particle, a preparation method and application thereof. BACKGROUND

[0002] The rapid development of biomedical research and diagnostic technology greatly depends on the efficient and specific means of biomolecular separation and detection. As a highly stable tetramer protein, streptavidin can form a very firm non-covalent bond with small molecule biotin. This property makes it an indispensable tool molecule in biosensing, molecular biology and clinical diagnosis. Although traditional streptavidin conjugated magnetic micro-particles have been applied to the capture and separation of various biomolecules, they still face several key challenges. First, the limited binding sites caused by single-layer streptavidin coverage limit the maximum binding capacity. Second, the spatial structure of single-layer streptavidin easily produces high steric hindrance effect, which makes it difficult to bind large molecules. Third, the imperfect surface modification technology of magnetic micro-particles affects the biocompatibility and specific binding of target molecules. Therefore, how to successfully construct a stable and efficient multi-layer streptavidin structure on the surface of magnetic micro-particles while maintaining their magnetic responsiveness and good biocompatibility is the focus and difficulty of current research.

[0003] Therefore, it is of great significance to develop a new type of streptavidin magnetic micro-particle that can overcome the limitations of existing technology, has high biotin loading capacity, low steric hindrance, high dispersibility and low non-specificity, and promote the progress of biomedical research and clinical practice. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a multi-layer streptavidin coated magnetic micro-particle, a preparation method and application thereof.

[0005] The present application provides a multi-layer streptavidin magnetic micro-particle, which is obtained by sequentially coating streptavidin magnetic beads with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide and streptavidin; the number of sequential coating with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide and streptavidin is m, and m is an integer from 1 to 10. In a specific embodiment of the present application, the number of coating layers is 3.

[0006] Further, n is an integer from 10 to 30; specifically, n is 12 is optimal.

[0007] The present application provides a preparation method of the multi-layer streptavidin magnetic micro-particle, which comprises the following steps:

[0008] Step 1: First reaction of streptavidin magnetic beads with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide to obtain a first reaction product;

[0009] Step 2, the first reactant is reacted with streptavidin to complete a layer of streptavidin coating;

[0010] Step 3, after repeating steps 1 to 2 m times, the coating is closed to obtain the multi-layer streptavidin magnetic microparticles.

[0011] In the preparation method of the application,

[0012] The streptavidin magnetic beads are obtained by activating the magnetic beads and coating with streptavidin.

[0013] The activated reagent is EDC, NHS and glutaraldehyde;

[0014] Further, the activated reagent is a mixture of EDC and NHS or glutaraldehyde;

[0015] In the mixture of EDC and NHS, the mass ratio of EDC to NHS is 1:1;

[0016] The mass ratio of the magnetic beads, EDC and NHS is (25-35):1:1, and in a specific embodiment of the application, the mass ratio is 30:1:1.

[0017] The mass ratio of the magnetic beads and glutaraldehyde is (25-35):(1-3), and in a specific embodiment of the application, the mass ratio is 30:1.5.

[0018] In a specific embodiment of the application, the performance of the magnetic microparticles prepared by using the mixture of EDC and NHS as the activating agent is slightly better than that of the magnetic microparticles activated by glutaraldehyde.

[0019] The magnetic beads have at least one of an amino group (-NH2), a carboxyl group -COOH, a tosyl group, an epoxy group (Epoxy) and / or a hydroxyl group -OH, and specifically, the carboxyl group;

[0020] The mass ratio of the magnetic beads, streptavidin and N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is (25-35):1:1, and in a specific embodiment of the application, the mass ratio is 30:1:1.

[0021] The first reaction is performed at room temperature for 0.5-1.5 hours, and specifically, the first reaction is performed at room temperature for 1 hour.

[0022] The second reaction is performed at room temperature for 0.2-1 hour, and specifically, the second reaction is performed at room temperature for 0.5 hour.

[0023] In the application, the carboxyl magnetic beads are activated by EDC and NHS, and then reacted with streptavidin to obtain streptavidin magnetic beads.

[0024] The streptavidin magnetic beads are combined with streptavidin through the structure of NHS-PEG (n) -NHS arm, and the combination is repeated to obtain the multi-layer streptavidin magnetic microparticles.

[0025] The NHS-PEG (n) -NHS arm is obtained by the first reaction of the streptavidin magnetic beads with N-hydroxysuccinimide-polyethylene glycol (n) -N-hydroxysuccinimide.

[0026] In a specific embodiment of the present application, the preparation method comprises the following steps:

[0027] Step 1: After the carboxyl magnetic beads are washed and activated (EDC and NHS activation) for 1h, the supernatant is removed by magnetic attraction to obtain activated magnetic beads, and the activated magnetic beads are reacted with streptavidin in MES buffer to obtain streptavidin magnetic beads;

[0028] Step 2: The streptavidin magnetic beads are washed for the third time, the supernatant is removed by magnetic attraction, and the first reaction is performed with N-hydroxysuccinimide-polyethylene glycol (n) -N-hydroxysuccinimide to obtain a first reaction product;

[0029] Step 3: The first reaction product is reacted with streptavidin for the second time to obtain double-layer co-affinity coated magnetic beads;

[0030] Step 4: Steps 2 and 3 are repeated multiple times, and a layer of streptavidin coating is obtained each time to obtain a coating reaction product;

[0031] Step 5: The coating reaction product is blocked to obtain the multi-layer streptavidin magnetic microparticles;

[0032] The reagent for the first washing is PBS buffer, the concentration of PBS in the PBS buffer is 0.01M~0.5M, and the concentration is specifically 0.01M; the number of first washing is 2~5 times, and the number is specifically 3 times;

[0033] The reagent for the second washing is MES buffer, the concentration of MES in the MES buffer is 0.01M~0.5M, and the concentration is specifically 0.05M; the number of second washing is 2~5 times, and the number is specifically 3 times;

[0034] The reagent for the third washing is MES buffer, the concentration of MES in the MES buffer is 0.01M~0.5M, and the concentration is specifically 0.05M; the number of second washing is 2~5 times, and the number is specifically 3 times;

[0035] The blocking reagent is a blocking solution, which is a PBS buffer containing 0.5 wt% to 2 wt% BSA; in a specific embodiment of the present invention, the blocking solution is a 0.01 M PBS buffer containing 1 wt% BSA.

[0036] This invention provides a reagent containing multilayer streptavidin magnetic microparticles, characterized in that it comprises excipients and at least one of the following: I) to II)

[0037] I) The multilayer streptavidin magnetic microparticles described in this invention;

[0038] II) Multilayer streptavidin magnetic microparticles prepared by the preparation method described in this invention.

[0039] Furthermore, the excipients are used to maintain the stability of the multilayer streptavidin magnetic microparticles, facilitate their preservation, or assist them in their function. The excipients include, but are not limited to, stabilizers, preservatives, antioxidants, and / or buffer solutions.

[0040] This invention provides an immunoassay kit comprising an immunoassay reagent and at least one of the following: i) to iii)

[0041] i) The multilayer streptavidin magnetic microparticles described in this invention;

[0042] ii) Multilayer streptavidin magnetic microparticles prepared by the method described in this invention;

[0043] iii) The reagents described in this invention.

[0044] Furthermore, the immunoassay reagent includes antigens or antibodies, chromogenic reagents, and / or cleaning agents, etc.

[0045] This invention provides the application of at least one of the following A) to D) in immune detection:

[0046] A) The multilayer streptavidin magnetic microparticles described in this invention;

[0047] B) Multilayer streptavidin magnetic microparticles prepared by the method described in this invention;

[0048] C) The reagents described in this invention;

[0049] D) The reagent kit described in this invention.

[0050] This invention provides a method for immunoassay, comprising detecting a sample using at least one of the methods shown in a) to c) :

[0051] a) The multilayer streptavidin magnetic microparticles described in this invention;

[0052] b) Multilayer streptavidin magnetic microparticles prepared by the method described in this invention;

[0053] c) The reagents described in this invention;

[0054] d) The reagent kit described in this invention.

[0055] This invention employs a self-assembly method, using N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide [NHS-PEG(n)-NHS] as a linker arm to connect streptavidin to streptavidin magnetic beads. Each end of the [NHS-PEG(n)-NHS] linker arm contains an NHS molecule; one NHS molecule binds to the amino group of streptavidin on the surface of the initial magnetic microparticle, and the other NHS molecule binds to the amino group of the outer streptavidin layer. EG(n)-NHS, as the connecting arm between streptavidin molecular layers, plays an important role in supporting the streptavidin molecular layers. Meanwhile, the connecting arm of this invention was obtained through multiple experimental screenings. Experimental results show that multilayer streptavidin magnetic beads prepared using other connecting arms have poor dispersibility, and the number n of PEG(n) in the connecting arm [NHS-PEG(n)-NHS] also affects the performance of the prepared multilayer streptavidin magnetic microparticles. Experimental results show that the effect is optimal when the number n of PEG(n) is 12. Furthermore, compared with other existing streptavidin magnetic beads, the magnetic beads of this invention significantly improve the sensitivity for detecting low-value samples.

[0056] This invention employs a self-assembly method, using NHS-PEG(n)-NHS as the connecting arms between streptavidin layers. As the number of streptavidin molecular layers increases, the biotin binding capacity of the magnetic microparticles is significantly enhanced. The PEG connecting arms between streptavidin molecular layers increase the spatial structure, effectively reducing steric hindrance between streptavidin molecules, thereby significantly reducing the difficulty of capturing target macromolecules. Simultaneously, the magnetic microparticles coated using this process exhibit significant advantages in dispersibility. The streptavidin magnetic microparticles coated using this method can be applied in magnetic microparticle chemiluminescence detection platforms for use in immunoassay kits. Attached Figure Description

[0057] Figure 1 Schematic diagram of N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide;

[0058] Figure 2 Schematic diagram of double-layered streptavidin magnetic microparticles;

[0059] Figure 3 Schematic diagram of multilayer streptavidin magnetic microparticles;

[0060] Figure 4Schematic diagram of streptavidin magnetic microparticles;

[0061] Figure 5 The dispersibility of magnetic microparticles was measured, where A is Merck carboxylated magnetic beads, B is the bilayer streptavidin magnetic microparticles prepared in Example 1, and C is the multilayer streptavidin magnetic microparticles prepared in Example 1.

[0062] Figure 6 The dispersibility of multilayer streptavidin magnetic microparticles prepared with different amounts of polyethylene glycol (n varies) is shown in Figure A, where n is 10; B is 12; C is 30; and D is 50.

[0063] Figure 7 Schematic diagram of di(N-succinimide) sebacate;

[0064] Figure 8 The dispersibility of di(N-succinimide) sebacate-coated magnetic beads is shown; Detailed Implementation

[0065] This invention provides multilayer streptavidin-coated magnetic microparticles, their preparation method, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0066] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0067] Example 1: Preparation of streptavidin-coated magnetic microparticles

[0068] I. Preparation of double-layer streptavidin (coated) magnetic microparticles

[0069] 1. Take Merck carboxyl magnetic beads (100 mg / ml, 30 μl, surface modified only with carboxyl groups), wash 3 times (300 µl of 0.01 M PBS each time) to remove the supernatant, add EDC solution (20 mg / ml, 50 µl) and NHS solution (20 mg / ml, 50 µl), and shake at room temperature for 1 h.

[0070] 2. After removing the supernatant by magnetic attraction, wash three times with MES solution (0.05M, 300μl);

[0071] 3. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0072] 4. After magnetic attraction, remove the supernatant and wash 3 times (0.05M MES 300µl / time) to remove the supernatant (this step yields the initial streptavidin magnetic microparticles). Add N-hydroxysuccinimide-polyethylene glycol (12)-N-hydroxysuccinimide solution (1mg / ml, 100μl, where the structural schematic diagram of N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is shown in the figure). Figure 1 (As shown) React at room temperature with shaking for 0.5 hours;

[0073] 5. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0074] 6. After magnetic attraction, remove the supernatant, wash 3 times (300µl / time with 0.05M MES), remove the supernatant again, block 3 times with blocking buffer (1% BSA, 300µl / time with 0.01M PBS), and then adjust the volume to 3ml to obtain bilayer streptavidin magnetic microparticles (e.g. Figure 2 ).

[0075] II. Preparation of multilayer streptavidin (coated) magnetic microparticles

[0076] 1. Take the bilayer streptavidin magnetic microparticles (100 mg / ml, 30 μl) obtained in "I. Preparation of bilayer streptavidin magnetic microparticles", wash 3 times (300 µl of 0.01 M PBS each time) to remove the supernatant;

[0077] 2. Add N-hydroxysuccinimide-polyethylene glycol (12)-N-hydroxysuccinimide solution (1 mg / ml, 100 μl) and shake at room temperature for 0.5 hours;

[0078] 3. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0079] 4. After magnetic attraction, remove the supernatant, wash 3 times (300µl / time with 0.05M MES), remove the supernatant again, block 3 times with blocking buffer (1% BSA, 300µl / time with 0.01M PBS), and then adjust the volume to 3ml to obtain multilayer streptavidin magnetic microparticles (see schematic diagram of multilayer streptavidin magnetic microparticles). Figure 3 As shown, there are 3 layers.

[0080] III. Performance Determination of Double-Layer Streptavidin-Coated Magnetic Microparticles and Multi-Layer Streptavidin-Coated Magnetic Microparticles

[0081] 1. Free biotin binding amount

[0082] With initial streptavidin magnetic particles (structure as follows) Figure 4 As a control, 100 μl (1 mg / ml) of each of the three types of magnetic beads (the bilayer streptavidin magnetic microparticles and multilayer streptavidin magnetic microparticles prepared in this example, and the initial streptavidin magnetic microparticles) were added to Biotin-FITC solution (3 nmol / ml, 1.9 ml) and reacted at room temperature with shaking for 0.5 hours. After centrifugation, the supernatant was collected and the fluorescence value was measured. The free biotin binding amount of the three types of magnetic microparticles was calculated, and the results are as follows:

[0083] The results are shown in Table 1. The results indicate that the amount of free biotin bound by the bilayer streptavidin magnetic microparticles was 1.73 times that of the initial magnetic microparticles, and the amount of free biotin bound by the multilayer streptavidin magnetic microparticles was 1.27 times that of the bilayer. The amount of free biotin bound by the multilayer streptavidin magnetic microparticles was 2.22 times that of the initial magnetic microparticles.

[0084] surface Determination of the amount of free biotin bound to magnetic microparticles

[0085]

[0086] 2. Particle size

[0087] Using the initial streptavidin magnetic microparticles as a control, the hydrated particle size of the three magnetic microparticles was determined using a laser particle size analyzer (manufacturer: Malven, model: MasterSize 3000). The results are shown in the table below:

[0088] surface Magnetic microparticle size determination

[0089]

[0090] The results are shown in Table 2. The results indicate that the initial magnetic microparticle size was 1.2 μm, the size of the bilayer streptavidin magnetic microparticles was 1.5 μm, and the size of the multilayer streptavidin magnetic microparticles was 1.8 μm. The particle size increased with the increase of the number of streptavidin layers.

[0091] 3. Dispersion

[0092] Merck carboxylated magnetic beads were selected as a control. The dispersion of the magnetic particles was observed using a microscope (manufacturer: OLYMPUS, model: XN43F). The results are shown in [Figure number missing]. Figure 5 The figure shows that the three magnetic particles are well dispersed.

[0093] 4. Sensitivity Test

[0094] The milk allergen detection kit (magnetic microparticle chemiluminescence method, abbreviated as f2 sIgE) uses a double-antibody sandwich method to detect antigens. Streptavidin magnetic microparticles capture biotin-labeled antibodies, and the biotin binding amount of the magnetic microparticles is directly proportional to the signal value. Simultaneously, the spatial arrangement of streptavidin on the surface of the magnetic microparticles is crucial to the influence of steric hindrance. Sera-mag SpeedBeads streptavidin magnetic beads (biotin binding amount 3500 to 4500 pmol / mg) were selected as the control magnetic microparticles. Samples in the low-value region of the f2 sIgE test were detected using control magnetic microparticles, bilayer streptavidin magnetic microparticles, and multilayer streptavidin magnetic microparticles, respectively. The results are as follows:

[0095] The results are shown in Table 3. The results indicate that the reactivity of the bilayer streptavidin magnetic microparticles, multilayer streptavidin magnetic microparticles, and control magnetic microparticles is basically the same, and the detection signal value is higher in the low value region, indicating higher sensitivity.

[0096] Table 3. Sensitivity tests of streptavidin magnetic beads prepared from double-layer and multi-layer streptavidin magnetic microparticles.

[0097]

[0098] Example 2: Optimization effect of magnetic beads with different amounts (degrees of polymerization) of polyethylene glycol in the connecting arm N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide.

[0099] The amounts of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide were selected as 10, 12, 30, and 50, respectively, and multilayer streptavidin magnetic beads were coated on them. The coating steps are as follows:

[0100] 1. Take Merck carboxylated magnetic beads (100 mg / ml, 30 μl), wash 3 times (300 µl of 0.01 M PBS each time) to remove the supernatant, add EDC solution (20 mg / ml, 50 µl) and NHS solution (20 mg / ml, 50 µl), and shake at room temperature for 1 h;

[0101] 2. After removing the supernatant by magnetic attraction, wash three times with MES solution (0.05M, 300μl);

[0102] 3. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0103] 4. Remove the supernatant by magnetic attraction, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, add N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide solution (1mg / ml, 100μl), and shake at room temperature for 0.5 hours.

[0104] 5. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0105] 6. Remove the supernatant by magnetic attraction, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, add N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide solution (1mg / ml, 100μl), and shake at room temperature for 0.5 hours.

[0106] 7. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0107] 8. After magnetic attraction, remove the supernatant, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, block 3 times with blocking buffer (1% BSA, 300µl / time with 0.01M PBS), and then bring the volume to 3ml to obtain multilayer streptavidin-coated magnetic microparticles.

[0108] The dispersibility of coated magnetic beads, such as Figure 6 Particle size is shown in Table 4, and free biotin binding amount is shown in Table 5;

[0109] Table 4. Particle size of magnetic beads with different amounts of polyethylene glycol

[0110]

[0111] Table 5. Free biotin binding amount of magnetic beads with different amounts of polyethylene glycol coating

[0112]

[0113] When the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is 10 or 12, the prepared magnetic beads have good dispersibility and high biotin loading. When the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is 30, the prepared magnetic beads agglomerate and the biotin loading is significantly reduced, showing no significant advantage compared with bilayer streptavidin magnetic beads. In particular, when the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is 50, the magnetic beads agglomerate severely, and the biotin loading is no longer significantly superior to the initial biotin loading of the magnetic microparticles. Based on the principle of cost reduction and efficiency improvement, the present invention preferably uses an amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide of 10 to 30, with 12 being the optimal amount.

[0114] Example 2: Preparation method of multilayer streptavidin (coated) magnetic microparticles

[0115] The specific steps of method 2 for preparing multilayer streptavidin-coated magnetic microparticles are as follows:

[0116] 1. Take Merck amino magnetic beads (100 mg / ml, 30 μl), wash 3 times (300 µl of 0.01 M PBS each time) to remove the supernatant, add glutaraldehyde solution (15 mg / ml, 100 µl) and shake at room temperature for 1 h;

[0117] 2. After removing the supernatant by magnetic attraction, wash three times with MES solution (0.05M, 300μl);

[0118] 3. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0119] 4. Remove the supernatant by magnetic attraction, wash 3 times (300µl / time with 0.05M MES) to remove the supernatant, add N-hydroxysuccinimide-polyethylene glycol (12)-N-hydroxysuccinimide solution (1mg / ml, 100μl) and shake at room temperature for 0.5 hours;

[0120] 5. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0121] 6. Remove the supernatant by magnetic attraction, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, add N-hydroxysuccinimide-polyethylene glycol (12)-N-hydroxysuccinimide solution (1mg / ml, 100μl) and shake at room temperature for 0.5 hours;

[0122] 7. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0123] 8. After magnetic attraction, remove the supernatant, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, block 3 times with blocking buffer (1% BSA, 300µl / time with 0.01M PBS), and then bring the volume to 3ml to obtain multilayer streptavidin-coated magnetic microparticles.

[0124] II. Sensitivity Testing of the Multilayer Streptomycin Magnetic Particles Prepared in Examples 1 and 2

[0125] The above milk allergen detection kit (magnetic microparticle chemiluminescence method, abbreviated as f2 sIgE) was used for detection, and the results are shown in Table 6:

[0126] The results in Table 6 show that the sensitivity of the multilayer streptavidin magnetic microparticles prepared in Example 1 and Example 2 is similar, but the multilayer streptavidin magnetic microparticles prepared in Example 1 are slightly better than those prepared in Example 2.

[0127] Table 6. Sensitivity tests of the multilayer streptavidin magnetic microparticles prepared in Examples 1 and 2

[0128]

[0129] Comparative Example 1: Comparison of the coating effects of di(N-succinimide) sebacate and N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide.

[0130] di(N-succinimide) sebacate (structural formula as follows) Figure 7 The steps for coating magnetic beads are as follows:

[0131] 1. Take Merck carboxylated magnetic beads (100 mg / ml, 30 μl), wash 3 times (300 µl of 0.01 M PBS each time) to remove the supernatant, add EDC solution (20 mg / ml, 50 µl) and NHS solution (20 mg / ml, 50 µl), and shake at room temperature for 1 h;

[0132] 2. After removing the supernatant by magnetic attraction, wash three times with MES solution (0.05M, 300μl);

[0133] 3. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0134] 4. Remove the supernatant by magnetic attraction, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, add di(N-succinimide) sebacate solution (1mg / ml, 100μl), and shake at room temperature for 0.5 hours.

[0135] 5. After removing the supernatant by magnetic attraction, add streptavidin (5 mg / ml, 20 μl of 0.01 M PBS) and 80 μl of MES (0.05 M) buffer and shake at room temperature for 1 h.

[0136] 6. After magnetic attraction, remove the supernatant, wash 3 times (300µl / time with 0.05M MES), remove the supernatant, block 3 times with blocking buffer (1% BSA, 300µl / time with 0.01M PBS), and bring the volume to 3ml to obtain di(N-succinimide) sebacate (coated) magnetic beads.

[0137] The dispersibility of di(N-succinimide) sebacate-coated magnetic beads is as follows: Figure 8 Particle size is shown in Table 7, and free biotin binding amount is shown in Table 8;

[0138] Table 7. Particle size of di(N-succinimide) sebacate-coated magnetic beads

[0139]

[0140] Table 8. Free biotin binding capacity of di(N-succinimide) sebacate-coated magnetic beads

[0141]

[0142] Based on the analysis of dispersibility and particle size results, the magnetic beads coated with di(N-succinimide) sebacate showed significant agglomeration, leading to an increase in particle size and increased steric hindrance of streptavidin, resulting in a decrease in the amount of free biotin bound.

[0143] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Multilayer streptavidin magnetic microparticles, characterized in that, It was obtained by sequentially encapsulating streptavidin magnetic beads with N-hydroxysuccinimide-polyethylene glycol nN-hydroxysuccinimide and streptavidin; The number of times the package is delivered is m, where m is an integer from 1 to 10; The n is an integer from 10 to 30; The degree of aggregation is n.

2. The method for preparing multilayer streptavidin magnetic microparticles according to claim 1, characterized in that, Includes the following steps: Step 1: Streptavidin magnetic beads are reacted with N-hydroxysuccinimide-polyethylene glycol nN-hydroxysuccinimide to obtain the first reactant; Step 2: The first reactant undergoes a second reaction with streptavidin to complete a streptavidin coating; Step 3: Repeat steps 1 to 2 m times to obtain the multilayer streptavidin magnetic microparticles; m is an integer from 1 to 10.

3. The preparation method according to claim 2, characterized in that, The streptavidin magnetic beads are obtained by encapsulating magnetic beads with streptavidin after activation.

4. The preparation method according to claim 3, characterized in that, The activating reagent is EDC, NHS, and / or glutaraldehyde.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the magnetic beads, streptavidin, and N-hydroxysuccinimide-polyethylene glycol nN-hydroxysuccinimide is (25~35):1:

1.

6. A reagent containing multilayer streptavidin magnetic microparticles, characterized in that, Includes excipients and at least one of the following: (I) to (II) I) The multilayer streptavidin magnetic microparticles as described in claim 1; II) Multilayer streptavidin magnetic microparticles prepared by the preparation method according to any one of claims 2 to 5.

7. An immunoassay kit, characterized in that, Includes immunoassay reagents and at least one of the following: i) to iii) i) The multilayer streptavidin magnetic microparticles as described in claim 1; ii) Multilayer streptavidin magnetic microparticles prepared by the preparation method according to any one of claims 2 to 5; iii) The reagent as described in claim 6.

8. The application of at least one of the following (A) to (D) in immunological detection for purposes other than disease diagnosis and treatment: A) The multilayer streptavidin magnetic microparticles as described in claim 1; B) Multilayer streptavidin magnetic microparticles prepared by the preparation method according to any one of claims 2 to 5; C) The reagent according to claim 6; D) The kit according to claim 7.

9. A method for immune detection not for disease diagnosis and treatment purposes, characterized in that, This includes detecting the sample using at least one of the methods shown in a) to d): a) The multilayer streptavidin magnetic microparticles as described in claim 1; b) Multilayer streptavidin magnetic microparticles prepared by the preparation method according to any one of claims 2 to 5; c) The reagent according to claim 6; d) The kit according to claim 7.

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