Multilayer streptavidin-coated magnetic particle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to multi-layer streptavidin coated magnetic particles and a preparation method and application thereof. A self-assembly mode is adopted, NHS-PEG (n)-NHS is used as a connecting arm between streptavidin layers, when streptavidin molecular layers are increased, the biotin binding amount of the magnetic particles is remarkably increased, the space structure is enlarged through the PEG connecting arm between the streptavidin molecular layers, the steric hindrance between streptavidin molecules can be effectively reduced, and the biotin binding capacity of the magnetic particles is improved. Therefore, the difficulty of capturing target macromolecules is obviously reduced. Meanwhile, the magnetic particles coated by the process have obvious advantages in the aspect of dispersity. The streptavidin magnetic particles coated by the method can be applied to a magnetic particle chemiluminescence detection platform and used in an immunodetection kit.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to multi-layer streptavidin-coated magnetic particles, and a preparation method and application thereof. Background Art

[0002] The rapid development of biomedical research and diagnostic technology greatly relies on efficient and highly specific methods for separation and detection of biomolecules. Streptavidin, as a highly stable tetrameric protein, can form extremely strong non-covalent bonds with small molecules of biotin, which makes it an indispensable tool molecule in biosensing, molecular biology and clinical diagnosis. Although traditional streptavidin-coupled magnetic microparticles have been used to capture and separate a variety of biomolecules, they still face several key challenges: first, the limited binding sites caused by the monolayer streptavidin coverage limit its maximum binding capacity; second, the spatial structure of the monolayer streptavidin is prone to high steric hindrance, resulting in difficulty in binding of large molecules; third, the imperfection of magnetic microparticle surface modification technology affects its biocompatibility and specific binding of target molecules. How to successfully construct a stable and efficient multilayer streptavidin structure on the surface of magnetic microparticles while maintaining its magnetic responsiveness and good biocompatibility is the focus and difficulty of current research.

[0003] Therefore, developing a new type of streptavidin magnetic microparticle that can overcome the limitations of existing technologies and have high biotin loading capacity, low steric hindrance, high dispersibility, and low nonspecificity is of great significance for promoting the progress of biomedical research and clinical practice. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide multi-layer streptavidin-coated magnetic particles, and a preparation method and application thereof.

[0005] The present invention provides multilayer streptavidin magnetic microparticles, which are obtained by sequentially coating streptavidin magnetic beads with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide and streptavidin; the number of times of sequentially coating with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide and streptavidin is m, and m is an integer of 1 to 10. In a specific embodiment of the present invention, the number of coating layers is 3.

[0006] Furthermore, the n is an integer between 10 and 30; specifically, n is optimal when it is 12;

[0007] The present invention provides a method for preparing the multilayer streptavidin magnetic particles, which comprises the following steps:

[0008] Step 1, performing a first reaction between streptavidin magnetic beads and N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide to obtain a first reactant;

[0009] Step 2, the first reactant and streptavidin undergo a second reaction to complete a layer of streptavidin coating;

[0010] Step 3, repeat step 1 to 2 m times and then seal to obtain the multilayered streptavidin magnetic particles.

[0011] In the preparation method of the present invention,

[0012] The streptavidin magnetic beads are obtained by activating the magnetic beads and then encapsulating them with streptavidin.

[0013] The activated reagents are EDC, NHS and / or glutaraldehyde;

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

[0015] In the mixed solution 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; in the specific embodiment of the present invention, it is 30:1:1.

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

[0018] In a specific embodiment of the present invention, the performance of magnetic particles prepared by using a mixture of EDC and NHS as an activator is slightly better than that of magnetic particles activated by glutaraldehyde.

[0019] The active groups of the magnetic beads include at least one of amino (-NH2), carboxyl -COOH, toluenesulfonyl (Tosyl), epoxy (Epoxy) and / or hydroxyl -OH; specifically, carboxyl;

[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 present invention, it is 30:1:1;

[0021] The first reaction condition is shaking at room temperature for 0.5 h to 1.5 h, specifically shaking at room temperature for 1 h.

[0022] The second reaction is carried out under the condition of shaking at room temperature for 0.2 h to 1 h, specifically shaking at room temperature for 0.5 h.

[0023] In the present invention, 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 NHS-PEG (n) -NHS arm structure, and this process is repeated to obtain multilayer streptavidin magnetic particles.

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

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

[0027] Step 1: After the carboxyl magnetic beads are first washed and activated (EDC and NHS activated) for 1 hour, the supernatant is removed by magnetic suction to obtain activated magnetic beads, and the activated magnetic beads are second washed and 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 suction, and then a first reaction is carried out with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide to obtain a first reaction product;

[0029] Step 3, the first reaction product is subjected to a second reaction with streptavidin to obtain a double-layered avidin-coated magnetic bead;

[0030] Step 4, repeating steps 2 and 3 multiple times, obtaining a layer of streptavidin coating each time, and obtaining a coating reaction product;

[0031] Step 5, the encapsulated reactants are sealed to obtain the multilayered streptavidin magnetic particles;

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

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

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

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

[0036] The present invention provides a reagent containing multilayer streptavidin magnetic particles, characterized in that it comprises auxiliary materials and at least one of the following I) to II):

[0037] I), the multilayer streptavidin magnetic particles of the present invention;

[0038] II), multilayer streptavidin magnetic particles prepared by the preparation method of the present invention.

[0039] Furthermore, the auxiliary materials are used to maintain the stability of the multilayered streptavidin magnetic particles, facilitate their preservation or assist them in functioning, and the auxiliary materials include but are not limited to stabilizers, preservatives, antioxidants and / or buffers.

[0040] The present invention provides an immunoassay kit, which comprises an immunoassay reagent and at least one of the following i) to iii):

[0041] i), the multilayered streptavidin magnetic particles of the present invention;

[0042] ii), the preparation method of the present invention obtained multilayer streptavidin magnetic particles;

[0043] iii), the reagent described in the present invention.

[0044] Furthermore, the immunoassay reagent includes an antigen or an antibody, a colorimetric reagent and / or a cleaning agent, etc.

[0045] The present invention provides at least one of the following A) to D) for use in immunoassay:

[0046] A), the multilayered streptavidin magnetic particles of the present invention;

[0047] B), the preparation method of the present invention obtained multilayer streptavidin magnetic particles;

[0048] C), the reagent of the present invention;

[0049] D), the kit of the present invention.

[0050] The present invention provides an immunoassay method, which comprises detecting a sample using at least one of a) to c):

[0051] a), the multilayered streptavidin magnetic particles of the present invention;

[0052] b), the preparation method of the present invention obtained multilayer streptavidin magnetic particles;

[0053] c), the reagent of the present invention;

[0054] d), the kit of the present invention.

[0055] The present invention adopts a self-assembly method, uses N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide [NHS-PEG (n)-NHS] as a connecting arm, and connects streptavidin to the streptavidin magnetic beads through the connecting arm; there is an NHS molecule at each end of the [NHS-PEG (n)-NHS] connecting arm, one of the NHS molecules is combined with the amino group of streptavidin on the surface of the initial magnetic particle, and the other NHS molecule is combined with the amino group of streptavidin on the outer layer, and the NHS-PEG (n)-NHS connecting arm is connected to the streptavidin magnetic beads. EG (n) -NHS serves as a connecting arm between the streptavidin molecular layers, and plays an important role in supporting the streptavidin molecular layers; at the same time, the connecting arm of the present invention is obtained through multiple test screenings, and the test results show that the multilayered streptavidin magnetic beads prepared with other connecting arms have poor dispersibility, and the number n of PEG (n) in the connecting arm [NHS-PEG (n) -NHS] will also affect the performance of the prepared multilayered streptavidin magnetic particles. The test results show that the effect is best when the number n of PEG (n) is 12; in addition, compared with other existing streptavidin magnetic beads, the magnetic beads of the present invention have significantly improved sensitivity for low-value sample detection.

[0056] The present invention adopts a self-assembly method, and uses NHS-PEG (n) -NHS as a connecting arm between streptavidin layers. When the number of streptavidin molecular layers increases, the biotin binding amount of the magnetic microparticles is significantly increased. The PEG connecting arm between the streptavidin molecular layers increases the spatial structure, which can effectively reduce the steric hindrance between streptavidin molecules, thereby significantly reducing the difficulty of capturing target macromolecules. At the same time, the magnetic microparticles coated by this process have obvious advantages in dispersibility. The streptavidin magnetic microparticles coated by this method can be applied to a magnetic microparticle chemiluminescence detection platform and used in an immunoassay kit. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0058] Figure 2 Schematic diagram of double-layer streptavidin magnetic particles;

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

[0060] Figure 4Schematic diagram of streptavidin magnetic particles is shown;

[0061] Figure 5 Magnetic particle dispersibility determination is shown, wherein A is Merck carboxyl magnetic beads, B is double-layered streptavidin magnetic particles prepared in Example 1, and C is multi-layered streptavidin magnetic particles prepared in Example 1;

[0062] Figure 6 The figure shows the dispersibility of multilayered streptavidin magnetic particles prepared with different amounts of polyethylene glycol (n is different), where A is n=10; B is n=12; C is n=30; and D is n=50.

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

[0064] Figure 8 The dispersion of magnetic beads coated with di(N-succinimidyl) sebacate is shown; DETAILED DESCRIPTION

[0065] The present invention provides multilayer streptavidin-coated magnetic microparticles, and the preparation method and application thereof can be achieved by those skilled in the art by referring to the contents of this article and appropriately improving the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0066] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0067] Example 1 Preparation of Streptavidin-coated Magnetic Microparticles

[0068] 1. Preparation of double-layer streptavidin (coated) magnetic particles

[0069] 1. Take Merck carboxyl magnetic beads (100 mg / ml, 30 μl, only carboxyl modified on the surface), wash 3 times (0.01M PBS 300 μl / time), 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 hour;

[0070] 2. Remove the supernatant after magnetic attraction using a magnet, and add MES solution (0.05M, 300μl) to wash three times;

[0071] 3. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0072] 4. Remove the supernatant after magnetic attraction using a magnet, wash 3 times (0.05M MES 300µl / time) to remove the supernatant (after this step, the initial streptavidin magnetic particles are obtained), add N-hydroxysuccinimide-polyethylene glycol (12)-N-hydroxysuccinimide solution (1mg / ml, 100μl, where the structural diagram of N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is as shown in Figure 1 The reaction was performed by shaking at room temperature for 0.5 hours.

[0073] 5. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0074] 6. Remove the supernatant after magnetic attraction with a magnet, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, block 3 times with blocking solution (1% BSA, 0.01M PBS 300µl / time) and adjust the volume to 3ml to obtain double-layer streptavidin magnetic particles (such as Figure 2 ).

[0075] 2. Preparation of multilayer streptavidin (coated) magnetic particles

[0076] 1. Take the double-layer streptavidin magnetic particles (100 mg / ml, 30 μl) obtained in "I. Preparation of double-layer streptavidin magnetic particles", wash them three times (0.01 M PBS 300 μl / time) and 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. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0079] 4. Remove the supernatant after magnetic attraction with a magnet, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, use blocking solution (1% BSA, 0.01M PBS 300µl / time) to block 3 times and then adjust the volume to 3ml to obtain multilayer streptavidin magnetic particles (schematic diagram of multilayer streptavidin magnetic particles is shown in Figure 3 As shown, there are 3 layers specifically).

[0080] 3. 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 Figure 4 ) was used as a control, 100 μl (1 mg / ml) of each of the three magnetic beads (double-layered streptavidin magnetic particles and multi-layered streptavidin magnetic particles prepared in this example, as well as initial streptavidin magnetic particles) was added to Biotin-FITC solution (3 nmol / ml, 1.9 ml) and reacted at room temperature for 0.5 hours. After centrifugation, the supernatant was taken to measure the fluorescence value, and the free biotin binding amount of the three magnetic particles was calculated. The results are as follows:

[0083] The results are shown in Table 1. The results show that the free biotin binding capacity of double-layer streptavidin magnetic particles is 1.73 times that of the initial magnetic particles, and the free biotin binding capacity of multi-layer streptavidin magnetic particles is 1.27 times that of the double-layer. The free biotin binding capacity of multi-layer streptavidin magnetic particles is 2.22 times that of the initial magnetic particles.

[0084] surface . Determination of free biotin binding amount of magnetic microparticles

[0085]

[0086] 2. Particle size

[0087] Using the initial streptavidin magnetic particles as a control, the hydrated particle sizes of the three magnetic particles were measured using a laser particle size analyzer (manufacturer: Malven, model: MasterSize 3000). The results are as follows:

[0088] surface .Magnetic particle size determination

[0089]

[0090] The results are shown in Table 2. The results show that the initial magnetic particle size is 1.2 μm, the double-layer streptavidin magnetic particle size is 1.5 μm, and the multi-layer streptavidin magnetic particle size is 1.8 μm. The particle size increases with the increase in the number of streptavidin layers.

[0091] 3. Dispersibility

[0092] Merck carboxyl magnetic beads were selected as the control, and the dispersion of magnetic particles was observed using a microscope (manufacturer: OLYMPUS, model: XN43F). The results are shown in Figure 5 , the figure shows that the three types of magnetic particles have good dispersion.

[0093] 4. Sensitivity test

[0094] The milk allergen detection kit (magnetic particle chemiluminescence method, referred to as: f2 sIgE) uses a double antibody sandwich method to measure antigens. Streptavidin magnetic particles capture biotin-labeled antibodies. The biotin binding amount of the magnetic particles is proportional to the signal value. At the same time, the spatial arrangement of streptavidin on the surface of the magnetic particles is crucial to the effect of steric hindrance. The control magnetic particles select Sera-mag SpeedBeads streptavidin magnetic beads (biotin binding amount 3500 to 4500 pmol / mg). The control magnetic particles, double-layer streptavidin magnetic particles, and multi-layer streptavidin magnetic particles were used to detect samples in the low-value area of ​​the f2 sIgE project, and the results are as follows:

[0095] The results are shown in Table 3, which show that the reactivity of the double-layered streptavidin magnetic particles, the multi-layered streptavidin magnetic particles and the control magnetic particles is basically the same, and the detection signal value is higher in the low value area, indicating a higher sensitivity.

[0096] Table 3. Preparation of double-layered streptavidin magnetic particles and multi-layered streptavidin magnetic particles and sensitivity test of streptavidin magnetic beads

[0097]

[0098] Example 2 Optimization effect of magnetic beads with different amounts of polyethylene glycol (degree of polymerization) in the linker N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide

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

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

[0101] 2. Remove the supernatant after magnetic attraction using a magnet, and add MES solution (0.05M, 300μl) to wash three times;

[0102] 3. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0103] 4. Remove the supernatant after magnetic attraction with a magnet, wash 3 times (0.05M MES 300µl / time), 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. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0105] 6. Remove the supernatant after magnetic attraction using a magnet, wash 3 times (0.05M MES 300µl / time), 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. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0107] 8. Use a magnet to remove the supernatant, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, use blocking solution (1% BSA, 0.01M PBS 300µl / time) to block 3 times and then adjust the volume to 3ml to obtain multilayer streptavidin-coated magnetic particles.

[0108] The dispersion of coated magnetic beads is as follows Figure 6 , particle size is shown in Table 4 , free biotin binding amount is shown in Table 5 ;

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

[0110]

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

[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 capacity. When the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is 30, the prepared magnetic beads agglutinate and the biotin loading capacity is significantly reduced, which has no obvious advantage over double-layer streptavidin magnetic beads. In particular, when the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide is 50, the magnetic beads agglutinate severely, and the biotin loading capacity has no obvious advantage over the biotin loading capacity of the initial magnetic microparticles. Based on the principle of reducing costs and increasing efficiency, the present invention preferably sets the amount of polyethylene glycol in N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide to be 10 to 30, with 12 being the best.

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

[0115] 1. The specific steps of method 2 for preparing multilayer streptavidin-coated magnetic particles are as follows:

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

[0117] 2. Remove the supernatant after magnetic attraction using a magnet, and add MES solution (0.05M, 300μl) to wash three times;

[0118] 3. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0119] 4. Remove the supernatant after magnetic attraction using a magnet, wash 3 times (0.05M MES 300µl / time), 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. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0121] 6. Remove the supernatant after magnetic attraction using a magnet, wash 3 times (0.05M MES 300µl / time), 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. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0123] 8. Use a magnet to remove the supernatant, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, use blocking solution (1% BSA, 0.01M PBS 300µl / time) to block 3 times and then adjust the volume to 3ml to obtain multilayer streptavidin-coated magnetic particles.

[0124] II. Sensitivity test of multilayered streptavidin magnetic particles prepared in Example 1 and Example 2

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

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

[0127] Table 6. Sensitivity test of multilayer streptavidin magnetic particles prepared in Example 1 and Example 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-succinimidyl) sebacate (structural formula: Figure 7 ) The steps for coating magnetic beads are as follows:

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

[0132] 2. Remove the supernatant after magnetic attraction using a magnet, and add MES solution (0.05M, 300μl) to wash three times;

[0133] 3. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

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

[0135] 5. Remove the supernatant after magnetic attraction using a magnet, add streptavidin (5 mg / ml, 0.01 M PBS 20 μl) and 80 μl MES (0.05 M) buffer and shake at room temperature for 1 h;

[0136] 6. Remove the supernatant after magnetic attraction with a magnet, wash 3 times (0.05M MES 300µl / time) to remove the supernatant, use blocking solution (1% BSA, 0.01M PBS 300µl / time) to block 3 times and then adjust the volume to 3ml to obtain di(N-succinimidyl) sebacate (coated) magnetic beads;

[0137] Dispersibility of di(N-succinimidyl) sebacate coated magnetic beads Figure 8 , particle size as shown in Table 7, free biotin binding amount as shown in Table 8;

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

[0139]

[0140] Table 8. Free biotin binding amount of magnetic beads coated with di(N-succinimidyl) sebacate

[0141]

[0142] Combining the analysis of the dispersion and particle size results, the magnetic beads coated with di(N-succinimidyl) sebacate were severely agglomerated, resulting in an increase in particle size and a greater steric hindrance of streptavidin, which led to a decrease in the amount of free biotin bound.

[0143] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Multilayer streptavidin magnetic particles, characterized in that The streptavidin magnetic beads are coated with N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide and streptavidin in sequence; The number of packages is m, where m is an integer from 1 to 10.

2. The multilayered streptavidin magnetic particle according to claim 1, characterized in that The n is an integer of 10 to 30.

3. The method for preparing multilayered streptavidin magnetic particles according to claim 1 or 2, characterized in that: The steps include: Step 1, performing a first reaction between streptavidin magnetic beads and N-hydroxysuccinimide-polyethylene glycol (n)-N-hydroxysuccinimide to obtain a first reactant; Step 2, the first reactant and streptavidin undergo a second reaction to complete a layer of streptavidin coating; Step 3, after repeating step 1 to 2 m times, the multilayered streptavidin magnetic particles are obtained; The m is an integer from 1 to 10.

4. The preparation method according to claim 3, characterized in that: The streptavidin magnetic beads are obtained by activating the magnetic beads and then encapsulating them with streptavidin.

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

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

1.

7. A reagent containing multilayer streptavidin magnetic particles, characterized in that Including auxiliary materials and at least one of the following I) to II): 1), the multilayered streptavidin magnetic particles according to claim 1 or 2; II), multilayer streptavidin magnetic particles prepared by the preparation method according to any one of claims 3 to 6.

8. An immunoassay kit, characterized in that: It includes an immunoassay reagent and at least one of the following i) to iii): i), the multilayered streptavidin magnetic particles according to claim 1 or 2; ii), the preparation method of any one of claims 3 to 6 obtained by multilayer streptavidin magnetic particles; iii) The reagent according to claim 7.

9. Application of at least one of the following A) to D) in immunoassay: A), the multilayered streptavidin magnetic particles according to claim 1 or 2; B), the preparation method of any one of claims 3 to 6 obtained by multilayer streptavidin magnetic particles; C) the reagent according to claim 7; D) The kit according to claim 8.

10. An immunoassay method, characterized in that: The method comprises detecting the sample using at least one of a) to d): a), the multilayered streptavidin magnetic particles according to claim 1 or 2; b), the preparation method of any one of claims 3 to 6 obtained by multilayer streptavidin magnetic particles; c) The reagent according to claim 7; d) The kit according to claim 8.

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