A "sandwich type" cross-linked polystyrene analysis magnetic bead based on swelling and layer-by-layer assembly, and a preparation method and application thereof

Abstract

The application discloses a kind of " sandwich type " crosslinked polystyrene analysis magnetic beads based on swelling and layer-by-layer assembly and its preparation method and application, the preparation method of magnetic bead is: with carboxylated crosslinked polystyrene microspheres as template, Fe3O4 is loaded to the microsphere template of swelling and forms microsphere assembly;Then in containing PEI poor solvent, assemble oleic acid modified hydrophobic Fe3O4;After that, microsphere assembly is phase transfer from organic phase to water phase, then grow silica shell layer, finally sequentially modified and carboxylated by amino, obtain carboxylated " sandwich type " crosslinked polystyrene analysis magnetic bead.The application uses carboxylated crosslinked polystyrene microspheres as template, loads Fe3O4 particle by swelling method and layer-by-layer self-assembly method, realizes biocompatibility by microsphere outer package silicon, provides a kind of analysis magnetic bead with uniform size, fast magnetic separation speed, slow settling time, good biocompatibility, good detection performance, applied to the detection combined with ELISA has good technical effect.

Description

Technical Field

[0001] This invention relates to a "sandwich-type" cross-linked polystyrene analytical magnetic bead based on swelling and layer-by-layer assembly, its preparation method, and its application. Background Technology

[0002] PCT (procalcitonin) is a protein whose plasma levels rise in severe bacterial, fungal, and parasitic infections, as well as in sepsis and multiple organ failure. PCT does not rise in autoimmune diseases, allergies, and viral infections, thus it can help differentiate between bacterial and viral infections. PCT may also rise in some non-infectious conditions, such as after surgery, severe trauma, burns, severe pancreatitis, and persistent cardiogenic shock, requiring differentiation from bacterial infections. PCT reflects the activity of the systemic inflammatory response. Factors affecting PCT levels include the size and type of the infected organ, the type of bacteria, the degree of inflammation, and the state of the immune response.

[0003] Pathogen detection remains the gold standard for diagnosing bacterial infections, but this method is time-consuming. Timely PCT testing in patients clinically suspected of having a bacterial infection helps in the early diagnosis of the disease, and PCT can guide the clinical use of antibiotics.

[0004] Accurately distinguishing PCT levels within the low concentration range is crucial for guiding rational antibiotic treatment. The "Expert Consensus on the Emergency Clinical Application of Procalcitonin PCT" and the "National Antibiotic Guidelines and Procalcitonin Antibiotic Prescription Guidelines for Lower Respiratory Tract Infections" state that: when serum PCT levels are below 0.1 ng / mL, antibiotic use is strongly discouraged; when serum PCT levels are between 0.1 and 0.25 ng / mL, antibiotic use is not recommended; when serum PCT levels are between 0.25 and 0.5 ng / mL, antibiotic use is suggested; and when serum PCT levels are above 0.5 ng / mL, antibiotic use is strongly recommended.

[0005] Immunological detection methods for PCT include immunofluorescence, immunochemiluminescence, and enzyme-linked immunosorbent assay (ELISA). Immunofluorescence, as a quantitative immunochemical detection method, offers high specificity for both antigens and antibodies. A monoclonal antibody pre-coated with gold-labeled procalcitonin (PCT) binds to the PCT in the sample. The detection area is then scanned using relevant instruments to further identify the luminescent signal. By comparing the luminescent signal intensity with PCT, a standard PCT concentration is determined, and a curve is prepared to obtain the PCT concentration value of the sample. This method is generally simple, highly accurate, and repeatable, and is widely used in laboratory procedures. However, this detection method has limitations, primarily in the possibility of false negatives or false positives. Immunochemiluminescence uses an optically labeled antibody, with another antibody fixed to the inner wall of the test tube. This diagnostic method has certain specificity and high sensitivity, is simple to operate, and has a low detection threshold. However, the luminescent reagents typically have poor thermal stability, making long-term storage inconvenient. Enzyme-linked immunosorbent assay (ELISA) involves binding the test sample (containing antibodies or antigens) and enzyme-labeled antigens or antibodies to the antigens or antibodies on the surface of a solid-phase carrier in different steps. After the substrate for the enzyme reaction is added, the substrate is catalyzed by the enzyme into a colored product. The amount of product is directly related to the amount of the analyte in the sample, so qualitative or quantitative analysis can be performed based on the intensity of the color reaction. Because enzymes have high catalytic efficiency, the reaction effect can be greatly amplified, thus enabling the assay method to achieve very high sensitivity.

[0006] Immunomagnetic beads have been widely used in the field of immunoassay due to their ease of separation. Compared with conventional detection methods, the use of immunomagnetic beads can achieve magnetic enrichment of target substances to improve signal sensitivity. Capture antibodies can be coupled to magnetic beads, simplifying the steps required in traditional immunoassays where antibodies need to be coupled to ELISA plates or test tubes. At the same time, it can also achieve specific binding of antigen-antibody and rapid separation of complexes in complex solutions, reducing test time and simplifying separation steps.

[0007] Currently, immunomagnetic beads are generally classified into three types: diffuse, core-shell, and sandwich. In diffuse immunomagnetic beads, magnetic particles are distributed within the bead. Their preparation method typically involves creating porous microspheres from polymer microspheres and then growing the magnetic material in situ. Alternatively, the magnetic material can be embedded during polymer synthesis via in-situ polymerization. Core-shell immunomagnetic beads are synthesized by first synthesizing single magnetic particles and then coating them with a hydrophilic polymer coating. Sandwich immunomagnetic beads have a polymer microsphere core, a magnetic particle-loaded middle layer, and an outermost hydrophilic polymer coating.

[0008] Well-known international magnetic bead brands include Thermo Scientific (USA) and JSR (Japan). Thermo Scientific's sub-brand related to immunomagnetic beads includes Thermo Scientific. TM Invitrogen TM Among them, Invitrogen's Dynabeads is its flagship immunomagnetic bead product, My One. TM Magnetic beads are widely used in scientific research and practical immunoassay detection. They are primarily made by using porous microspheres as templates to grow magnetic particles in situ within the pores, forming diffuse immunomagnetic beads with uniformly loaded magnetic microparticles within the microspheres. However, some drawbacks exist, such as the inability to control the loading of magnetic microparticles, and the complexity of the porous microsphere preparation process and the difficulty of in-situ growth, leading to higher costs and selling prices.

[0009] Currently, many companies in China are developing and producing immunomagnetic beads, mainly divided into silicon-based magnetic beads and polymer magnetic beads. However, due to the large batch-to-batch variability, non-uniform microsphere size, and poor monodispersity of silicon-based magnetic beads, they are mainly used for nucleic acid extraction. Leading domestic immunomagnetic bead companies include Boyue, Suzhou Nanomicro, and Beaver. Boyue magnetic beads have a similar diffuse structure to Thermo Dynabeads, with superparamagnetic γ-Fe2O3 nanoparticles as the magnetic core material and an iron oxide content of 15%-20%. Suzhou Nanomicro's magnetic polymer microspheres have a sandwich structure, with a porous polymer microsphere core inside and different polymer coatings on the outside to meet different application requirements. Magnetic materials fill the pores between the two. The diameter of this series of microspheres is between 1.0-5.0 μm. However, the preparation of porous polymer microspheres requires pore-forming operations, increasing production costs and making it impossible to guarantee uniform pore formation. BeaverBeaver... TM Mag COOH series magnetic beads use Fe3O4 as the magnetic core and polymer as the outer shell. However, this requires a high degree of dispersion of the synthesized single Fe3O4 particles. Otherwise, the Fe3O4 magnetic cores are prone to agglomeration, resulting in uneven microsphere size.

[0010] Therefore, addressing the shortcomings of the aforementioned immunomagnetic bead technology, this invention introduces a method for swelling and layer-by-layer assembly of Fe3O4-loaded microspheres. Utilizing the swelling and shrinkage properties of cross-linked polystyrene microspheres in good solvents and poor solvents, Fe3O4 is swollen within the microspheres, imparting magnetism to them. Assembly is then driven by the metal-ligand affinity between PEI and Fe3O4, enhancing the magnetic separation performance of the microspheres. Subsequently, the assembly is surface-modified to ensure stability in aqueous media, and further used for enzyme-linked immunosorbent assay (ELISA) to detect PCT. The prepared cross-linked polystyrene analytical magnetic beads exhibit uniform size, rapid magnetic separation, slow settling time, and good biocompatibility. This provides a powerful preparation method for industrial-scale analytical magnetic beads. Summary of the Invention

[0011] To address the aforementioned technical problems in existing technologies, the present invention aims to provide a "sandwich-type" cross-linked polystyrene analytical magnetic bead based on swelling and layer-by-layer assembly, along with its preparation method and applications. This invention uses carboxylated cross-linked polystyrene microspheres as templates, loads Fe3O4 particles using a swelling method and a layer-by-layer self-assembly method, and achieves biocompatibility through silicon coating of the microspheres. This results in analytical magnetic beads with uniform size, fast magnetic separation speed, slow sedimentation time, good biocompatibility, and excellent detection performance.

[0012] The technical solution adopted in this invention is as follows:

[0013] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized by using carboxylated cross-linked polystyrene microspheres as templates. After swelling the microsphere templates overnight at room temperature in a good solvent, Fe3O4 nanoparticles are added and sonicated, allowing the Fe3O4 nanoparticles to be loaded onto the swollen microsphere templates to form microsphere assemblies PS-MNPs. Then, the microspheres shrink in a poor solvent. Subsequently, in a poor solvent containing PEI, oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles are assembled using the metal-ligand affinity between PEI and Fe3O4, forming the first... The microspheres were loaded with two layers of Fe3O4 nanoparticles to enhance their magnetism. Then, OTMS was used to induce a hydrolysis-condensation reaction on the surface of the microsphere assembly, achieving a phase transfer from the organic phase to the aqueous phase. Next, a silica shell was grown on the surface using TEOS hydrolysis, resulting in cross-linked polystyrene analytical magnetic beads with a silica layer. Finally, the microspheres were further modified by aminopropyltriethoxysilane for ammoniation and by succinic anhydride for carboxylation, resulting in carboxylated "sandwich-type" cross-linked polystyrene analytical magnetic beads, thus completing the preparation process.

[0014] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized by the following steps: Polyvinylpyrrolidone is dissolved in an ethanol-water mixture with a volume ratio of 8-10:1 at a solid-liquid ratio of 1g:60-80mL. Under a nitrogen atmosphere, the mixture is heated to 65-75℃, and then styrene, azobisisobutyronitrile, and acrylic acid are added. The mixture is stirred at 250-300rpm for 3-5 hours. Then, a solution of... After the anhydrous ethanol of divinylbenzene is added dropwise, the reaction solution is stirred for 20-30 hours. After the reaction is completed, the solid is separated by centrifugation and washed several times with ethanol. Finally, the synthesized polystyrene microspheres are dried to complete the preparation. The feeding ratio of polyvinylpyrrolidone, styrene, azobisisobutyronitrile, acrylic acid and divinylbenzene is (0.6-0.8) g: (6-7) mL: (0.07-0.1) g: (0.15-0.3) mL: (2-4) mL.

[0015] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized in that the good solvent is toluene, the mass ratio of the microsphere template to the first layer of Fe3O4 nanoparticles is 10:6 to 10, preferably 10:8; and the bad solvent is ethanol.

[0016] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized by the following steps in the process of loading a second layer of Fe3O4 nanoparticles:

[0017] 1) The microsphere assembly PS-MNPs loaded with the first layer of Fe3O4 nanoparticles was dispersed in an ethanol solution containing PEI. The mass ratio of the microsphere assembly PS-MNPs to PEI was 1.2-1.5:1. After rotating and incubating for 1-3 hours, magnetic separation was performed.

[0018] 2) The microspheres obtained in step 1) are dispersed in a toluene-hexanol mixture with a volume ratio of 1:2 to 4. Oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles are added. After rotating and incubating for 1-3 hours, magnetic separation is performed to obtain a microsphere assembly loaded with two layers of Fe3O4 nanoparticles.

[0019] In step 2), the mass ratio of microspheres to oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles is 1.5-2.5:1.

[0020] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized by the following steps: hydrolysis-condensation reaction occurs on the surface of the microsphere assembly via OTMS, and a silica shell layer is grown on the surface via hydrolysis.

[0021] S1: Add OTMS to the microsphere assembly, mix with ultrasonication, then add methanol and ammonia, mix with ultrasonication for 20-60 min to transfer the microsphere assembly from the oil phase to the aqueous phase, centrifuge to remove the supernatant, and wash once with methanol to remove excess OTMS.

[0022] S2: Further add ultrapure water and ammonia to the precipitate, stir the reaction at room temperature for 15-20 h, and wash several times with ethanol after the reaction to obtain cross-linked polystyrene magnetic microspheres modified with organosilicon on the surface.

[0023] S3: Add ultrapure water and ammonia to the cross-linked polystyrene magnetic microspheres modified with organosilane obtained in step S2, and add TEOS under stirring to grow a silica shell. After the reaction is complete, centrifuge and magnetically separate the product, and wash it several times with ethanol to obtain cross-linked polystyrene magnetic microspheres coated with a silica shell.

[0024] The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly is characterized in that, in step S1, the volume ratio of OTMS to the mass of the microsphere assembly is (8-20) μL:1 mg, preferably (10-18) μL:1 mg, and the volume ratio of OTMS, methanol and ammonia is 1:70-80:1.5-2.

[0025] The volume ratio of OTMS in step S1 to ammonia in step S2 is 2.5–3.5:1;

[0026] In step S3, the volume ratio of TEOS to ammonia is 1:35-45, and the mass concentration of ammonia is 25-30%. The volume ratio of TEOS in step S3 to the mass ratio of the microsphere assembly in step S1 is (10-18) μL:(10-18) mg.

[0027] This invention provides an application of "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly in homogeneous analysis of PCT, characterized by the following steps in the application method:

[0028] 1) Preparation of cross-linked polystyrene immunomagnetic microsphere probe PMPMS-mAb1: Carboxylated cross-linked polystyrene magnetic beads were dispersed in phosphate-buffered saline (PBS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide sodium salt (Sulfo-NHS) were added and stirred for 20-40 min to activate the carboxyl groups on the surface of the carboxylated magnetic beads in the buffer. The activated magnetic beads were centrifuged, the supernatant was discarded, and the mixture was dispersed in PBS buffer. Then, procalcitonin (PCT) monoclonal antibody was added, and the mixture was reacted at room temperature for 1-3 h. Bovine serum albumin (BSA) was then added for blocking for 1-3 h. After the reaction was completed, the product was washed several times with PBS buffer and collected by magnetic separation. The obtained cross-linked polystyrene immunomagnetic microsphere probe PMPMS-mAb1 was dispersed in PBS buffer and stored.

[0029] 2) PMPMS-mAb1-based enzyme-linked immunosorbent assay (ELISA): Prepare a series of PCT standard solutions of different concentrations. Add the PBS buffer containing the probe PMPMS-mAb1 obtained in step 1) to a 96-well plate, and add a series of PCT standard sample solutions of different concentrations. Incubate at 37℃ for 30 min. After incubation, rinse several times. After removing the supernatant from the well plate, add diluted mAb2-HRP to the remaining microspheres in the well plate and incubate at 37℃ for 60 min. After incubation, rinse several times. After magnetic separation and washing, add the chromogenic substrate solution and develop the color for 5-20 min. Finally, add H2SO4 solution to terminate the reaction. Read the absorbance results using an ELISA reader and construct a standard curve with absorbance as the ordinate and PCT concentration as the abscissa for quantitative analysis.

[0030] 3) Following the steps in step 2), replace the PCT standard sample solution with the sample solution for detection. Substitute the final absorbance result into the standard curve plotted in step 2) to deduce the PCT content in the sample solution.

[0031] The present invention has the following technical advantages:

[0032] 1. A method for preparing cross-linked polystyrene microspheres based on dispersion polymerization, wherein the prepared microspheres have good monodispersity, high sphericity, simple operation, easy synthesis, and good repeatability and stability.

[0033] 2. A cross-linked polystyrene magnetic microsphere prepared based on a swelling method and a layer-by-layer assembly method. Utilizing the property of cross-linked polystyrene microspheres swelling in good solvents and shrinking in poor solvents, nano-Fe3O4 particles are loaded inside the cross-linked polystyrene microspheres, simplifying the loading process and enabling oil-phase assembly. Assembly is driven by the metal-ligand affinity between PEI and Fe3O4, loading nano-Fe3O4 particles onto the microsphere surface. This method is simple to operate and solves the problem of low nano-Fe3O4 particle loading on the microspheres that may occur with the swelling method. The high nano-Fe3O4 loading on the microspheres of this invention enables rapid separation of magnetic beads, and the uniform distribution of nano-Fe3O4 on the microspheres provides good suspension for homogeneous immunoassay. In contrast, existing technologies using microspheres loaded with a single layer of nano-Fe3O4 or with low Fe3O4 loading result in longer magnetic separation times, which is detrimental to the magnetic separation steps in the analytical process.

[0034] 3. A method for thiol modification on the surface of silica-coated cross-linked polystyrene magnetic microspheres. 3-Mercaptopropyltrimethoxysilane is hydrolyzed and grafted onto the silica surface via an ethanol / ammonia / hydrolysis system.

[0035] 4. A detection method combining immunomagnetic beads and ELISA, which, compared to the heterogeneous reaction process of traditional ELISA, reduces the steps of antigen or antibody fixation and washing on the enzyme-labeled plate. Simultaneously, the immunomagnetic beads provide magnetic enrichment and magnetic separation washing for analytical assays, simplifying the experimental procedures and improving analytical sensitivity.

[0036] Compared with the prior art, the technical effects achieved by the present invention are as follows:

[0037] 1. Monodisperse functional microspheres with cross-linked polystyrene as the backbone have excellent non-biodegradability, thermal stability and chemical stability, uniform particle size and light weight.

[0038] 2. Using cross-linked polystyrene microspheres with swelling and shrinkage properties as a carrier, the microspheres swell but do not dissolve in readily soluble solvents and shrink in insoluble solvents. This provides a simple method for encapsulating magnetic materials within the microspheres, eliminating the need to modify oil-soluble Fe3O4 nanoparticles and simplifying the experimental process. Compared to conventional loading methods, this method achieves magnetic material loading more easily. A second layer of Fe3O4 nanoparticles is then loaded onto the microsphere surface via layer-by-layer assembly using PEI, simplifying the process and solving the problem of low Fe3O4 particle loading on the microspheres caused by the swelling method. The prepared cross-linked polystyrene magnetic microspheres can be rapidly separated under an external magnetic field.

[0039] 3. Unlike most polymer microspheres that are encapsulated using polymer encapsulation methods, this experiment uses colloidal chemistry to encapsulate the microspheres with silicon. This makes it easier to modify the surface of the microspheres with different functional groups. The external functional groups of the microspheres can be changed according to different practical application requirements to achieve multiple biochemical applications of the material. Attached Figure Description

[0040] Figure 1 This diagram illustrates the preparation of the cross-linked polystyrene magnetic microspheres (PMPM-SiO2) of this invention and the detection method of the enzyme-linked immunosorbent assay (MELISA) based on magnetic beads.

[0041] Figure 2 Scanning electron microscope (SEM) images and transmission electron microscope (TEM) images of the products of the cross-linked polystyrene magnetic microsphere synthesis process of this invention.

[0042] Figure 3 This is a graph showing the feed ratio and loading rate of nano-Fe3O4 particles-crosslinked polystyrene microspheres tested in Example 2 of this invention.

[0043] Figure 4 Hysteresis loop diagrams of Fe3O4 nanoparticles loaded with one layer by the swelling method, Fe3O4 nanoparticles loaded with two layers by the layer-by-layer assembly method, and cross-linked polystyrene magnetic beads after being coated with a silicon layer, according to the present invention.

[0044] Figure 5 This is a performance characterization diagram of the cross-linked polystyrene carboxylated magnetic microspheres (PMPMS-COOH) prepared in Example 1 of the present invention;

[0045] Figure 6 The results of PCT detection using magnetic bead-based ELISA were obtained for the cross-linked polystyrene magnetic microsphere probe (PMPMS-mAb1) prepared in this invention.

[0046] Figure 7 The results are for testing the cross-linked polystyrene magnetic microsphere probe (PMPMS-mAb1) prepared in this invention. Detailed Implementation

[0047] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0048] In this embodiment of the invention, the mass concentration of ammonia water is 25%. The hydrophobic Fe3O4 magnetic particles modified with oleic acid are existing technology, and can be found in Example 3 of Chinese Patent CN114591726A.

[0049] Example 1:

[0050] The schematic diagrams for the preparation of cross-linked polystyrene magnetic microspheres (PMPM-SiO2) and the detection schematic diagrams for the enzyme-linked immunosorbent assay (MELISA) based on magnetic beads of this invention are shown below. Figure 1 As shown.

[0051] 1. Synthesis of cross-linked polystyrene carboxyl microspheres (PS-COOH)

[0052] Dissolve 0.7g of polyvinylpyrrolidone in 50ml of ethanol aqueous solution (V 乙醇 / V 水 In a mixture of 45:5, under a nitrogen atmosphere, the temperature was raised to 70℃, and then 6.5 mL of styrene, 0.09 g of azobisisobutyronitrile (AIBN), and 0.2 mL of acrylic acid were added. The mixture was stirred at 300 rpm and the reaction time was set to 4 h. Then, a divinylbenzene ethanol solution (prepared by dissolving 3.05 mL of divinylbenzene in 2.4 mL of anhydrous ethanol) was added dropwise over 2 h. After the addition was complete, the reaction solution was stirred continuously for a total reaction time of 24 h. After the reaction was complete, the mixture was washed with ethanol solution and centrifuged at 4400 rpm for 10 min. This process was repeated three times to remove the ethanol. The synthesized polystyrene microspheres were then placed in a vacuum drying oven and dried at 40℃ for 24 h to obtain cross-linked polystyrene carboxyl microspheres (PS-COOH).

[0053] The scanning electron microscope (SEM) image of the cross-linked polystyrene carboxyl microspheres (PS-COOH) product obtained in step 1 of Example 1 is shown below. Figure 2 As shown in (a).

[0054] 2. Synthesis of Fe3O4-loaded cross-linked polystyrene magnetic microspheres (PMPM) via swelling method + layer-by-layer self-assembly method

[0055] At room temperature, 10 mg of cross-linked polystyrene carboxyl microspheres were swollen overnight in 1 mL of methyl methacrylate (MTA). Then, using sonication, 0.5 mL of Fe3O4 solution (16 mg / mL) was immersed in the swollen polystyrene carboxyl microspheres for 15 min. After the reaction was complete, the microspheres were magnetically separated and shrunk with ethanol to synthesize Fe3O4-loaded polystyrene magnetic cores (PS-MNPs). Approximately 20 mg of the Fe3O4-loaded microspheres (PS-MNPs) were dispersed in an ethanol solution containing 14 mg of PEI (700 kDa), and incubated by rotation for 2 h. Then, approximately 20 mg of the magnetically separated microspheres were dispersed in a toluene:hexanol mixture (1:3, containing 16 mg of oleic acid-modified hydrophobic Fe3O4 magnetic particles, 20 mL volume), and incubated by rotation for 2 h. After magnetic separation, cross-linked polystyrene magnetic microspheres (PMPM) were obtained.

[0056] The scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the polystyrene magnetic cores (PS-MNPs) loaded with a layer of Fe3O4 obtained in step 2 of Example 1 are shown below. Figure 2 (b) and Figure 2 As shown in (c). The transmission electron microscope image of the microsphere-loaded PEI obtained in step 2 of Example 1 is shown below. Figure 2 (d). Example 1, Step 2: The cross-linked polystyrene magnetic microspheres (PMPM) formed by assembling oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles using the metal-ligand affinity of PEI and Fe3O4 are shown in the transmission electron microscope image. Figure 2 (e) and Figure 2 As shown in (g).

[0057] The hysteresis loop diagrams of the polystyrene magnetic cores (PS-MNPs) loaded with one layer of Fe3O4 obtained in step 2 of Example 1 and the cross-linked polystyrene magnetic microspheres (PMPM) assembled layer by layer loaded with two layers of Fe3O4 are shown below. Figure 4 Curves a and curve b are shown in the figure.

[0058] 3. Synthesis of sandwich-type polystyrene magnetic beads (PMPM-SiO2)

[0059] After magnetic separation of the obtained cross-linked polystyrene magnetic microspheres (PMPM), the supernatant was removed. 200 μL of n-octyltrimethoxysilane (OTMS) was added to 10 mg of the solid precipitate, and the mixture was ultrasonically mixed. Then, a mixture of 15 mL of methanol and 375 μL of ammonia was added, and the mixture was ultrasonically mixed for 30 min to transfer the outer layer of the cross-linked polystyrene magnetic beads from the oil phase to the aqueous phase. The reactants were centrifuged, the supernatant was removed, and the mixture was washed once with methanol to remove excess n-octyltrimethoxysilane. After magnetic separation, 33 mL of ultrapure water and 66 μL of ammonia were added to the precipitate, and the mixture was stirred at room temperature for 18 h. After the reaction, the mixture was washed three times with ethanol and dispersed in 10 mL of ultrapure water to obtain cross-linked polystyrene magnetic beads modified with organosilane. 0.625 mL of ammonia and 7.7 μL of TEOS were added to the obtained liquid, and after a 3-h interval, another 7.7 μL of TEOS was added and the reaction was continued for 3 h, for a total TEOS content of 15.4 μL, to grow a silica shell. After the reaction is complete, the product is magnetically separated and washed three times with ethanol to obtain sandwich polystyrene magnetic beads (PMPM-SiO2), abbreviated as PMPMS.

[0060] The transmission electron microscope (TEM) image of the sandwich-type polystyrene magnetic beads (PMPM-SiO2) obtained in step 3 of Example 1 is shown below. Figure 2 (f) and Figure 2 As shown in (h).

[0061] Figure 2 The characterization results (a)-(b) show that the microspheres can maintain good monodispersity during the synthesis of magnetic beads. Figure 2 The characterization results (c)-(h) demonstrate the successful modification of the magnetic particles and silicon layer.

[0062] The hysteresis loop diagram of the sandwich-type polystyrene magnetic beads (PMPM-SiO2) obtained in step 3 of Example 1 is shown below. Figure 4 As shown by curve c in the figure.

[0063] Figure 4 The results show that the coercivity and remanence of PS-MNPs, PMPM, and PMPM-SiO2 are negligible, indicating that the synthesized MNPs (Fe3O4) possess superparamagnetism. Therefore, the prepared PS-MNPs, PMPM, and PMPM-SiO2 all exhibit superparamagnetism. The saturation magnetizations of the prepared PS-MNPs, PMPM, and PMPM-SiO2 materials are 3.79, 7.64, and 6.33 emu / g, respectively.

[0064] 4. Surface functionalization of sandwich polystyrene magnetic beads (PMPMS-COOH)

[0065] Approximately 20 mg of the obtained PMPM-SiO2 microspheres (i.e., PMPMS microspheres) were dissolved in 40 mL of ethanol solution, followed by the addition of 1 mL of ammonia and 40 μL of 3-aminopropyltrimethoxysilane. The mixture was stirred for 12 h to obtain amino-modified PMPMS microspheres. These microspheres were purified by centrifugation with ethanol, then dispersed in 20 mL of N,N-dimethylformamide containing 5 mg / mL succinic anhydride and reacted for 4 h to obtain carboxyl-modified PMPMS microspheres. After washing several times with ethanol and water, the microspheres were dispersed in PB buffer (0.01 M, pH = 6.0) for further use. The concentration of the carboxyl-modified PMPMS microspheres in the PB buffer was 4 mg / mL.

[0066] The performance characterization results of the microsphere PMPMS-COOH product obtained in step 4 of Example 1 are shown in the figure. Figure 5 . Figure 5 In the image: (a) Magnetic separation time diagram of PMPMS-COOH; (b) Sedimentation time diagram of PMPMS-COOH (the sedimentation rate of the magnetic beads was characterized by measuring the absorbance of the supernatant); (c) Colloidal stability diagram of PMPMS-COOH at 35 days.

[0067] Figure 5 (a) is the magnetic separation time diagram of the magnetic bead. It can be seen that the magnetic separation of the magnetic bead is basically complete at 25s. Figure 5 (b) shows the sedimentation rate of the magnetic beads. Sedimentation rate is an effective means of characterizing the suspension of magnetic beads, and the quality of magnetic bead suspension directly affects the efficiency of the immunoassay reaction. The sedimentation rate was characterized by aspirating the supernatant of the PMPMS-COOH magnetic bead solution every 10 minutes over 360 minutes and reading the absorbance at 500 nm. When the sedimentation time was 60 minutes, the absorbance of the supernatant did not change significantly, indicating that the magnetic beads were uniformly dispersed and sufficient to ensure that the magnetic beads would not settle significantly during the immunoassay incubation period (<60 minutes), which is beneficial for the effective homogenization of the immunoassay reaction. When the sedimentation time reached 300 minutes, the absorbance of the supernatant only decreased by half, indicating that the prepared PMPMS-COOH had good suspension properties.

[0068] 5. Preparation of cross-linked polystyrene immunomagnetic microsphere probes (PMPMS-mAb1)

[0069] Take 1 mL of PBS buffer containing carboxyl-modified PMPMS microspheres (approximately 4 mg of carboxylated PMPMS microspheres), add 4 mg EDC and 4 mg Sulfo-NHS, and stir for 30 min to activate the surface carboxyl groups of the PMPMS microspheres in the buffer. Magnetically separate and redisperse the activated microspheres in the same volume of PBS buffer (0.01 M, pH 7.4), add 200 μg of PCT monoclonal antibody (MJG 03, purchased from Hangzhou Qitai Biotechnology Co., Ltd.), and react at room temperature for 2 h. Then, add BSA to a final concentration of 10 mg / mL for blocking for 2 h. Wash the product twice with PBS buffer (0.01 M, pH 7.4), collect by magnetic separation, and disperse the resulting analytical magnetic beads in PBS buffer (0.01 M, pH 7.4, containing 0.5% BSA and 0.5% PEG) and store at 4 °C.

[0070] 6. ELISA detection based on PMPMS-mAb1

[0071] Take the PBS buffer containing 40 μg of analytical magnetic beads obtained in step 5 and add it to a 4 mL centrifuge tube. Add 2 mL of PCT standard samples of different concentrations (PCT standard solutions are prepared by serial dilution, with concentrations of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 ng / mL). Incubate at 37°C for 30 min. After incubation, wash five times with 150 μL of PBS buffer (0.01 M, pH = 7.4) containing 0.05% Tween 20 and 1% BSA. Then transfer the solution to a 96-well plate and add diluted mAb2-HRP (a monoclonal antibody conjugated with horseradish peroxidase (HRP)) to the wells. Incubate at 37°C for 60 min. After incubation, the sample was washed five times with 150 μL of PBS buffer (0.01 M, pH 7.4) containing 0.05% Tween 20 and 1% BSA. After magnetic separation washing, 100 μL of chromogenic substrate solution was added, and the solution was incubated for 10 min until it turned blue. Finally, 50 μL of 0.5 M H2SO4 solution (as a stop agent) was added to terminate the reaction, and the solution turned yellow. At this point, the absorbance of the liquid in the well plate was read at 450 nm using a microplate reader.

[0072] The above-mentioned substrate solutions were prepared by mixing 50 μL each of colorimetric solution A and solution B. Colorimetric substrate solution A: 13.6 g sodium acetate, 1.6 g citric acid, 0.3 mL 30% H₂O₂, and distilled water were added to a final volume of 500 mL to prepare solution A. Colorimetric substrate solution B: 0.2 g disodium ethylenediaminetetraacetate, 0.95 g citric acid, 50 mL glycerol, 0.15 g 3,3',5,5'-tetramethylbenzidine, 3 mL dimethyl sulfoxide, and distilled water were added to a final volume of 500 mL to prepare solution B.

[0073] The cross-linked polystyrene magnetic microsphere probe (PMPMS-mAb1) prepared in Example 1 of this invention was used for PCT detection using a magnetic bead-based ELISA method. (See attached image for details.) Figure 6 . Figure 6 In the image: (a) Photograph of the color development of the solution under fluorescent light when detecting gradient concentrations of PCT (a1), where the asterisks in (a1) indicate the color change at the lowest visible concentration, and the color change of the solution after adding the stop agent (a2); (b) Four-parameter nonlinear fitting curve corresponding to a; (c) Linear fitting graph corresponding to a. Absorbance is expressed as the absorbance value at 450 nm after adding the stop agent, measured by an ELISA reader.

[0074] Figure 6 The results showed that the concentration was 0.1 ng / mL (Figure a), which met the minimum clinical guidance concentration threshold for PCT. Figures b and c showed that magnetic enrichment at 20-fold resulted in good correlation (R0.01) within the range of 0.01-200 ng / mL. 2 =0.99435), exhibiting good linear response in the range of 0.01-5 ng / mL (y = 0.441x + 0.151, ...).

[0075] R 2 =0.981), and the limit of detection (LOD) = 0.031 ng / mL (3σ / m, where σ is the standard deviation of 10 blank samples and m is the slope). This demonstrates that PMPMS-mAb1 immunomagnetic beads can be further used for practical immunoassay analysis.

[0076] In step 6 of Example 1 of this invention, several common blood inflammatory markers and proteins, including C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), ferritin (FER), human serum albumin (HSA), and IgG antibody (IgG), were selected as interfering substances to verify the specificity of the probe. In this study, the concentration of the interfering substance was set at 0.5 μg / mL, the PCT concentration was 0.5 ng / mL, and a blank control (i.e., no interfering substance was added) was added. The test results are as follows: Figure 7 As shown in (a). From Figure 7 (a) It can be seen that when the concentration of the interfering sample is 1000 times that of the PCT sample, the detected absorbance is comparable to that of the blank sample, and the detected PCT sample signal value is significantly higher than that of the blank and interfering samples. These results demonstrate that the PMPMS-mAb1 probe prepared in this study exhibits excellent specificity for detecting PCT in the MELISA system. Figure 7 The (a) specificity experiment shows that the prepared immunoassay probe has excellent specificity and can be used for immunoassay detection research.

[0077] Furthermore, the correlation graph between the enzyme-linked immunosorbent assay (ELISA) of magnetic beads used in this invention for detecting PCT and existing commercial chemiluminescence methods for detecting procalcitonin concentration in clinical diagnosis is shown in the figure below. Figure 7 As shown in (b). Figure 7 (b) The linear correlation between the enzyme-linked immunosorbent assay based on magnetic beads and the gold standard chemiluminescence method for diagnosing clinical procalcitonin concentration showed a good correlation, which is beneficial to the clinical application of the immunoprobe.

[0078] Example 2:

[0079] The experimental procedure of Example 1 was repeated, except that the amount of Fe3O4 added in step 2 of Example 1 was changed. Specifically, 10 mg of cross-linked polystyrene carboxyl microspheres were swollen overnight in 1 mL of toluene at room temperature. Then, using ultrasonic treatment, 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, and 1 mL of Fe3O4 (16 mg / mL) were impregnated into the swollen polystyrene carboxyl microspheres for 15 min. After the reaction was completed, magnetic separation was performed, and the supernatant was subjected to UV testing to calculate the loading rate. Iron-loaded microspheres (PS-MNPs) (feed ratio 0.8:1) were dispersed in an ethanol solution containing 7 mg of PEI (700 kDa). After rotational incubation for 2 h, the microspheres were magnetically separated. The microspheres were then dispersed in a mixed solvent of toluene:hexanol = 1:3 (the solvent contained 2, 4, 6, 8, 10, 12, and 16 mg of oleic acid-modified hydrophobic Fe3O4 magnetic particles). After rotational incubation for 2 h, the supernatant of cross-linked polystyrene magnetic microspheres (PMPM) was obtained after magnetic separation and subjected to UV testing to calculate the loading rate.

[0080] The feed ratio-loading rate of the nano-Fe3O4 particles-crosslinked polystyrene microspheres obtained in the experiment is shown in the figure below. Figure 3 As shown. Figure 3 Subplot a corresponds to the test results of polystyrene magnetic cores (PS-MNPs) with a layer of Fe3O4 swollen and loaded, reflecting the trend of the first layer of Fe3O4 loading rate with the feed ratio. Figure 3 Figure b corresponds to the test results of cross-linked polystyrene magnetic microspheres (PMPM) loaded with a second layer of Fe3O4 using the layer-by-layer assembly method. It reflects the trend of the second layer Fe3O4 loading rate as a function of the feed ratio.

[0081] The feeding ratio is calculated as follows: (Amount of nano-Fe3O4 particles added / Mass of cross-linked polystyrene microspheres) × 100%. The loading ratio is calculated as follows: (Loading amount of nano-Fe3O4 particles / Mass of cross-linked polystyrene microspheres) × 100%. The loading amount of Fe3O4 is calculated by the ultraviolet absorption of Fe3O4 in the supernatant before and after assembly.

[0082] Figure 3The results in a show that when the first layer of magnetic nanoparticles is loaded using the swelling method, the loading rate of the microspheres increases with the increase of the feed ratio. The loading rate plateaus when the feed ratio reaches 0.8, and ultimately reaches approximately 10%. The inset also shows that the color of the PS-MNPs after magnetic separation gradually deepens with the increase of the feed ratio, indicating that the magnetic particles were successfully loaded onto the polystyrene microspheres. Figure 3 As shown in b, during the assembly of the second layer of magnetic nanoparticles, the loading pattern followed a similar pattern to that of the swelling method. With increasing feed ratio, the loading rate also increased, and the color of the resulting PMPM magnetic beads gradually darkened. When the feed ratio reached 0.8, the loading rate of the magnetic beads ceased to increase. These phenomena demonstrate the successful loading of the magnetic particles. After two loading operations, the loading rate of the magnetic beads reached approximately 35%.

[0083] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.

Claims

1. A method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly, characterized in that... Using carboxylated cross-linked polystyrene microspheres as templates, the microsphere templates were swollen overnight in a good solvent at room temperature. Fe3O4 nanoparticles were then added and sonicated to load the Fe3O4 nanoparticles onto the swollen microsphere templates, forming microsphere assemblies (PS-MNPs). Subsequently, the microspheres shrank in a poor solvent. Then, in a poor solvent containing polyethyleneimine (PEI), oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles were assembled using the metal-ligand affinity between PEI and Fe3O4, forming a second layer of Fe3O4 nanoparticles to enhance the magnetic properties of the microspheres. Subsequently, octyltrimethoxysilane (OTMS) was used to perform a hydrolysis-condensation reaction on the surface of the microsphere assembly, realizing the phase transfer from the organic phase to the aqueous phase. Then, a silica shell was grown on its surface by hydrolysis of tetraethyl silicate (TEOS), thus preparing cross-linked polystyrene analytical magnetic beads with a silica layer on the surface. Finally, the microspheres were further modified by aminopropyltriethoxysilane for amination and by succinic anhydride for carboxylation, thus obtaining carboxylated "sandwich-type" cross-linked polystyrene analytical magnetic beads, and the preparation was completed. The process of loading a second layer of Fe3O4 nanoparticles includes the following steps: 1) The microsphere assembly PS-MNPs loaded with the first layer of Fe3O4 nanoparticles was dispersed in an ethanol solution containing PEI. The mass ratio of the microsphere assembly PS-MNPs to PEI was 1.2-1.5:

1. After rotating and incubating for 1-3 h, magnetic separation was performed. 2) The microspheres obtained in step 1) are dispersed in a toluene-hexanol mixture with a volume ratio of 1:2~4. Oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles are added. After rotating and incubating for 1-3 h, magnetic separation is performed to obtain a microsphere assembly loaded with two layers of Fe3O4 nanoparticles. In step 2), the mass ratio of microspheres to oleic acid-modified hydrophobic Fe3O4 magnetic nanoparticles is 1.5-2.5:

1.

2. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 1, characterized in that... The preparation method of the carboxylated cross-linked polystyrene microspheres is as follows: Polyvinylpyrrolidone is dissolved in an ethanol-water mixture with a volume ratio of 8-10:1 at a solid-liquid ratio of 1 g: 60-80 mL. Under a nitrogen protective atmosphere, the temperature is raised to 65-75℃, and then styrene, azobisisobutyronitrile, and acrylic acid are added. The mixture is stirred at 250-300 rpm for 3-5 h. Then, anhydrous ethanol containing divinylbenzene is added dropwise. After the addition is complete, the reaction solution is stirred for 20-30 h. After the reaction is completed, the solid is separated by centrifugation and washed several times with ethanol. Finally, the synthesized polystyrene microspheres are dried to complete the preparation. The feeding ratio of polyvinylpyrrolidone, styrene, azobisisobutyronitrile, acrylic acid, and divinylbenzene is (0.6-0.8) g: (6-7) mL: (0.07-0.1) g: (0.15-0.3) mL: (2-4) mL.

3. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 1, characterized in that... The good solvent is toluene, the mass ratio of the microsphere template to the first layer of Fe3O4 nanoparticles is 10:6~10, and the bad solvent is ethanol.

4. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 3, characterized in that... The mass ratio of the microsphere template to the first layer of Fe3O4 nanoparticles is 10:

8.

5. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 1, characterized in that... The specific process of hydrolysis and condensation reaction on the surface of the microsphere assembly via OTMS, and the hydrolysis growth of a silica shell on the surface, includes the following steps: S1: Add OTMS to the microsphere assembly, mix with sonication, then add methanol and ammonia, mix with sonication for 20-60 min to transfer the microsphere assembly from the oil phase to the aqueous phase, centrifuge to remove the supernatant, and wash once with methanol to remove excess OTMS. S2: Further add ultrapure water and ammonia to the precipitate, stir the reaction at room temperature for 15-20 h, and wash several times with ethanol after the reaction to obtain cross-linked polystyrene magnetic microspheres modified with organosilicon on the surface. S3: Add ultrapure water and ammonia to the cross-linked polystyrene magnetic microspheres modified with organosilane obtained in step S2, and add TEOS under stirring to grow a silica shell. After the reaction is complete, centrifuge and magnetically separate the product, and wash it several times with ethanol to obtain cross-linked polystyrene magnetic microspheres coated with a silica shell.

6. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 5, characterized in that... In step S1, the volume ratio of OTMS to the mass of the microsphere assembly is (8~20)ul : 1mg, and the volume ratio of OTMS, methanol and ammonia is 1:70~80:1.5~2. The volume ratio of OTMS in step S1 to ammonia in step S2 is 2.5~3.5:1; In step S3, the volume ratio of TEOS to ammonia is 1:35~45, and the mass concentration of ammonia is 25~30%; the volume ratio of TEOS in step S3 to the mass ratio of the microsphere assembly in step S1 is (10~18)ul : (10~18)mg.

7. The method for preparing "sandwich-type" cross-linked polystyrene analytical magnetic beads based on swelling and layer-by-layer assembly as described in claim 6, characterized in that... In step S1, the ratio of the volume of OTMS to the mass of the microsphere assembly is (10~18)ul :1mg.

8. A "sandwich" cross-linked polystyrene analytical magnetic bead based on swelling and layer-by-layer assembly prepared by the method according to any one of claims 1-7.

9. The application of a "sandwich-type" cross-linked polystyrene analytical magnetic bead based on swelling and layer-by-layer assembly as described in claim 8 in homogeneous immunoassay of PCT, characterized in that... The application method includes the following steps: 1) Preparation of cross-linked polystyrene immunomagnetic microsphere probe PMPMS-mAb1: Carboxylated cross-linked polystyrene analytical magnetic beads were dispersed in PBS buffer, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide sodium salt were added and stirred for 20-40 min to activate the carboxyl groups on the surface of the carboxylated magnetic beads in the buffer. The activated magnetic beads were centrifuged to remove the supernatant and dispersed in PBS buffer. Then, procalcitonin PCT monoclonal antibody was added and reacted at room temperature for 1-3 h. Bovine serum albumin (BSA) was added for blocking for 1-3 h. After the reaction was completed, the product was washed several times with PBS buffer and collected by magnetic separation. The obtained cross-linked polystyrene immunomagnetic microsphere probe PMPMS-mAb1 was dispersed in PBS buffer and stored. 2) PMPMS-mAb1-based enzyme-linked immunosorbent assay (ELISA): A series of PCT standard solutions of different concentrations were prepared. PBS buffer containing the probe PMPMS-mAb1 obtained in step 1) was added to a 96-well plate, followed by a series of PCT standard sample solutions of different concentrations. The incubation conditions were 37 ℃ for 30 min. After incubation, the plate was rinsed several times. After removing the supernatant, diluted mAb2-HRP was added to the remaining microspheres in the plate, and the incubation conditions were 37 ℃ for 60 min. After incubation, the plate was rinsed several times. After magnetic separation and washing, the chromogenic substrate solution was added, and the reaction was incubated for 5-20 min. Finally, H2SO4 solution was added to terminate the reaction. The absorbance results were read using an ELISA reader, and a standard curve was constructed with absorbance as the ordinate and PCT concentration as the abscissa for quantitative analysis. 3) Following the steps in step 2), replace the PCT standard sample solution with the sample solution for detection. Substitute the final absorbance result into the standard curve plotted in step 2) to deduce the PCT content in the sample solution.

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