Temperature-sensitive magnetic bead, preparation method thereof and related application of temperature-sensitive magnetic bead in early cancer screening

Abstract

The invention discloses a temperature-sensitive magnetic bead, a preparation method thereof and related application of the temperature-sensitive magnetic bead in early cancer screening, and the temperature-sensitive magnetic bead is prepared by taking an unsaturated superparamagnetic bead with vinyl on the surface as a matrix through an in-situ free radical polymerization mode. N-isopropylacrylamide, acrylamide and oligomeric (ethylene glycol) methyl ether methacrylate are initiated to be copolymerized on the surfaces of the magnetic beads to form a temperature-sensitive polymer brush layer with an adjustable low critical solution temperature (LCST). The temperature-sensitive magnetic beads are in a hydrophobic collapsing state under the condition of 37-39 DEG C, are maintained in a viscoelastic fluid state beneficial to dynamic replacement of low-abundance proteins, and can efficiently adsorb the low-abundance proteins in plasma; the temperature-sensitive magnetic beads are rapidly converted into a hydrophilic stretching state at the temperature lower than 36 DEG C, and the multi-point interaction between the proteins and the temperature-sensitive magnetic beads is synchronously weakened by utilizing micromechanical ejection force generated by phase change, so that the proteins are completely eluted from the surfaces of the magnetic beads.

Description

Technical Field

[0001] This invention relates to the fields of biomedical new materials, proteomics, and in vitro diagnostics, specifically to a temperature-sensitive magnetic bead, its preparation method, and its application in early cancer screening. Background Technology

[0002] Cancer incidence and mortality rates continue to rise globally, making it a significant disease type that seriously threatens human health. Numerous studies have shown that the occurrence and development of tumors are accompanied by systemic changes in protein expression levels, modification states, and interaction networks, and these changes often precede the appearance of obvious clinical symptoms. Therefore, developing technologies that enable reliable detection in the early stages of disease is of great importance for improving early cancer diagnosis rates and treatment outcomes.

[0003] Current tumor detection methods mainly include imaging examinations, histopathological analysis, and serological biomarker detection. Among these, imaging methods have limited sensitivity to early, small lesions and molecular-level changes; while tissue biopsies provide direct histological evidence, they are highly invasive, have limited sampling capabilities, and are difficult to repeat; and traditional serological biomarkers have a limited variety, insufficient sensitivity and specificity, making them unsuitable for early tumor screening. Against this backdrop, liquid biopsy, due to its minimally invasive nature, repeatable sampling, and ability to reflect systemic molecular changes, is gradually becoming an important development direction in the field of tumor detection.

[0004] Among the various analytes in liquid biopsy, circulating proteins directly participate in and reflect tumor-related biological processes, and are considered a highly promising source of biomarkers. Mass spectrometry-based proteomics technology enables large-scale protein identification and quantification, offering advantages in high throughput and high information density, thus attracting widespread attention in liquid biopsy research. However, in bodily fluids such as blood, high-abundance proteins account for the vast majority of the total protein content, severely limiting the detection space for low-abundance, tumor-related proteins, becoming a key factor restricting the sensitivity and coverage depth of mass spectrometry proteomics.

[0005] In existing liquid biopsy procedures using magnetic beads to separate proteins, different proteins simultaneously adsorb onto the bead surface, forming complex protein crown structures. Protein molecules often adsorb stably onto the solid-phase surface simultaneously through multiple weak interaction sites. Traditional magnetic beads typically rely on denaturants or surfactants to gradually weaken the protein-solid phase interaction for elution. The elution process struggles to achieve simultaneous dissociation of multiple interactions within a limited time, resulting in some proteins, especially low-abundance proteins, remaining at the solid-phase interface after elution. This residue significantly affects the proportion of low-abundance proteins and can even cause the systematic loss of key low-abundance proteins at the detection level, thus limiting the ability of liquid biopsy to capture and detect key early tumor signals. To overcome the limitations of traditional "chemical elution," temperature-responsive smart polymers (such as poly(N-isopropyl acrylamide), or PNIPAM) offer a new technological opportunity to construct controllable adsorption / desorption interfaces. However, when the Low Critical Solution Temperature (LCST) is approximately 32°C, directly applying conventional PNIPAM to highly complex plasma proteomics systems has significant theoretical limitations: On the one hand, the phase transition temperature of 32°C is too close to the room temperature operating environment, making it easy for adsorption to be initiated unexpectedly before the magnetic beads and plasma are evenly mixed, resulting in high-abundance proteins "preemptively occupying" the space due to their concentration advantage; on the other hand, the strong hydrophobic rigid interface formed after the phase transition of pure PNIPAM will hinder the dynamic exchange of protein molecules, making it difficult for low-abundance proteins with stronger affinity to replace the adsorbed high-abundance impurities through the "Vroman effect".

[0006] Therefore, how to improve the interfacial properties of magnetic beads themselves, avoid the interference of "premature adsorption" and improve the current situation of "difficulty in protein replacement", and increase the relative proportion of low-abundance proteins in eluted proteins has become a technical problem that urgently needs to be solved in the field of liquid biopsy proteomics. Summary of the Invention

[0007] This invention provides a temperature-sensitive magnetic bead, its preparation method, and its application in early cancer screening. In some embodiments, the temperature-sensitive magnetic bead uses a vinyl-containing superparamagnetic magnetic bead as a matrix, and copolymerizes N-isopropylacrylamide (NIPAM), acrylamide (AM), and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) on its surface via free radical polymerization to form a stable temperature-sensitive copolymer layer on the magnetic bead surface. By controlling the ratio and degree of polymerization of the three comonomers, the lower critical solution temperature (LCST) of the resulting magnetic bead is controlled at 36°C, thereby establishing a clear "safety valve" between room temperature operation (20-30°C) and adsorption enrichment (37-39°C), ensuring that the magnetic bead remains hydrophilic and inert before uniform mixing, and avoiding premature adsorption of impurities.

[0008] When the system temperature is above LCST (37~39℃), the copolymer segments undergo hydrophobic collapse, and the surface of the magnetic beads exhibits a flexible hydrophobic interface, allowing proteins to be reversibly adsorbed onto the surface of the magnetic beads. This invention specifically selects 38℃ as the adsorption operating temperature. This temperature not only matches the conventional temperature control equipment in most biochemical laboratories, facilitating operation and promotion without damaging protein activity, but also, at this temperature (slightly above LCST), the copolymer interface maintains a water-rich "viscoelastic fluid" state. Compared to traditional rigid hydrophobic magnetic beads, this flexible interface lowers the interfacial energy barrier, allowing protein molecules within the adsorption layer to remain dynamic. This enables low-abundance proteins with stronger affinity to effectively displace non-specifically adsorbed high-abundance proteins through the "Vroman effect (displacement effect)," thereby achieving a dual improvement in application convenience and enrichment specificity.

[0009] When the system temperature drops below the LCST (25~30℃), the copolymer segments change from a hydrophobic collapsed state to a hydrophilic extended state. The overall free energy of the magnetic bead surface decreases significantly, and the explosive swelling of the polymer layer generates mechanical thrust at the micro-interface, causing the multi-point weak interactions between the protein and magnetic beads to become unstable simultaneously within a short period of time. Unlike traditional magnetic beads that rely on denaturants to gradually weaken the interactions, this temperature-responsive process avoids the situation where the protein is in a semi-desorbed state and re-adsorbs, thus significantly promoting the complete elution of proteins, especially low-abundance proteins, from the magnetic bead surface and their entry into the solution phase.

[0010] In some implementations, the above mechanism effectively reduces the systemic residual risk of low-abundance proteins during the elution process and increases the relative proportion of low-abundance proteins in the eluted proteins. This invention helps to increase the detection capability of key low-abundance proteins in subsequent mass spectrometry analysis and improves the accuracy of early cancer detection based on plasma proteomics.

[0011] To achieve the above objectives, the present invention is accomplished through the following aspects: On one hand, the present invention provides a temperature-sensitive magnetic bead, the temperature-sensitive magnetic bead comprising a magnetic core and a temperature-sensitive copolymer layer modified on the surface of the magnetic core, wherein: The magnetic core comprises vinyl group-modified iron oxide (sometimes called Fe3O4) particles with a particle size of 50 nm to 500 nm. The temperature-sensitive copolymer layer is a polymer layer modified on the surface of the magnetic core by free radical polymerization, wherein: The thermosensitive copolymer layer is formed by using the vinyl group-modified iron oxide particles as the initiating matrix to initiate the free radical copolymerization reaction of the following monomers: N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate, wherein the initiator used in the free radical copolymerization reaction is potassium persulfate or azobisisobutylamidine hydrochloride, and the crosslinking agent is N,N'-methylenebisacrylamide.

[0012] In some embodiments, the magnetic core is a superparamagnetic magnetic particle modified with vinyl groups, preferably a magnetite particle modified with vinyl groups, more preferably, the magnetic core particle size is 50 nm to 500 nm, and most preferably, the magnetic core particle size is 100 nm to 300 nm, so as to balance magnetic response speed and specific surface area.

[0013] In some embodiments, the magnetic core consists of or is substantially composed of magnetite particles modified with vinyl groups.

[0014] In some embodiments, the thermosensitive copolymer layer is a polymer layer grafted onto the surface of the magnetic core via free radical polymerization, wherein: In some embodiments, the thermosensitive copolymer layer is formed by using vinyl-modified superparamagnetic magnetic particles as an initiating matrix to initiate a free radical copolymerization reaction of the following monomers: N-isopropylacrylamide (or NIPAM), acrylamide (or AM), and oligo(ethylene glycol) methyl ether methacrylate (or OEGMA). In one embodiment, the molar ratio of N-isopropylacrylamide (or NIPAM), acrylamide (or AM), and oligo(ethylene glycol) methyl ether methacrylate (or OEGMA) is NIPAM:AM:OEGMA = (20~70):(0.5~5):(0.5~3), preferably (30~60):(1~3):(1~2), and the total concentration of NIPAM, AM, and OEGMA is 20 mg / mL to 50 mg / mL, preferably 20 mg / mL to 25 mg / mL. By introducing AM to improve the hydrophilicity of the system and introducing OEGMA to adjust the steric hindrance and chain segment flexibility, the copolymer layer exhibits a water-rich "viscoelastic fluid" state rather than a dense, rigid glassy state after the phase transition, thereby achieving precise control of temperature-sensitive behavior.

[0015] In some embodiments, the temperature-sensitive magnetic beads of the present invention are used to enrich low-abundance proteins in plasma.

[0016] In another embodiment, the vinyl group-modified iron oxide particles are prepared by coupling iron oxide nanoparticles with a vinyl-containing silane coupling agent, preferably 3-(trimethoxysilyl)propyl methacrylate (MPS), wherein the mass ratio of iron oxide to MPS is 1:(1.0~3.0), preferably 1:2.

[0017] In another embodiment, the initiator for initiating the free radical copolymerization reaction is potassium persulfate (KPS) or azobisisobutylamidine hydrochloride (V50), and the amount of initiator is 1% to 2% of the total monomer mass. The crosslinking agent is N,N'-methylenebisacrylamide (MBA), and the amount is 1% to 3% of the total monomer molar mass. Alternatively, the initiator may be potassium persulfate (KPS) at 2% of the total monomer mass, or N,N'-methylenebisacrylamide (MBA) at 2% of the total monomer molar mass.

[0018] In some embodiments, the number-average molecular weight (Mn) of OEGMA is 200 Da to 2000 Da, preferably 400 Da to 1000 Da, and more preferably 500 Da to 800 Da. This molecular weight range is advantageous for controlling the hydrophilicity-hydrophobicity balance of the copolymer and improving protein desorption efficiency without significantly increasing steric hindrance. The average particle size of the temperature-sensitive magnetic beads is 100 nm to 500 nm.

[0019] In some embodiments of the present invention, by adjusting the above-mentioned monomer molar ratio and the overall degree of polymerization of the copolymer, the low critical solution temperature (LCST) of the resulting thermosensitive copolymer layer is set at 35.5~36.5°C, preferably 35.8~36.2°C, and most preferably 36.0°C.

[0020] In some implementations, the temperature range of the present invention is highly matched with the commonly used temperature for plasma protein enrichment and elution. More importantly, the temperature setting is higher than the normal laboratory room temperature and operator hand temperature (30℃~34℃), which creates a safe operating window to avoid impurities "preemptively occupying" the pretreatment stage, and is conducive to achieving precise temperature control of the "adsorption-desorption" process.

[0021] When the ambient temperature exceeds the LCST, the thermosensitive copolymer segments undergo hydrophobic collapse. However, thanks to the introduction of OEGMA side chains, a flexible hydrophobic interface (viscoelastic fluid state) with dynamic exchange capacity is formed on the magnetic bead surface. This creates a multi-point interaction interface on the magnetic bead surface that facilitates protein adsorption, thereby achieving efficient adsorption of low-abundance proteins in plasma. This flexible interface lowers the energy barrier for protein adsorption, allowing lower-abundance proteins with stronger affinity to displace non-specifically adsorbed higher-abundance proteins through the "Vroman effect (displacement effect)".

[0022] When the ambient temperature drops below LCST, the thermosensitive copolymer segments rapidly transform into a hydrophilic and extended state, generating microscopic mechanical thrust. The hydrophobic and non-specific interactions between the protein and the magnetic beads are significantly weakened, enabling the rapid removal of adsorbed proteins.

[0023] On the other hand, the present invention provides a method for preparing temperature-sensitive magnetic beads, which includes magnetic core synthesis, surface vinylization modification, in-situ copolymerization reaction of the temperature-sensitive layer, and purification steps. In a specific embodiment, the magnetic core synthesis step is as follows: ferric chloride hexahydrate, trisodium citrate, and anhydrous sodium acetate are dissolved in ethylene glycol and reacted in a high-temperature and high-pressure reactor, wherein the amount of ferric chloride hexahydrate is 1.0 g to 1.5 g, trisodium citrate is 0.3 g to 0.5 g, anhydrous sodium acetate is 1.0 g to 3.0 g, and ethylene glycol is 10 mL to 60 mL; the reaction temperature is 190℃ to 210℃, and the reaction time is 8 to 12 hours. In a preferred embodiment, the amount of ferric chloride hexahydrate is 1.5 g, trisodium citrate is 0.48 g, anhydrous sodium acetate is 1.68 g, and ethylene glycol is 10 mL; the reaction temperature is 200℃, and the reaction time is 12 hours.

[0024] In a further embodiment, the product obtained from the reaction is ultrasonically washed with alternating ethanol and deionized water at least three times until the supernatant is colorless, magnetically separated and collected, and vacuum dried to obtain iron oxide nanoparticles.

[0025] In a more specific embodiment, the surface vinylization modification step (introducing reaction sites) includes dispersing the prepared iron oxide in a mixed solvent of ethanol, water, and concentrated ammonia (volume ratio 40:1:1), ultrasonically dispersing until uniform dispersion, and adding a vinyl-containing silane coupling agent, preferably 3-(trimethoxysilyl)propyl methacrylate (MPS), for coupling. Preferably, the mass ratio of iron oxide to MPS is 1:(1.0~3.0), more preferably 1:2. The reaction is mechanically stirred at 60°C~80°C for 6~12 hours.

[0026] In another embodiment, the preparation method of the present invention may further include magnetic separation after the reaction is completed, and vigorous washing with ethanol to remove physically adsorbed silane hydrolysates to obtain vinylated magnetic beads (Fe3O4-CH=CH2) with double bonds on the surface.

[0027] The in-situ copolymerization reaction steps of the temperature-sensitive layer may include: Dispersion: The obtained vinylated magnetic beads were dispersed in deionized water and nitrogen gas was bubbled through to remove dissolved oxygen; Feeding: Under nitrogen protection, monomers NIPAM, AM and OEGMA are added sequentially, with the total monomer concentration controlled at 20 mg / mL to 50 mg / mL, preferably 20 to 25 mg / mL; Crosslinking and initiation: Add crosslinking agent N,N'-methylenebisacrylamide (MBA) at a rate of 1% to 3% of the total molar mass of the monomer, preferably 2%. Heat to 70°C and add initiator potassium persulfate (KPS) or azobisisobutylamidine hydrochloride (V50), preferably potassium sulfate (KPS), at a rate of 1% to 2% of the total mass of the monomer, preferably 2%. React at a stirring speed of 200 to 400 rpm, preferably 300 rpm, for 4 to 6 hours, preferably 6 hours.

[0028] Preferably, the amount of N,N'-methylenebisacrylamide (MBA) is 2% of the total molar amount of the monomer; the initiator is potassium persulfate (KPS), and the amount of initiator is 2% of the total mass of the monomer, and the reaction is carried out at a stirring speed of 300 rpm for 6 hours.

[0029] The purification step may include magnetic separation after the reaction, repeated washing with deionized water at a temperature below the low critical solution temperature (LCST) of the resulting temperature-sensitive magnetic beads, preferably 30°C, to remove unreacted monomers and free polymers, and then freeze-drying for storage.

[0030] On the other hand, the present invention provides a method for processing low-abundance plasma proteins for early cancer screening. The method includes contacting a diluted plasma sample with temperature-sensitive magnetic beads of the present invention, or temperature-sensitive magnetic beads prepared by the method of the present invention, at a temperature higher than the LCST of the magnetic beads to adsorb proteins; eluting the adsorbed plasma proteins from the magnetic beads with a solution at a temperature lower than the LCST, allowing the proteins to elute from the surface of the magnetic beads and enter the solution phase; adding a protease, preferably trypsin, to the solution phase for enzymatic hydrolysis; and recovering peptides. Preferably, the adsorption temperature is 37–39°C, more preferably 38°C, and / or the elution temperature is room temperature, more preferably 25–30°C. In some embodiments, reversible switching of the surface state of the magnetic beads is achieved through temperature control, which can increase the relative proportion of low-abundance proteins in the eluted proteins.

[0031] In one embodiment, the solution used for protein elution performs unfolding, reduction of protein disulfide bonds, and alkylation operations on the protein, preferably using urea buffer (UA buffer), an aqueous solution of dithiothreitol (DTT), and an aqueous solution of iodoacetamide (IAA) in sequence to perform the above unfolding, reduction of protein disulfide bonds, and alkylation operations.

[0032] Thermosensitive magnetic beads are applied to the pretreatment of plasma samples for protein adsorption and enrichment under conditions above the LCST. Preferably, the adsorption temperature is 37℃~39℃, and more preferably 38℃. This invention selects 38℃ as the preferred adsorption temperature because, on the one hand, this temperature matches conventional laboratory thermostats and does not damage protein activity, making operation convenient; on the other hand, at this temperature, the surface of the magnetic beads is maintained in a specific viscoelastic fluid state, promoting the replacement of high-abundance proteins by low-abundance proteins. Protein desorption is then performed under conditions below the LCST. Preferably, the desorption temperature is room temperature, and more preferably 25~30℃, so that the protein is completely eluted from the surface of the magnetic beads and enters the solution phase.

[0033] By switching between adsorption and desorption within the aforementioned temperature range, the temperature-sensitive magnetic beads reduce multi-point interactions of low-abundance proteins during the protein adsorption stage and significantly weaken hydrophobic and non-specific interactions between proteins and magnetic beads during the desorption stage. This reduces the residue of low-abundance proteins during elution and increases the proportion of low-abundance proteins in the eluted protein. The eluted proteins can then be processed using conventional methods and used for mass spectrometry detection.

[0034] After mass spectrometry sample loading, mass spectrometry analysis was performed. MaxQuant software was used to search the database and compare the results with the UniProt human proteome database to screen for differentially expressed low-abundance proteins as cancer biomarkers. Furthermore, a machine learning model was used to perform statistical analysis on the tested plasma samples.

[0035] On the other hand, the present invention provides a kit for enriching low-abundance plasma proteins for early cancer screening, comprising thermosensitive magnetic beads prepared by the method of the first aspect or the method of the second aspect, reagents for achieving plasma protein adsorption, desorption and enzymatic digestion, and optional instructions for use (a booklet or insert typically found in kits).

[0036] On the other hand, the present invention provides a system for early cancer screening, which includes enriching plasma low-abundance proteins in plasma samples of specific types of cancer with different types of magnetic beads, performing mass spectrometry detection on the enriched plasma low-abundance proteins, performing deep learning on the obtained data to establish a prediction model, and applying receiver operating characteristic curve (ROC curve) analysis to evaluate the accuracy of the established prediction model.

[0037] The positive advancements of this application are: Compared with the prior art, the present invention has at least one of the following beneficial effects: 1. In some embodiments of the present invention, the LCST is precisely controlled to 36°C to establish an operational safety valve: through multi-monomer synergistic copolymerization, the phase transition temperature of the magnetic beads is precisely locked at 36°C, which is higher than the normal room temperature and hand temperature. This effectively avoids the hydrophobic collapse of the magnetic beads due to accidental heating before uniform mixing, prevents high-abundance impurities from occupying positions in advance, and ensures the uniformity of the enrichment initiation point; 2. In some embodiments of the present invention, a "viscoelastic fluid" interface is constructed to facilitate Vroman replacement: by introducing OEGMA to adjust the flexibility, the magnetic beads exhibit a metastable viscoelastic fluid interface at a preferred operating temperature of 38°C (slightly above LCST). This flexible interface lowers the protein adsorption energy barrier, allowing lower-abundance tumor markers with stronger affinity to dynamically replace non-specifically adsorbed higher-abundance proteins, significantly improving enrichment specificity. Simultaneously, the 38°C condition is compatible with conventional laboratory equipment and ensures protein activity. 3. In some embodiments of the present invention, low-temperature “ejection-style” non-destructive elution is achieved: by utilizing the micro-mechanical thrust generated by the explosive hydrophilic swelling of the polymer layer during cooling, combined with the drastic decrease in surface free energy, the adsorbed proteins (especially strongly interacting low-abundance proteins) are rapidly and completely removed, solving the residue problem of traditional chemical elution. 4. In some embodiments of the present invention, cancer detection performance is significantly improved: Based on the above mechanism, the present invention significantly improves the mass spectrometry detection capability of low-abundance key proteins, thereby improving the sensitivity and accuracy of early cancer detection. Attached Figure Description

[0038] Figure 1 This invention provides a schematic diagram of the structure of the temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) and a schematic diagram of the high-temperature (38°C) adsorption and low-temperature (25°C) desorption mechanism. PNIPAM is poly(N-isopropylacrylamide), PAM is polyacrylamide, and POEG is poly(oligomeric (ethylene glycol) methyl ether methacrylate).

[0039] Figure 2 The particle size variation diagram of the thermosensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) of this invention in the temperature-sensitive response range of 20℃~50℃.

[0040] Figure 3 The graph shows a comparison of the adsorption quantity of low-abundance proteins by the thermosensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) at adsorption equilibrium at 38℃ compared to pure PNIPAM magnetic beads.

[0041] Figure 4A comparison chart of the detection quantities of low-abundance proteins by the thermosensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) of this invention under low-temperature elution (25°C) and high-temperature elution (38°C).

[0042] Figure 5 This invention compares the number of proteins detected after elution in liver cancer samples using temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) and control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). PDMAPMA is poly(dimethylaminopropyl methacrylamide); PEGOMA is poly(ethylene glycol) methyl ether methacrylate.

[0043] Figure 6 This invention compares the detection of low-abundance proteins in liver cancer samples after elution using temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) and control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA).

[0044] Figure 7 The present invention compares the detection levels of key low-abundance proteins in liver cancer samples after elution using temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) and control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA).

[0045] Figure 8 The ROC curves of the temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) and the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA) in the present invention are compared for cancer detection in liver cancer samples.

[0046] Figure 9 Confusion matrix for cancer detection in advanced liver cancer samples using the thermosensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) of this invention.

[0047] Figure 10 Confusion matrix for early liver cancer sample detection using temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) of this invention. Detailed Implementation

[0048] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of protection of the present invention.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0050] In addition, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the raw materials, reagents, and other materials used in the following embodiments are commercially available products.

[0051] This invention discloses a temperature-sensitive magnetic bead, its preparation method, and its application in early cancer screening. The temperature-sensitive magnetic bead can enrich low-abundance proteins in plasma based on its responsive adsorption and desorption functions. The bead uses unsaturated superparamagnetic magnetic beads with vinyl groups on their surface as a matrix. Through in-situ free radical polymerization, NIPAM, AM, and OEGMA are copolymerized on the bead surface to form a temperature-sensitive polymer brush layer. By controlling the ratio and degree of polymerization of the comonomers, the LCST of the magnetic beads is precisely located at 36±0.5℃. This establishes a clear "safety valve" between room temperature operation (20℃~30℃) and adsorption enrichment (37℃~39℃), effectively avoiding premature non-specific adsorption of high-abundance impurities during the pretreatment mixing stage. The magnetic beads exhibit a hydrophobic collapse state at 37℃~39℃, but thanks to side chain regulation, the interface remains in a metastable "viscoelastic fluid" state rather than a dense glassy state. Utilizing the dynamic exchange properties of the flexible interface, the energy barrier is lowered, promoting the replacement of high-abundance proteins with more potent proteins through the "Vroman effect," achieving efficient adsorption. At room temperature, such as 25℃~30℃, they rapidly transform into a hydrophilic expanded state. The micro-mechanical thrust (ejection effect) generated by the explosive swelling of the polymer chains simultaneously weakens the multi-point interactions between the protein and the temperature-sensitive magnetic beads, thereby promoting complete elution of the protein from the bead surface. Compared to traditional magnetic beads, the temperature-sensitive magnetic beads of this invention solve the problems of high residue and poor adsorption specificity associated with traditional chemical elution, helping to improve the detection quantity of low-abundance proteins based on plasma proteomics and the accuracy of early cancer detection. This invention is applicable to early cancer screening based on liquid biopsy and has promising clinical application prospects.

[0052] The present application will be further described below with reference to the embodiments.

[0053] Example

[0054] Example 1: Preparation of temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) Step 1: Synthesis of magnetite nanoparticles Weigh 1.35 g of ferric chloride hexahydrate, 0.48 g of trisodium citrate, and 1.68 g of anhydrous sodium acetate, and completely dissolve them in 10 mL of ethylene glycol. Stir magnetically for 30 minutes to form a transparent yellowish-brown solution. Transfer the mixture to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE), seal it, and place it in an oven. Heat the reactor to 200 °C and maintain the temperature for 12 hours. Allow it to cool naturally to room temperature. The black product is then ultrasonically washed three times alternately with ethanol and deionized water, and vacuum dried to obtain magnetite beads with an average particle size of approximately 200 nm.

[0055] Step 2: Surface vinylation modification Accurately weigh 200 mg of the iron(III) oxide prepared in step 1 and ultrasonically disperse it in 105 mL of a mixed solvent of ethanol, water, and concentrated ammonia (volume ratio 100:2.5:2.5). Add 0.4 mL of propyl 3-(trimethoxysilyl)methacrylate (MPS) and react with mechanical stirring in an oil bath at 70 °C for 8 hours. After the reaction is complete, wash three times with ethanol by centrifugation to remove unreacted silane, yielding Fe3O4-CH=CH2 with double bonds on its surface.

[0056] Step 3: Coating with a temperature-sensitive polymer layer 100 mg of Fe3O4-CH=CH2 was redispersed in 50 mL of deionized water and transferred to a three-necked flask. High-purity nitrogen was bubbled through the flask for 30 minutes to remove dissolved oxygen. Under nitrogen protection, the following reagents were added sequentially (molar ratios strictly controlled): NIPAM: 450 mg, AM: 15.4 mg, OEGMA (Mn=500): 75 mg, MBA: 15 mg. Mechanical stirring was started at 300 rpm, and the system was heated to 70°C. 15 mg of initiator was dissolved in 1 mL of water using KPS and injected into the reaction system. The reaction was maintained at 70°C for 6 hours. After the reaction, the mixture was washed five times with deionized water at 30°C using a magnetic separator, and then freeze-dried to obtain the thermosensitive magnetic beads Fe3O4-PNIPAM-PAM-POEG.

[0057] Step 4: Verification of temperature-sensitive properties To verify whether the material possesses a "safety valve" characteristic to prevent early adsorption at room temperature, dynamic light scattering (DLS) was used to measure the particle size variation of the magnetic beads within the temperature range of 20℃ to 50℃. Figure 2 The results showed that the low critical solution temperature (LCST) of the magnetic beads was precisely located at 36.0℃. Below 34℃ (normal room temperature and hand temperature range), the magnetic beads remained in a hydrophilic swollen state, and the steric hindrance formed by the large particle size effectively prevented non-specific protein adsorption; a phase transition only occurred when the temperature rose to the set operating temperature (>36℃).

[0058] Example 2: Enrichment and elution process of low-abundance proteins in plasma 1. Sample pretreatment Take 10 μL of human plasma sample and dilute it to 100 μL with 90 μL of binding buffer (such as phosphate buffer solution, or PBS, pH 7.4).

[0059] 2. High-temperature capture Add 100 μL of the magnetic beads prepared in Example 1. Place the centrifuge tube in a constant-temperature metal bath at 38°C and incubate with shaking for 30 minutes. At this time, the polymer on the surface of the magnetic beads collapses, exposing the hydrophobic groups, resulting in efficient adsorption of low-abundance proteins. After incubation, maintain the 38°C environment, place the centrifuge tube on a magnetic rack for separation, and discard the supernatant. Wash the magnetic beads twice with a washing buffer preheated to 38°C (such as 10 mM Tris(Hydroxymethyl)AminomethaneHydrochloride, abbreviated as Tris-HCl) to remove non-specifically adsorbed high-abundance proteins.

[0060] 3. Low-temperature elution At room temperature (25°C), urea buffer (prepared by dissolving urea in Tris-HCl buffer), aqueous solutions of DTT and IAA were added to the magnetic beads to perform unfolding, reduction of protein disulfide bonds and alkylation, followed by magnetic separation, and the supernatant was collected.

[0061] 4. Enzymatic hydrolysis Add 2 μg of trypsin to the eluent. Place the reaction system in a constant temperature shaker at 37°C and react at 800 rpm for 12 hours.

[0062] 5. Peptide recovery After the reaction was completed, direct magnetic separation was performed, and the supernatant was collected. The supernatant was desalted using a C18 StageTip and then vacuum evaporated for analysis.

[0063] Comparative Example 1: Synthesis of Fe3O4-PNIPAM 100 mg of Fe3O4-CH=CH2 was redispersed in 50 mL of deionized water and transferred to a three-necked flask. High-purity nitrogen was bubbled through the flask for 30 minutes to remove dissolved oxygen. Under nitrogen protection, 450 mg of NIPAM and 15 mg of MBA were added. Mechanical stirring was started at 300 rpm, and the system was heated to 70°C. 15 mg of initiator was dissolved in 1 mL of water using KPS and injected into the reaction system. The reaction was maintained at 70°C for 6 hours. After the reaction, the mixture was washed five times with deionized water at 30°C using a magnetic separator, and then freeze-dried to obtain the temperature-sensitive magnetic beads Fe3O4-PNIPAM.

[0064] Comparative Example 2: The temperature-sensitive magnetic beads prepared in Example 1 of this invention were eluted using conventional high-temperature elution. The mass spectrometry pretreatment of Example 2 was performed, except that the elution temperature in step 2 was 38°C.

[0065] Comparative Example 3: Polymer-free magnetic beads (Fe3O4) for protein adsorption and elution The preparation method is the same as in Example 1. The same pretreatment process as in Example 2 is then performed before mass spectrometry.

[0066] Comparative Example 4: Temperature-sensitive magnetic beads used for protein adsorption and elution Proteins were enriched using magnetic beads made from Fe3O4 modified with poly(dimethylaminopropylmethacrylamide) (Fe3O4-PDMAPMA) and Fe3O4 modified with polyethylene glycol (Fe3O4-PEGOMA) (see patent CN113412274A). The pre-treatment for mass spectrometry as described in Example 2 was then performed.

[0067] Synthesis of reference material Fe3O4-PDMAPMA 20 mg of vinyl magnetic spheres (Fe3O4-CH=CH2) were uniformly dispersed in 50 mL of deionized water. After bubbling under nitrogen for 30 minutes, 0.4 g of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) and 0.04 g of divinylbenzene (DVB) were added to the Fe3O4-CH=CH2 suspension under nitrogen protection. The resulting mixture was heated to 75°C, and 8 mg of ammonium persulfate (APS) dissolved in 5 mL of deionized water was added. The mixture was stirred overnight at 75°C. After cooling, Fe3O4-DMAPMA was separated using a magnet and washed three times with water. The final product was stored in an ethanol solution.

[0068] Synthesis of reference material Fe3O4-PEGOMA 20 mg of vinyl magnetic spheres (Fe3O4-CH=CH2) were uniformly dispersed in 50 mL of deionized water. After bubbling under nitrogen for 30 minutes, 0.4 g of oligo(ethylene glycol) methyl ether methacrylate (OEGMA, molecular weight 500 Da) and 10 mg of N,N'-methylenebisacrylamide (MBA) were added to the Fe3O4-CH=CH2 suspension under nitrogen protection. The resulting mixture was heated to 75°C, and 10 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA) dissolved in 5 mL of ethanol was added, followed by stirring overnight at 75°C. After cooling, Fe3O4-PEGOMA was separated using a magnet and washed three times with water. The final product was stored in an ethanol solution.

[0069] Example 3: Mass Spectrometry Detection Conditions and Data Analysis Testing conditions: After the sample was evaporated to dryness, peptide quantification was performed. Then, a certain amount of ultrapure water was added to reconstitute the sample according to the concentration. After high-speed centrifugation, the supernatant was collected and loaded with a sample volume of 10 μL. A Bruker timsTOF ion mobility high-resolution time-of-flight mass spectrometer was used, with a CaptiveSpray connected to a nanoElute LC. The HPLC conditions were: C18 reversed-phase column (1.7 μm, 75 μm × 15 cm). The mobile phases A and B were 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile, respectively.

[0070] Data Analysis: The ".d" files generated by the tempsTOF HT mass spectrometer were downloaded using MaxQuant software, selecting the Human Protein Database (Taxonomy: Human), and then using FASTA format reference proteins. The raw data were compared with the UniProt database to identify peptides and proteins. Furthermore, a machine learning model was used to perform statistical analysis on the tested plasma samples.

[0071] Example 4: Analysis of the detection quantity of low-abundance proteins in the present invention and comparison magnetic beads (pure PNIPAM) To verify the displacement enhancement effect of the "viscoelastic fluid interface" constructed under the adsorption condition of 38℃ in this invention on low-abundance proteins, the mass spectrometry data of the temperature-sensitive magnetic beads obtained in Example 1 of this invention and the control magnetic beads (pure PNIPAM) were compared after processing liver cancer samples under the same conditions. Figure 3As shown, at 38°C (slightly above LCST), the surface of the temperature-sensitive magnetic beads in Example 1 of this invention did not form a dense, glassy hard shell like pure PNIPAM. Instead, it remained in a "viscoelastic fluid" state thanks to the introduction of OEGMA side chains. This flexible interface lowers the protein adsorption energy barrier, allowing low-abundance tumor markers with stronger affinity to dynamically replace non-specifically adsorbed high-abundance proteins through the "Vroman effect," which is significantly superior to the contrast magnetic beads with stronger interface rigidity.

[0072] Example 5: Analysis of the number of low-abundance proteins eluted by low-temperature and high-temperature elution methods using temperature-sensitive magnetic beads prepared in Example 1 of this invention. To verify the contribution of the "micromechanical ejection" generated by the explosive swelling of polymer chains under low-temperature conditions to residue removal, the detection of low-abundance proteins using the temperature-sensitive magnetic beads of Example 1 of this invention was compared between "low-temperature elution (25°C)" and "high-temperature elution (maintaining 38°C with reagents only)". Mass spectrometry quantitative analysis showed ( Figure 4 The number of low-abundance proteins detected in the 25°C low-temperature elution group was significantly higher than that in the high-temperature elution group. This demonstrates that relying solely on chemical reagents (such as urea) is insufficient to completely destroy the strong multi-point interactions between low-abundance proteins and magnetic beads. In contrast, this invention, by cooling to below LCST, utilizes the dramatic conformational transition of polymer chains from a collapsed to an extended state, generating mechanical thrust at the microscopic interface. This "ejects" deeply buried or strongly adsorbed low-abundance proteins into the solution, thereby minimizing elution residue and improving the detection sensitivity of key biomarkers.

[0073] Example 6: Analysis of the number of low-abundance proteins detected by the temperature-sensitive magnetic beads prepared in Example 1 of this invention compared with conventional comparative magnetic beads (without polymer modification and previously reported magnetic beads). To demonstrate the superior performance of the temperature-sensitive magnetic beads in Example 1 of this invention, the low-abundance protein adsorption capacity of the temperature-sensitive magnetic beads prepared in Example 1 of this invention was further compared with that of traditional magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). The temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) prepared in Example 1 of this invention identified an average of 515 plasma proteins per sample, significantly higher than the control group (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). Figure 5 Furthermore, the proportion of low-abundance proteins was 82%, significantly higher than that of the control magnetic bead group (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). Figure 6 ).

[0074] Example 7: Analysis of the detection of key low-abundance proteins in the temperature-sensitive magnetic beads prepared in Example 1 of this invention compared with reported comparative magnetic beads. In-depth analysis of the detected low-abundance proteins revealed that the thermosensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) prepared in Example 1 of this invention could capture 29 liver cancer biomarkers, significantly higher than the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). This demonstrates that the magnetic beads provided by this invention can not only increase the number of low-abundance proteins but also significantly increase the number of cancer-related tumor biomarkers. This further confirms that the synergistic effect of "viscoelastic adsorption" and "ejection elution" significantly enhances the enrichment capacity for trace key proteins compared to traditional magnetic beads. Figure 7 ).

[0075] Example 8: Construction of Machine Learning Models To further verify the improved accuracy of liver cancer detection using the pH-responsive magnetic beads provided in this invention, 200 liver cancer samples were tested using different magnetic beads and randomly divided into training, validation, and test sets in a 7:2:1 ratio to ensure a balanced distribution of liver cancer and healthy samples within each dataset. For the binary classification task of early liver cancer screening, deep learning prediction models integrating biological prior knowledge and attention mechanisms were constructed for the proteomics datasets corresponding to each group of nanomaterials. The model architecture consisted of an input layer, a learnable grouping feature extraction layer, a graph attention enhancement layer, and a classification output layer. The model consists of an input layer that receives preprocessed multidimensional protein abundance features; a learnable grouping feature extraction layer that sets prior constraints based on core biological pathways of liver cancer development, automatically dividing protein features into multiple functional association groups and configuring independent sub-encoders, thereby enhancing the extraction of low-abundance liver cancer biomarkers through parameter sharing while mining local functional associations between proteins; a graph attention enhancement layer that maps features of each functional group to graph nodes, dynamically calculating the association weights between pathways through a multi-head graph attention mechanism, strengthening the core pathway features related to liver cancer pathogenesis, and suppressing non-specific background noise; and finally, a fully connected decoding layer and a sigmoid activation function output the probability of liver cancer in the sample. Focal Loss is used as the loss function to alleviate the sample class imbalance problem during model training. The AdamW optimizer is used, combined with a cosine annealing learning rate adjustment strategy and an early stopping mechanism. Hyperparameter optimization is performed based on validation set performance to avoid overfitting and ensure the model's generalization ability and predictive stability. The accuracy of the established predictive model is evaluated using Receiver Operating Characteristic (ROC) curves and confusion matrix analysis.

[0076] like Figure 8As shown, the model obtained from the detection data of the magnetic beads (Fe3O4-PNIPAM-PAM-POEG) provided by this invention achieved an AUC (Area Under Curve) value of 0.9294 in the training set. The AUC values ​​of this model are superior to those of the control magnetic bead (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA) group, demonstrating excellent diagnostic performance. This proves the application potential of this invention in cancer detection.

[0077] Example 9: Plasma sample testing of patients with advanced disease The plasma of 40 randomly selected patients with advanced liver cancer was analyzed. Figure 9 As shown, the model obtained from the detection data of the temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) provided by this invention has a positive prediction value of 95.23% and a negative prediction value of 94.73%, which is significantly better than the prediction accuracy of the comparison magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). The temperature-sensitive magnetic beads of this invention are significantly higher than the comparison magnetic beads group, which proves that this invention has excellent cancer detection accuracy.

[0078] Example 10: Detection of plasma samples from early-stage cancer patients The plasma of 40 patients with early-stage liver cancer was tested. Figure 10 As shown, the positive predictive value of the model obtained from the detection data of the temperature-sensitive magnetic beads (Fe3O4-PNIPAM-PAM-POEG) provided by this invention is 94.44%, and the negative predictive value is 94.73%, which is significantly better than the prediction accuracy of the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). The temperature-sensitive magnetic beads of this invention are significantly higher than the control magnetic beads group, which proves that this method has great application potential in early cancer screening.

[0079] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A temperature-sensitive magnetic bead, characterized in that, The temperature-sensitive magnetic bead comprises a magnetic core and a temperature-sensitive copolymer layer modified on the surface of the magnetic core, wherein: The magnetic core comprises magnetite particles modified with vinyl groups, and the particle size of the magnetic core is 50 nm to 500 nm. The temperature-sensitive copolymer layer is a polymer layer modified on the surface of the magnetic core by free radical polymerization, wherein: The thermosensitive copolymer layer is formed by using the vinyl group-modified iron oxide particles as the initiating matrix to initiate the free radical copolymerization reaction of the following monomers: N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate, wherein the initiator used in the free radical copolymerization reaction is potassium persulfate or azobisisobutylamidine hydrochloride, and the crosslinking agent is N,N'-methylenebisacrylamide.

2. The temperature-sensitive magnetic bead as described in claim 1, characterized in that, The vinyl group-modified iron oxide particles are prepared by coupling iron oxide nanoparticles with propyl 3-(trimethoxysilyl)methacrylate, wherein the mass ratio of iron oxide to propyl 3-(trimethoxysilyl)methacrylate is 1:(1.0~3.0).

3. The temperature-sensitive magnetic bead as described in claim 1 or 2, characterized in that, The molar ratio of N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate is (20~70):(0.5~5):(0.5~3), and the total concentration of N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate is 20 mg / mL~50 mg / mL.

4. The temperature-sensitive magnetic bead as described in claim 3, characterized in that, The molar ratio of N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate is (30~60):(1~3):(1~2), and the total concentration of N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate is 20 mg / mL~25 mg / mL.

5. The temperature-sensitive magnetic bead as described in claim 1 or 2, characterized in that, The amount of potassium persulfate or azobisisobutylamidine hydrochloride is 1% to 2% of the total mass of the monomers, and the amount of N,N'-methylenebisacrylamide is 1% to 3% of the total molar mass of the monomers.

6. The temperature-sensitive magnetic bead as described in claim 1 or 2, characterized in that, The number average molecular weight of the oligo(ethylene glycol) methyl ether methacrylate is Mn = 200 Da ~ 2000 Da.

7. The temperature-sensitive magnetic bead as described in claim 6, characterized in that, The average particle size of the temperature-sensitive magnetic beads is 100 nm to 500 nm, and the number average molecular weight of the oligo(ethylene glycol) methyl ether methacrylate is 500 Da to 800 Da.

8. The temperature-sensitive magnetic bead as described in claim 1 or 2, characterized in that, The lower critical melting temperature of the thermosensitive copolymer layer is 35.5℃~36.5℃.

9. A method for preparing the temperature-sensitive magnetic bead according to any one of claims 1-8, characterized in that, This includes magnetic core synthesis, surface vinylization modification, in-situ copolymerization of the temperature-sensitive layer, and purification steps. The magnetic nucleus synthesis step includes: dissolving ferric chloride hexahydrate, trisodium citrate, and anhydrous sodium acetate in ethylene glycol and reacting them in a high-temperature and high-pressure reactor; then, ultrasonically washing the product with alternating ethanol and deionized water at least three times until the supernatant is colorless, collecting it through magnetic separation, and vacuum drying to obtain iron(III) oxide nanoparticles. The surface vinylization modification step includes: dispersing the iron oxide nanoparticles in a mixed solvent of ethanol, water, and concentrated ammonia, wherein the volume ratio of ethanol, water, and concentrated ammonia is 40:1:1; ultrasonically dispersing to achieve uniform dispersion; and adding a vinyl-containing silane coupling agent for coupling; after the reaction is complete, magnetic separation is performed, and the particles are washed with ethanol to remove physically adsorbed silane hydrolysates, yielding vinylized magnetic beads with double bonds on their surface. The in-situ copolymerization reaction step of the temperature-sensitive layer includes: Dispersion: The obtained vinylated magnetic beads were dispersed in deionized water, and nitrogen gas was bubbled through to remove dissolved oxygen; Feeding: Under nitrogen protection, add monomers N-isopropylacrylamide, acrylamide and oligo(ethylene glycol) methyl ether methacrylate in sequence, with the total concentration of monomers controlled at 20 mg / mL~50 mg / mL; Crosslinking and initiation: Add crosslinking agent N,N'-methylenebisacrylamide, wherein the amount of crosslinking agent is 1%~3% of the total molar amount of monomer; heat to 70℃, add initiator potassium persulfate or azobisisobutylamidine hydrochloride, wherein the amount of initiator is 1%~2% of the total mass of monomer, and react for 4~6 hours under stirring; The purification step includes: after the reaction is completed, magnetic separation, repeated washing with deionized water at a temperature lower than the low critical dissolution temperature of the obtained temperature-sensitive magnetic beads to remove unreacted monomers and free polymers, and freeze-drying for storage.

10. The preparation method according to claim 9, characterized in that, The silane coupling agent is propyl 3-(trimethoxysilyl)methacrylate.

11. The preparation method according to claim 9, characterized in that, In the feeding step, the total concentration of monomers N-isopropylacrylamide, acrylamide, and oligo(ethylene glycol) methyl ether methacrylate is controlled at 20 mg / mL to 25 mg / mL; in the crosslinking and initiation step, the amount of N,N'-methylenebisacrylamide is 2% of the total molar amount of monomers, and the initiator is potassium persulfate, with the amount of initiator being 2% of the total mass of monomers.

12. A method for processing low-abundance plasma proteins for early cancer screening, characterized in that, include: Using the temperature-sensitive magnetic beads according to any one of claims 1-8, the temperature-sensitive magnetic beads are brought into contact with a diluted plasma sample under conditions higher than the lower critical dissolution temperature of the temperature-sensitive magnetic beads to perform protein adsorption. Under conditions below the lower critical dissolution temperature, the temperature-sensitive magnetic beads that adsorb low abundance proteins in plasma are used for protein elution, allowing the proteins to be eluted from the surface of the temperature-sensitive magnetic beads and enter the solution phase; trypsin is added to the solution phase for enzymatic hydrolysis, and peptides are recovered. The adsorption temperature during the protein adsorption process is 37℃~39℃ and / or the elution temperature during the protein elution process is 20℃-30℃.

13. The processing method as described in claim 12, characterized in that, The adsorption temperature is 38°C, the elution temperature is 25°C to 30°C, and the protein is eluted with urea buffer, dithiothreitol aqueous solution and iodoacetamide aqueous solution.

14. A kit for enriching low-abundance plasma proteins for early cancer screening, characterized in that, It includes the temperature-sensitive magnetic bead according to any one of claims 1-8.

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