Nanometer magnetic bead based on polyamino acid material, preparation method of nanometer magnetic bead and application of nanometer magnetic bead in early cancer screening

Abstract

The invention discloses a nano magnetic bead based on a polyamino acid material, a preparation method of the nano magnetic bead and application of the nano magnetic bead in early cancer screening, the nano magnetic bead takes a superparamagnetic bead with the surface modified with amino as an initiation center, glutamic acid (Glu) and lysine (Lys) are initiated to be copolymerized on the surface of the magnetic bead in a ring-opening polymerization (ROP) mode, and the nano magnetic bead is prepared. And a polyamino acid brush layer with pH response capability is formed. By utilizing the protonation / deprotonation characteristic of the side chain of the polyamino acid, the magnetic beads are in a semi-collapsed gel state under the condition of low pH (preferably 4.5), and low-abundance protein in plasma can be efficiently adsorbed; under the condition of high pH (preferably 8.5), the magnetic beads are converted into a full-hydrophilic strong-negative-electricity stretching state, and multi-point interaction between the protein and the magnetic beads is synchronously weakened by utilizing strong electrostatic repulsive force, so that the protein is promoted to be eluted from the surfaces of the magnetic beads.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a type of magnetic nanobead based on polyamino acid materials, 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 early changes at the molecular level, which typically precede the appearance of obvious clinical symptoms. Therefore, developing technologies capable of reliable detection in the early stages of disease is of great importance for improving early cancer diagnosis rates, enhancing patient prognosis, and reducing the social burden of healthcare.

[0003] Currently, clinical methods for tumor detection mainly include imaging examinations, histopathological analysis, and serological biomarker detection. Imaging methods have limited sensitivity to early, small lesions and molecular-level changes; while tissue biopsies provide direct histological information, they are highly invasive, have limited sampling capabilities, and are difficult to repeat; traditional serological biomarkers are limited in variety and often lack sufficient sensitivity and specificity in the early stages, making it difficult to meet the needs of large-scale early cancer screening. Against this backdrop, liquid biopsy, with its advantages of being minimally invasive and allowing for repeatable sampling, is gradually becoming an important development direction for early tumor detection.

[0004] Among the various analytes in liquid biopsy, circulating plasma proteins directly participate in and reflect the biological processes of tumor development and progression, and are considered a highly promising source of tumor biomarkers. Mass spectrometry-based proteomics technology enables high-throughput identification and quantitative analysis of proteins in complex samples, offering significant advantages in coverage and information depth, and is therefore considered an important technical pathway for achieving refined analysis in liquid biopsies. However, high-abundance proteins in the plasma system account for the vast majority of protein mass, significantly masking the detection signals of low-abundance, tumor-related proteins. This makes the effective enrichment of low-abundance proteins a core technical bottleneck for applying mass spectrometry proteomics to early cancer screening.

[0005] To address the aforementioned issues, existing low-abundance protein enrichment technologies primarily employ magnetic bead-based adsorption and separation strategies, resulting in a variety of commercially available kits. These kits typically rely on non-specific or semi-specific interactions between the hydrophobic, charged, or affinity ligands on the magnetic bead surface and the protein to achieve adsorption and enrichment of plasma proteins. However, in practical applications, different proteins often simultaneously adsorb onto the magnetic bead surface, forming complex protein crown structures. Protein molecules are usually highly stable bound to the solid-phase interface through multiple weak interaction sites. Existing magnetic bead materials lack a "smart response" mechanism; their surface properties remain static during adsorption and elution. This leads to a core contradiction: capturing low-abundance proteins often requires strong adsorption forces, but these strong interactions (especially multi-site adsorption) become obstacles during the elution stage. Existing magnetic bead systems rely on denaturants, salt concentrations, or surfactants to gradually weaken protein-magnetic bead interactions during the elution phase. This makes it difficult to achieve the simultaneous dissociation of multiple interactions within a limited time, resulting in some proteins, especially some key characteristic low-abundance proteins, remaining on the surface of the magnetic beads after elution. This leads to the systematic loss of key low-abundance proteins at the detection level, limiting the ability of mass spectrometry proteomics to capture and detect early molecular signals in tumors.

[0006] Therefore, how to introduce intelligent switches that can respond to the environment, starting from the magnetic bead material itself, and achieve controllable switching of protein adsorption and desorption processes by regulating its interface properties, thereby minimizing residues and increasing the relative proportion of low-abundance proteins in the eluted proteins, has become a key technical problem that urgently needs to be solved in the field of liquid biopsy proteomics. Summary of the Invention

[0007] This invention discloses a nanomagnetic bead based on polyamino acid material, its preparation method, and its application in early cancer screening.

[0008] In some implementations, the nanomagnetic beads are initiated by superparamagnetic beads with amino-modified surfaces. Glutamic acid and lysine are grafted onto their surfaces via ring-opening polymerization to form a polyamino acid brush layer, thus constructing an intelligent enrichment system of "acid trapping-alkaline release". On the one hand, utilizing the natural pH-smart switching properties of polyglutamic acid, a unique "semi-collapsed gel state" is constructed in an acidic environment of around pH 4.5: the retained protonated carboxyl groups provide the necessary hydrophobic anchors, while the dissociated carboxyl groups, together with the introduced lysine side chains, support a three-dimensional microgel structure with suitable pore size through inter-chain salt bridges. This structure avoids the steric hindrance of dense "glassy state" under strong acid conditions, achieving efficient "three-dimensional volume adsorption" of low-abundance plasma proteins. By introducing lysine units to adjust the monomer ratio and utilizing the polarity of lysine to precisely control interfacial properties, it effectively weakens the non-specific adhesion of high-abundance albumin at pH 4.5, achieving a synergistic effect of high capacity and high background removal. On the other hand, by utilizing the side chain deprotonation of polyamino acid chains at pH 8.5, the strong electrostatic repulsion force generated by the dense negative charge on the surface is used to completely overcome the residual adsorption of proteins by lysine side chains, actively and residuelessly stripping the adsorbed proteins. Moreover, this elution system has an "enzymatic hydrolysis buffer reserve" function, which can be directly compatible with downstream enzymatic hydrolysis processes without additional alkali adjustment.

[0009] In some implementations, the reversible protonation behavior and conformational changes of the polyamino acid side chains of the pH-responsive magnetic beads promote protein elution, effectively reducing low-abundance protein residue and increasing the proportion of low-abundance proteins in the eluted protein. This invention helps 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.

[0010] To achieve the above objectives, the present invention includes the following aspects: On one hand, the present invention provides a nanomagnetic bead based on polyamino acid material, comprising a magnetic core and a polyamino acid copolymer layer modified on the surface of the magnetic core. The magnetic core contains superparamagnetic iron oxide (sometimes called Fe3O4) nanoparticles with amino functional groups on their surface; the polyamino acid copolymer layer is modified on the surface of the magnetic core by surface-initiated ring-opening polymerization of glutamic acid structural units and lysine structural units. The magnetic nanobeads exhibit pH-responsive properties: under pH conditions of 4.2–4.8, the polyamino acid copolymer layer of the magnetic nanobeads adsorbs proteins; under pH conditions of 8.3–8.7, the polyamino acid copolymer layer releases proteins. Preferably, in the polyamino acid copolymer layer, the molar ratio of glutamic acid structural units to lysine structural units is 3:1 to 8:1.

[0011] In some embodiments, the magnetic core consists of or is substantially composed of superparamagnetic iron oxide nanoparticles with amino functional groups on their surface.

[0012] In some embodiments, the present invention provides a nanomagnetic bead based on polyamino acid materials for enriching low-abundance proteins in plasma, comprising a magnetic core and a polyamino acid copolymer layer modified on the surface of the magnetic core, wherein the magnetic core is a superparamagnetic iron oxide nanoparticle with amino functional groups on its surface; the polyamino acid copolymer layer is composed of glutamic acid (Glu) structural units and lysine (Lys) structural units and is modified on the surface of the magnetic core by surface-initiated ring-opening polymerization. The nano-magnetic beads exhibit pH-responsive characteristics: Under acidic conditions, preferably pH 4.2–4.8, the polyamino acid copolymer layer is in a partially protonated state, in equilibrium with partial dissociation, with hydrogen bonds and salt bridges forming between the side chains, exhibiting a semi-collapsed gel conformation to adsorb proteins through a three-dimensional volume effect; Under alkaline conditions, preferably pH 7.5–8.5, the polyamino acid copolymer layer undergoes complete deprotonation, exhibiting a fully hydrophilic, strongly negatively charged unfolded conformation, releasing proteins through electrostatic repulsion.

[0013] Furthermore, in the polyamino acid copolymer layer, the molar ratio of glutamic acid structural units to lysine structural units is 3:1 to 8:1, preferably 6:1. This specific ratio is used to reduce non-specific binding to high-abundance albumin while maintaining high adsorption capacity, utilizing the charge shielding effect of the lysine side chain.

[0014] Further, the average particle size of the magnetic core is 100 nm to 400 nm, preferably 200 nm to 300 nm, and the mass of the polyamino acid copolymer layer is 10% to 30% of the total mass of the magnetic beads, preferably 15% to 25%. Preferably, the average particle size of the magnetic core is 200 nm to 300 nm, and the mass is 15% to 25%.

[0015] Furthermore, the polyamino acid copolymer layer is a polymer brush formed by surface-initiated ring-opening polymerization (ROP) using the amino groups on the surface of the magnetic core as initiation points. Its average degree of polymerization (DP) is 50–200, preferably 80–150. This range of polymerization ensures that the polymer brush layer has sufficient thickness to form an effective three-dimensional trapping space.

[0016] On the other hand, the present invention provides a method for preparing the above-mentioned plasma low-abundance protein enrichment magnetic beads based on polyamino acid materials, comprising the following steps: (1) Preparation of aminated magnetic cores: Superparamagnetic nanoparticles were synthesized, dispersed in a solvent, and surface modifiers were added. The mixture was reacted at 50℃~90℃, washed and dried to obtain aminated magnetic beads with rich initiation sites on the surface. (2) Polyamino acid modification: Under anhydrous and oxygen-free conditions, aminated magnetic beads are dispersed in an aprotic polar solvent. Using the amino groups on the surface of the aminated magnetic beads as initiators, surface-initiated ring-opening polymerization (sometimes called ROP) of glutamic acid-N-carboxylic acid anhydride (sometimes called Glu-NCA) and lysine-N-carboxylic acid anhydride (sometimes called Lys-NCA) monomers is initiated to obtain polyamino acid modified magnetic beads. (3) Deprotection and purification: The polyamino acid modified magnetic beads were dispersed in a deprotection reagent to remove the protecting groups of the amino acid side chains. After magnetic separation, washing to neutrality and freeze drying, nano-magnetic beads were obtained.

[0017] Furthermore, the surface modifier is selected from amino-containing silane coupling agents.

[0018] Preferably, the amino-containing silane coupling agent is 3-aminopropyltriethoxysilane (APTES) or 3-aminopropyltrimethoxysilane (APTMS).

[0019] More preferably, in step (1), the reaction solvent is a mixture of ethanol and water (volume ratio 1:1 to 10:1), and the reaction time is 6 to 12 hours.

[0020] Preferably, the aprotic polar solvent in step (2) is N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Preferably, the reaction temperature is 25℃~40℃, and the reaction time is 24~48 hours. Preferably, the molar ratio of Glu-NCA to Lys-NCA is 4:1~8:1 (preferably 4:1~8:1), and the total monomer concentration is 0.1 M~0.5 M.

[0021] Preferably, the deprotecting agent in step (3) is a hydrogen bromide solution, preferably a 33 wt% hydrobromic acid solution dissolved in acetic acid.

[0022] Another aspect of the present invention provides a method for processing low-abundance plasma proteins for mass spectrometry detection. The method includes contacting the nanomagnetic beads of the present invention or the nanomagnetic beads prepared by the method of the present invention with plasma at an acidic pH, preferably pH 4.2-4.8, more preferably pH 4.5, to adsorb proteins, and then eluting the adsorbed proteins from the surface of the magnetic beads with an alkaline solution at a pH, preferably pH 8.3-8.7, more preferably pH 8.5. The eluent at pH 8.5, upon subsequent addition of acidic or reducing reagents, can act as a buffer to maintain the system within the appropriate enzymatic digestion range of around pH 8.0.

[0023] Another aspect of the present invention provides a kit for enriching low-abundance plasma proteins for early cancer screening, comprising magnetic nanobeads of the present invention or magnetic nanobeads prepared by the method of the present invention, a pH adjuster, and instructions for use. Preferably, the pH adjuster is an acid, a base, or a buffer. The buffer includes a capture buffer at pH 4.5 and an elution buffer at pH 8.5.

[0024] Another aspect of the present invention relates to the use of the magnetic nanobeads of the present invention or the magnetic nanobeads prepared by the preparation method of the present invention or the kit of the present invention in the enrichment of low-abundance plasma proteins for mass spectrometry detection.

[0025] Another aspect of the present invention provides a method for processing peptides of low-abundance plasma proteins for early cancer screening. The method includes contacting nanomagnetic beads from the kit of the present invention with plasma at an acidic pH, preferably pH 4.2-4.8, more preferably pH 4.5, to adsorb proteins; eluting the adsorbed proteins from the surface of the magnetic beads with an alkaline pH, preferably pH 8.3-8.7, more preferably pH 8.5; and directly performing unfolding, disulfide bond reduction, and alkylation treatments, followed by enzymatic hydrolysis to recover the peptides. The enzymatic hydrolysis step does not require additional pH adjustment or desalting steps.

[0026] Another aspect of the present invention provides a method for early cancer screening, which includes mass spectrometry detection of the peptides prepared in the present invention and cancer prediction using a machine learning model on the mass spectrometry data.

[0027] Another aspect of the present invention provides a system for early cancer screening, comprising the kit of the present invention, a mass spectrometer, and a device for predicting cancer from mass spectrometry data based on a machine learning model.

[0028] In one embodiment of the invention, the device for cancer prediction is a computer, and the machine learning model is deployed locally on the computer or online.

[0029] The positive advancements of this invention are: Compared with the prior art, the present invention has at least the following beneficial effects: 1. In some embodiments, the present invention constructs a unique pH 4.5 semi-collapsed gel interface, utilizing the pH response characteristics of polyamino acid materials and a specific monomer ratio to achieve adsorption of low-abundance proteins with both high capacity and high specificity. 2. In some embodiments, the present invention utilizes the threshold repulsion force of pH 8.5 to overcome the charge hysteresis effect of lysine, thereby achieving the release of adsorbed proteins and significantly increasing the number of low-abundance proteins detected by mass spectrometry. 3. In some embodiments, the present invention establishes a buffer reserve system that is seamlessly integrated with the downstream enzymatic hydrolysis process, which simplifies the operation process and improves the accuracy of early cancer detection. Attached Figure Description

[0030] Figure 1 This invention presents a schematic diagram of the structure of pH-responsive magnetic nanobeads (Fe3O4-PLG-PLL) and a schematic diagram of the "acidic capture-alkaline release" mechanism. PLG stands for poly(L-Glutamic acid), and PLL stands for poly(L-Lysine).

[0031] Figure 2 The hydration particle size variation curves of the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention at different pH values ​​(pH 3.0~10.0).

[0032] Figure 3 A comparison of the adsorption capacity of magnetic beads for high-abundance albumin at different glutamic acid / lysine (Glu / Lys) feed ratios (10:0, 8:1, 6:1, 4:1, 2:1).

[0033] Figure 4 A comparison of the number of proteins desorbed by the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention at different elution pH (pH 7.5, 8.0, 8.5, 9.0).

[0034] Figure 5 This invention compares the protein levels detected after elution in liver cancer samples using the nanomagnetic beads (Fe3O4-PLG-PLL) and control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). PEGOMA is polyethylene glycol methyl ether methacrylate; PDMAPMA is poly(dimethylaminopropyl methacrylamide).

[0035] Figure 6This paper compares the detection of low-abundance proteins in liver cancer samples after elution using the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention and the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA).

[0036] Figure 7 This invention compares the detection levels of key low-abundance proteins in liver cancer samples after elution using the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention with those of control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA).

[0037] Figure 8 The ROC curves of the present invention's nanomagnetic beads (Fe3O4-PLG-PLL) and the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA) for cancer detection in liver cancer samples are compared.

[0038] Figure 9 Confusion matrix for cancer detection in advanced liver cancer samples using the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention.

[0039] Figure 10 Confusion matrix for early liver cancer sample detection using the nanomagnetic beads (Fe3O4-PLG-PLL) of this invention. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] 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.

[0043] This invention discloses a nanomagnetic bead based on polyamino acid materials for enriching low-abundance proteins in plasma, its preparation method, and its application in early cancer screening. The nanomagnetic beads use superparamagnetic beads with amino-modified surfaces as the initiation center. Through ring-opening polymerization (ROP), Glu and Lys are grafted and copolymerized on the surface of the superparamagnetic beads, forming a polyamino acid brush layer with pH-sensitive reversible protonation switching capability. This invention utilizes the protonation / deprotonation properties of the polyamino acid side chains to achieve a smart switching function of "low pH (preferably 4.5) semi-collapsed gel capture and high pH (preferably 8.5) hydrophilic strong negatively charged release." This mechanism not only efficiently enriches low-abundance proteins in plasma using a three-dimensional trapping effect, but also allows the polyamino acid side chains to rapidly transform into a fully hydrophilic and extended state at the release pH environment (pH 8.5). The strong electrostatic repulsion overcomes the charge hysteresis effect of the lysine side chains, simultaneously weakening the multi-point interaction between the protein and the nanomagnetic beads, thereby promoting the elution of proteins from the surface of the nanomagnetic beads. Compared to conventional magnetic beads (i.e., contrast magnetic beads), the pH-responsive magnetic beads of the present invention effectively reduce the residue of low-abundance proteins during the elution process, increase the proportion of low-abundance proteins in the eluted proteins, and help improve the detection quantity of low-abundance proteins based on plasma proteomics and the accuracy of early cancer detection. The present invention will be further illustrated below with reference to embodiments.

[0044] Example

[0045] Example 1: Preparation of polyamino acid pH-responsive magnetic beads (Fe3O4-PLG-PLL) (1) Synthesis of Aminated Magnetic Beads 200 mg of iron(III) oxide was ultrasonically dispersed in 105 mL of a mixed solvent of ethanol, water, and concentrated ammonia (volume ratio 100:2.5:2.5). 0.4 mL of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was refluxed at 70 °C for 8 hours. Magnetic separation was performed, and the mixture was washed five times with ethanol to obtain amino-modified magnetic beads (Fe3O4-NH2).

[0046] (2) Modification of the glutamic acid / lysine copolymer layer (NCA method) 1. Monomer preparation: Purchase or synthesize γ-benzyl-L-glutamic acid-N-carboxylic anhydride (BLG-NCA) and ε-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride (ZLL-NCA).

[0047] 2. Polymerization reaction: 100 mg Fe3O4-NH2 was dispersed in anhydrous DMF. BLG-NCA and ZLL-NCA were added at a molar ratio of Glu:Lys = 6:1 (total monomer mass to magnetic bead mass ratio of 1:1). The reaction was stirred at room temperature for 48 hours, and ring-opening polymerization was initiated by the amino groups on the surface of the magnetic beads.

[0048] 3. Deprotection: The product was dispersed in a 33 wt% hydrogen bromide solution and stirred for 1 hour to remove the side chain protecting groups (benzyl and benzyloxycarbonyl) and restore the carboxyl group of glutamic acid and the amino group of lysine.

[0049] 4. Purification: Wash repeatedly with deionized water until neutral, freeze dry to obtain Fe3O4-PLG-PLL magnetic beads.

[0050] Example 2: Mass Spectrometry Detection Conditions and Data Analysis Methods 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 the sample at a 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.

[0051] Data Analysis: Using MaxQuant software, the ".d file" generated by the tempsTOF HT mass spectrometer was downloaded as a FASTA format reference protein database (Taxonomy: Human). The raw data was then compared with the UniProt database to identify peptides and proteins.

[0052] Example 3: Optimization of key process parameters and characterization of physical properties 1. Characterization of pH-responsive conformation The Fe3O4-PLG-PLL magnetic beads prepared in Example 1 were dispersed in buffer solutions of different pH values ​​(3.0~10.0), and their hydrated particle size was measured. The results are as follows: Figure 2 As shown, at pH 4.5, the magnetic bead size is approximately 198 nm, significantly larger than the dense glassy state at pH 3.0 and smaller than the fully expanded state (360 nm) at pH 8.5, confirming the existence of a "semi-collapsed gel state," which is most conducive to protein capture.

[0053] 2. Screening of monomer feed ratio To determine the optimal monomer ratio, a series of magnetic beads were prepared by varying the Glu:Lys feed ratio (10:0, 8:1, 6:1, 4:1, 2:1, respectively) according to the method in Example 1. The adsorption amount of high-abundance albumin in the eluent was then determined using the mass spectrometry detection method in Example 2. The results are as follows: Figure 3 As shown, the non-specific adsorption of high-abundance albumin first decreases and then increases with the increase of the lysine ratio. The adsorption of high-abundance albumin reaches its lowest point at a Glu:Lys ratio of 6:1, demonstrating that this ratio effectively utilizes the charge-shielding effect of lysine to suppress background interference, and is therefore determined to be the optimal ratio.

[0054] 3. Determination of the elution pH threshold Magnetic beads adsorbed with plasma proteins were eluted in buffer solutions at pH 7.5, 8.0, 8.5, and 9.0, respectively. The eluted beads were then subjected to strong denaturation treatment, and the amount of adsorbed protein was determined using the method described in Example 2. The results are as follows: Figure 4 As shown, at pH 8.0, the number of low-abundance proteins adsorbed was 460; while when the pH was increased to 8.5, the number of low-abundance proteins increased significantly to 528, proving that pH 8.5 is the key threshold for overcoming the adsorption of lysine residues.

[0055] Example 4: Protein adsorption and desorption based on optimal pH switch Based on the optimization results of Example 3, the following standard operating procedure was determined for subsequent comparative experiments: 1. Sample binding (pH 4.5) Take 10 μL of human plasma sample and dilute it to 100 μL with 90 μL of binding buffer (100 mM sodium acetate buffer, pH 4.5). Add 100 μg of the magnetic beads prepared in Example 1 (Glu:Lys = 6:1). Incubate at room temperature with shaking for 30 minutes. At this point, the pH 4.5 is close to the pKa of polyglutamic acid, and the polymer chains are in a semi-collapsed gel state, which avoids the steric hindrance of the glassy state under strong acid and provides suitable hydrophobic anchors and three-dimensional pores, thus efficiently capturing proteins in plasma.

[0056] 2. Cleaning Magnetic separation was performed, and the supernatant was discarded. The sample was washed twice with 200 μL of washing buffer at pH 4.5 to remove unbound, high-abundance contaminating proteins.

[0057] 3. High pH elution (pH 8.5) Add 100 μL of buffer solution (50 mM NH4HCO3) directly to the magnetic beads. 3，The pH was adjusted from 4.5 to 8.5 during the process. The polyglutamic acid side chains completely dissociated, carrying a strong negative charge, changing from a "semi-collapsed state" to a "fully extended state." The strong electrostatic repulsion across the entire surface overcame the residual adsorption of the lysine side chains, releasing the encapsulated protein and pushing it away from the surface. The adsorbed protein was rapidly released. Subsequently, urea buffer (UA buffer, prepared by dissolving urea in Tris-HCl buffer), dithiothreitol (DTT), and iodoacetamide (IAA) aqueous solutions were added to unfold the magnetic beads, reduce protein disulfide bonds, and alkylate them. The pH of the system spontaneously returned to approximately 8.0, and magnetic separation was performed. The supernatant was collected.

[0058] 4. Enzymatic hydrolysis After pH adjustment, 2 μg of trypsin was added directly to the elution buffer. Due to the reserve capacity of the aforementioned elution buffer, the system could be maintained within the optimal enzyme activity range without additional alkali adjustment. The reaction system was placed in a constant temperature shaker at 37°C and reacted at 800 rpm for 12 hours.

[0059] 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.

[0060] Comparative Example 1: Polymer-free magnetic beads (Fe3O4) for protein adsorption and elution The preparation method is the same as in Example 1, and the mass spectrometry pretreatment of Example 4 is performed.

[0061] Comparative Example 2: Magnetic beads with no pH response effect used for protein adsorption and elution Proteins were enriched using Fe3O4 magnetic beads modified with poly(dimethylaminopropylmethacrylamide) (Fe3O4-PDMAPMA) and Fe3O4 magnetic beads modified with polyethylene glycol (Fe3O4-PEGOMA) as described in the published patent (see CN 113412274 A), and then subjected to the mass spectrometry pretreatment described in Example 4.

[0062] 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.

[0063] Among them, Fe3O4-CH=CH2 synthesis 250 mg of Fe3O4 was uniformly dispersed under ultrasonic assistance in a mixture of ethanol (100 mL), deionized water (2.5 mL), and concentrated ammonia solution (2.5 mL, 28-30 wt%), followed by the addition of 0.5 mL of tetraethyl orthosilicate (TEOS). After stirring at 70 °C for 6 hours, 0.5 mL of 3-(trimethoxysilyl)methacrylate was added to the reaction mixture, and the mixture was stirred overnight at 70 °C. The product was washed three times with methanol and three times with water to obtain vinyl-functionalized Fe3O4 (Fe3O4-CH=CH2) nanoparticles, which were then stored in ethanol.

[0064] 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(ethyleneglycol) 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.

[0065] Furthermore, the machine learning model, as described in Example 7, was used to perform statistical analysis on the tested plasma samples.

[0066] Example 5: Analysis of the number of low-abundance proteins detected Analysis of liver cancer samples showed that the pH-responsive magnetic beads (Fe3O4-PLG-PLL) prepared in this invention, under optimal conditions (Glu:Lys=6:1, adsorption pH 4.5, elution pH 8.5), identified an average of 528 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 lower than that of the control group (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). Figure 6 This demonstrates that pH-responsive adjustment and monomer ratio optimization can promote the elution of low-abundance proteins and increase their proportion, showcasing the superiority of this invention.

[0067] Example 6: Analysis of the detection of key low-abundance proteins In-depth analysis of the detected low-abundance proteins revealed that the pH-responsive magnetic beads (Fe3O4-PLG-PLL) prepared in this invention could capture 28 liver cancer biomarkers, significantly higher than the control group of 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. Figure 7 ).

[0068] Example 7: Building a Machine Learning Model 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.

[0069] As attached Figure 8 As shown, the model obtained from the detection data of the magnetic beads (Fe3O4-PLG-PLL) provided by this invention achieved an AUC value of 0.9329 in the training set. Its AUC value was superior to that of the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA) group, demonstrating excellent diagnostic performance. This proves the application potential of this invention in cancer detection.

[0070] Example 8: Plasma sample testing of patients with advanced disease Plasma samples from 40 randomly selected patients with advanced liver cancer were analyzed. (See attached image) Figure 9As shown, the positive predictive value of the model obtained from the detection data of the pH-responsive magnetic beads (Fe3O4-PLG-PLL) provided by the present invention is 95.23%, and the negative predictive value is 89.47%, which is significantly better than the prediction accuracy of the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). The pH-responsive magnetic beads of the present invention are significantly higher than the control magnetic beads group, which proves that the present invention has excellent cancer detection accuracy.

[0071] Example 9: Detection of plasma samples from early-stage cancer patients Plasma samples from 40 patients with early-stage liver cancer were analyzed. (See attached image) Figure 10 As shown, the positive predictive value of the model obtained from the detection data of the pH-responsive magnetic beads (Fe3O4-PLG-PLL) provided by this invention is 94.44%, and the negative predictive value is 86.36%, which is significantly better than the prediction accuracy of the control magnetic beads (Fe3O4, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). The pH-responsive 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.

[0072] The above description is merely a specific embodiment 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 technical scope 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 type of magnetic nanobead based on polyamino acid material, characterized in that, The invention includes a magnetic core and a polyamino acid copolymer layer modified on the surface of the magnetic core. The magnetic core comprises superparamagnetic iron oxide nanoparticles with amino functional groups on their surface. The polyamino acid copolymer layer is modified on the surface of the magnetic core by surface-initiated ring-opening polymerization of glutamic acid structural units and lysine structural units. The nanomagnetic beads exhibit pH-responsive characteristics: under pH conditions of 4.2–4.8, the polyamino acid copolymer layer of the nanomagnetic beads adsorbs proteins; under pH conditions of 8.3–8.7, the polyamino acid copolymer layer releases proteins. In the polyamino acid copolymer layer, the molar ratio of glutamic acid structural units to lysine structural units is 3:1 to 8:

1.

2. The nanomagnetic beads as described in claim 1, characterized in that, In the polyamino acid copolymer layer, the molar ratio of glutamic acid structural units to lysine structural units is 6:

1.

3. The nanomagnetic beads as described in claim 1 or 2, characterized in that, The magnetic core has an average particle size of 100 nm to 400 nm, and the mass of the polyamino acid copolymer layer is 10% to 30% of the total mass of the magnetic beads.

4. The nanomagnetic beads as described in claim 3, characterized in that, The magnetic core has an average particle size of 200 nm to 300 nm, and the mass of the polyamino acid copolymer layer is 15% to 25% of the total mass of the magnetic beads.

5. The nanomagnetic beads as described in claim 1 or 2, characterized in that, The polyamino acid copolymer layer is a polymer brush formed by surface-initiated ring-opening polymerization using amino groups on the surface of a magnetic core as initiation points, with an average degree of polymerization of 50-200.

6. The nanomagnetic beads as described in claim 5, characterized in that, The average degree of polymerization is 80~150.

7. A method for preparing the nanomagnetic beads according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of aminated magnetic cores: Superparamagnetic nanoparticles were synthesized, dispersed in a solvent, and surface modifiers were added. The mixture was reacted at 50℃~90℃, washed and dried to obtain aminated magnetic beads with rich initiation sites on the surface. (2) Polyamino acid modification: Under anhydrous and oxygen-free conditions, the aminated magnetic beads are dispersed in an aprotic polar solvent. The amino groups on the surface of the aminated magnetic beads are used as initiators to initiate the surface-initiated ring-opening polymerization reaction of glutamic acid-N-carboxylic acid anhydride and lysine-N-carboxylic acid anhydride monomers to obtain polyamino acid modified magnetic beads. (3) Deprotection and purification: The polyamino acid modified magnetic beads are dispersed in a deprotection reagent to remove the protecting groups of the amino acid side chains. After magnetic separation, washing to neutrality and freeze drying, the nano magnetic beads are obtained.

8. The preparation method according to claim 7, characterized in that, The surface modifier is selected from amino-containing silane coupling agents.

9. The preparation method according to claim 8, characterized in that, The silane coupling agent is selected from 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane.

10. The preparation method according to any one of claims 7-9, characterized in that, The solvent in the preparation step of the amination magnetic core is a mixture of ethanol and water, with a volume ratio of ethanol to water of 1:1 to 10:1, and a reaction time of 6 to 12 hours. In the polyamino acid modification step, the aprotic polar solvent is N,N-dimethylformamide or dimethyl sulfoxide, the reaction temperature is 25℃~40℃, and the reaction time is 24~48 hours. The molar ratio of glutamic acid-N-carboxylic acid anhydride to lysine-N-carboxylic acid anhydride is 4:1 to 8:1, and the total monomer concentration is 0.1 M to 0.5 M; the deprotection reagent includes hydrogen bromide solution.

11. A method for processing low-abundance proteins in plasma, characterized in that, include: The magnetic nanobeads prepared by the preparation method according to any one of claims 7-10 are contacted with plasma at pH 4.2-4.8 to allow the surface of the magnetic nanobeads to be in a semi-collapsed gel state for protein adsorption; the adsorbed proteins are eluted from the surface of the magnetic nanobeads with a solution at pH 8.3-8.7; the eluted proteins are subjected to unfolding, disulfide bond reduction and alkylation treatment, and then enzymatically hydrolyzed to recover peptides.

12. A kit for enriching low-abundance proteins in plasma, characterized in that, It includes the nanomagnetic beads according to any one of claims 1-6.

13. Use of the magnetic nanobeads according to any one of claims 1-6, or the kit according to claim 12, in enriching low-abundance plasma proteins for early cancer screening.

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