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CN-122006677-A - Nano magnetic bead based on polyamino acid material, preparation method thereof and application thereof in early cancer screening

CN122006677ACN 122006677 ACN122006677 ACN 122006677ACN-122006677-A

Abstract

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

Inventors

  • SONG WANTONG
  • WANG DIANWEI
  • WANG HAO
  • CHEN XUESI

Assignees

  • 中国科学院长春应用化学研究所

Dates

Publication Date
20260512
Application Date
20260408

Claims (13)

  1. 1. The nano magnetic bead based on the polyamino acid material is characterized by comprising a magnetic inner core and a polyamino acid copolymer layer modified on the surface of the magnetic inner core, wherein the magnetic inner core comprises superparamagnetic ferroferric oxide nano particles with amino functional groups modified on the surface; the nano magnetic beads have pH response characteristics that the polyamino acid copolymer layer of the nano magnetic beads adsorbs protein under the condition of pH 4.2-4.8, and releases protein under the condition of pH 8.3-8.7, Wherein, in the polyamino acid copolymer layer, the molar ratio of the glutamic acid structural unit to the lysine structural unit is 3:1 to 8:1.
  2. 2. The nanomagnetic bead according to claim 1, wherein the molar ratio of glutamic acid structural units to lysine structural units in the polyamino acid copolymer layer is 6:1.
  3. 3. The nanomagnetic bead according to claim 1 or 2, wherein the average particle diameter of the magnetic core is 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 bead.
  4. 4. The nanomagnetic bead according to claim 3, wherein the average particle diameter of the magnetic core is 200nm to 300 nm, and the mass of the polyamino acid copolymer layer is 15% to 25% of the total mass of the magnetic bead.
  5. 5. The nano magnetic bead according to claim 1 or 2, wherein the polyamino acid copolymer layer is a polymer brush formed by surface-initiated ring-opening polymerization with an amino group on the surface of a magnetic core as an initiation point, and the average polymerization degree is 50-200.
  6. 6. The nanomagnetic bead according to claim 5 wherein the average degree of polymerization is 80 to 150.
  7. 7. A method of preparing the nanomagnetic beads according to any one of claims 1 to 6, comprising the steps of: (1) Synthesizing superparamagnetism nano particles, dispersing the nano particles in a solvent, adding a surface modifier, reacting at 50-90 ℃, washing and drying to obtain the aminated magnetic beads with the surfaces rich in initiation sites; (2) Dispersing the amino magnetic beads in an aprotic polar solvent under anhydrous and anaerobic conditions, and initiating glutamic acid-N-carboxyl cyclic anhydride and lysine-N-carboxyl cyclic anhydride monomers to perform surface-initiated ring-opening polymerization reaction by taking amino groups on the surfaces of the amino magnetic beads as initiators to obtain the magnetic beads modified by the amino acids; (3) And (3) deprotection and purification, namely dispersing the magnetic beads modified by the polyamino acid in a deprotection reagent, removing protecting groups of amino acid side chains, magnetically separating, washing to neutrality, and freeze-drying to obtain the nano magnetic beads.
  8. 8. The method of claim 7, wherein the surface modifier is selected from amino-containing silane coupling agents.
  9. 9. The method of preparing according to claim 8, wherein the silane coupling agent is selected from 3-aminopropyl triethoxysilane or 3-aminopropyl trimethoxysilane.
  10. 10. The preparation method of any one of claims 7 to 9, wherein the solvent in the preparation step of the aminated magnetic core is a mixed solution of ethanol and water, the volume ratio of ethanol to water is 1:1-10:1, and the reaction time is 6-12 hours; The aprotic polar solvent in the polyamino acid modification step is N, N-dimethylformamide or dimethyl sulfoxide, the reaction temperature is 25-40 ℃, and the reaction time is 24-48 hours; The feeding molar ratio of the glutamic acid-N-carboxyl cyclic anhydride to the lysine-N-carboxyl cyclic anhydride is 4:1-8:1, the total concentration of monomers is 0.1M-0.5M, and the deprotection reagent comprises a hydrogen bromide solution.
  11. 11. A method for treating low-abundance proteins in plasma is characterized by comprising the steps of contacting the nano magnetic beads prepared by the preparation method of any one of claims 7-10 with plasma with pH of 4.2-4.8, enabling the surfaces of the nano magnetic beads to be in a semi-collapsed gel state for adsorbing proteins, eluting the adsorbed proteins from the surfaces of the nano magnetic beads by using a solution with pH of 8.3-8.7, carrying out refolding, disulfide bond reduction and alkylation treatment on the eluted proteins, and recovering peptide fragments after enzymolysis.
  12. 12. A kit for enriching plasma low abundance proteins, characterized in that it comprises the nanomagnetic beads according to any one of claims 1-6.
  13. 13. Use of the nanomagnetic beads of any one of claims 1-6, or the kit of claim 12, for enriching low abundance proteins of plasma for early screening of cancer.

Description

Nano magnetic bead based on polyamino acid material, preparation method thereof and application thereof in early cancer screening Technical Field The invention relates to the technical field of biological medicine, in particular to a polyamino acid material-based nano magnetic bead, a preparation method thereof and application thereof in early cancer screening. Background Cancer morbidity and mortality continue to rise worldwide, becoming an important disease type that severely threatens human health. Numerous studies have shown that tumor development and progression is accompanied by early changes at the molecular level, which usually occur earlier than the apparent clinical symptoms. Therefore, development of a technical means capable of realizing reliable detection at an early stage of a disease is of great importance for improving early diagnosis rate of cancer, improving prognosis of a patient and reducing social medical burden. Currently, methods for clinical tumor detection mainly include imaging, histopathological analysis, and serological marker detection. The imaging method has limited sensitivity to early micro focus and molecular level change, the tissue biopsy can provide direct histological information, but has the defects of strong invasiveness, limited sampling, difficult repeated implementation and the like, while the traditional serological markers have limited types, and have insufficient sensitivity and specificity at the early stage, so that the requirement of large-scale early cancer screening is difficult to meet. In this context, liquid biopsies with the advantage of minimally invasive, resamplable are becoming an important development for early detection of tumors. Among the various analytical subjects of liquid biopsies, plasma circulating proteins are directly involved in and reflect the biological processes of tumorigenic development and are considered as a source of tumor biomarkers with great potential for application. Mass spectrometry-based proteomics technology can perform high-throughput identification and quantitative analysis on proteins in complex samples, has remarkable advantages in terms of coverage and information depth, and is considered as an important technical path for realizing liquid biopsy refinement analysis. However, the high-abundance proteins in the plasma system occupy most of the protein mass, and the detection signals of low-abundance and tumor-related proteins are obviously covered, so that the effective enrichment of the low-abundance proteins becomes a core technical bottleneck of the application of mass spectrometry proteomics in early cancer screening. Aiming at the problems, the existing low-abundance protein enrichment technology mostly adopts an adsorption separation strategy based on magnetic beads, and various commercial kits are formed. Such kits typically rely on non-specific or semi-specific interactions between hydrophobic, charge or affinity ligands on the surface of the magnetic beads and the proteins to achieve adsorption enrichment of plasma proteins. However, in practical applications, different proteins tend to adsorb to the surface of the beads simultaneously and form complex protein corona structures, and protein molecules are usually bound to solid phase interfaces with high stability through multiple weak interaction sites. The lack of "smart response" mechanisms for existing magnetic bead materials, whose surface properties remain static during adsorption and elution, has led to a core contradiction that a strong adsorption force is often required to capture low abundance proteins, but such strong interactions (especially multipoint adsorption) become a hindrance during the elution phase. The existing magnetic bead system gradually weakens the interaction of protein and magnetic beads under the conditions of denaturant, salt concentration or surfactant and the like in the elution stage, so that synchronous dissociation of various interactions is difficult to realize in a limited time, partial proteins, especially some key characteristic low-abundance proteins, are still remained on the surfaces of the magnetic beads after elution, thereby causing systematic loss of the key low-abundance proteins at the detection level and limiting the capture and detection capability of mass spectrum proteomics on early-stage tumor molecular signals. Therefore, how to start from the magnetic bead material, an intelligent switch capable of responding to the environment is introduced, and the controllable switching of the protein adsorption and desorption process is realized by regulating and controlling the interface property of the intelligent switch, so that the residual is reduced to the greatest extent, the relative proportion of low-abundance proteins in eluted proteins is improved, and the intelligent switch becomes a key technical problem to be solved in the field of liquid biopsy proteomics. Disclosure of Invention The invention discloses