CN-122011304-A - Thermo-sensitive magnetic bead, preparation method thereof and related application thereof in early cancer screening

CN122011304ACN 122011304 ACN122011304 ACN 122011304ACN-122011304-A

Abstract

The invention discloses a temperature-sensitive magnetic bead, a preparation method thereof and related application thereof in early cancer screening, wherein the temperature-sensitive magnetic bead takes unsaturated superparamagnetic beads with vinyl groups on the surfaces as a matrix, and N-isopropyl acrylamide, acrylamide and oligo (ethylene glycol) methyl ether methacrylate are initiated to copolymerize on the surfaces of the magnetic beads in an in-situ free radical polymerization mode to form a temperature-sensitive polymer brush layer with adjustable Low Critical Solution Temperature (LCST). The temperature-sensitive magnetic beads are in a hydrophobic collapse state at 37-39 ℃ and maintained in a viscoelastic fluid state which is favorable for dynamic replacement of low-abundance proteins, can efficiently adsorb the low-abundance proteins in blood plasma, quickly convert into a hydrophilic stretching state at a temperature lower than 36 ℃, and synchronously weaken multi-point interaction between the proteins and the temperature-sensitive magnetic beads by utilizing micro-mechanical ejection force generated by phase change, so that complete elution of the proteins from the surfaces 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 (14)

1. The temperature-sensitive magnetic bead is characterized by comprising a magnetic inner core and a temperature-sensitive copolymer layer modified on the surface of the magnetic inner core, wherein: The magnetic core comprises ferroferric oxide particles modified by vinyl groups, and the particle size of the magnetic core is 50 nm-500 nm; The temperature-sensitive copolymer layer is a high polymer layer modified on the surface of the magnetic inner core in a free radical polymerization mode, wherein: The temperature-sensitive copolymer layer is formed by taking the ferroferric oxide particles modified by the ethylene group as an initiating matrix and initiating the following monomers to carry out free radical copolymerization reaction, wherein the initiating agent used in the initiation of the free radical copolymerization reaction is potassium persulfate or azo diisobutylamidine hydrochloride, and the crosslinking agent is N, N' -methylene bisacrylamide.
2. The temperature-sensitive magnetic bead according to claim 1, wherein the vinyl group-modified ferroferric oxide particles are prepared by coupling reaction of ferroferric oxide nano particles and 3- (trimethoxysilyl) propyl methacrylate, wherein the mass ratio of ferroferric oxide to 3- (trimethoxysilyl) propyl methacrylate is 1 (1.0-3.0).
3. The temperature-sensitive magnetic bead according to claim 1 or 2, wherein the molar ratio of N-isopropylacrylamide, acrylamide to 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 to 50 mg/mL.
4. A temperature-sensitive magnetic bead as claimed in claim 3, wherein 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 according to claim 1 or 2, wherein the amount of potassium persulfate or azobisisobutylamidine hydrochloride is 1% -2% of the total mass of the monomer, and the amount of N, N' -methylenebisacrylamide is 1% -3% of the total molar amount of the monomer.
6. The temperature-sensitive magnetic bead according to claim 1 or 2, wherein the oligomeric (ethylene glycol) methyl ether methacrylate has a number average molecular weight Mn = 200 Da to 2000 Da.
7. The temperature-sensitive magnetic bead according to claim 6, wherein the average particle diameter of the temperature-sensitive magnetic bead is 100 nm to 500nm, and the number average molecular weight mn=500 Da to 800 Da of the oligo (ethylene glycol) methyl ether methacrylate.
8. The temperature-sensitive magnetic bead according to claim 1 or 2, wherein the low critical solution temperature of the temperature-sensitive copolymer layer is 35.5 ℃ to 36.5 ℃.
9. A method for preparing a temperature-sensitive magnetic bead according to any one of claims 1 to 8, comprising the steps of magnetic core synthesis, surface vinylation modification, temperature-sensitive layer in-situ copolymerization and purification, The magnetic core synthesis step comprises dissolving ferric chloride hexahydrate, trisodium citrate and anhydrous sodium acetate in ethylene glycol, reacting in a high-temperature high-pressure reaction kettle, then alternately ultrasonically cleaning the reaction product with ethanol and deionized water for at least 3 times until the supernatant is colorless, magnetically separating and collecting, vacuum drying to obtain ferroferric oxide nano particles, The surface vinylation modification step comprises dispersing the ferroferric oxide nano particles in a mixed solvent of ethanol, water and concentrated ammonia water, wherein the volume ratio of the ethanol to the water to the concentrated ammonia water is 40:1:1, carrying out ultrasonic dispersion uniformly, adding a silane coupling agent containing vinyl for coupling, carrying out magnetic separation after the reaction is finished, washing with ethanol to remove silane hydrolysate which is physically adsorbed, obtaining vinylation magnetic beads with double bonds on the surface, Wherein the temperature sensitive layer in-situ copolymerization reaction step comprises the following steps: Dispersing, namely dispersing the obtained vinyl magnetic beads in deionized water, and introducing nitrogen to bubble so as to remove dissolved oxygen; under the protection of nitrogen, sequentially adding monomers N-isopropyl acrylamide, acrylamide and oligo (ethylene glycol) methyl ether methacrylate, wherein the total concentration of the monomers is controlled to be 20 mg/mL-50 mg/mL; Crosslinking and initiation, namely adding a crosslinking agent N, N' -methylene bisacrylamide, wherein the dosage of the crosslinking agent is 1-3% of the total molar weight of the monomer, heating to 70 ℃, adding an initiator potassium persulfate or azo diisobutyl amidine hydrochloride, and reacting for 4-6 hours under stirring, wherein the dosage of the initiator is 1-2% of the total weight of the monomer; The purification step comprises the steps of magnetic separation after the reaction is finished, repeatedly washing with deionized water with the temperature lower than the low critical dissolution temperature of the obtained temperature-sensitive magnetic beads, removing unreacted monomers and free polymers, and freeze-drying for preservation.
10. The method of claim 9, wherein the silane coupling agent is propyl 3- (trimethoxysilyl) methacrylate.
11. The method according to claim 9, wherein the total concentration of the monomers N-isopropylacrylamide, acrylamide and oligo (ethylene glycol) methyl ether methacrylate in the feeding step is controlled to be 20 mg/mL-25 mg/mL, and the amount of N, N' -methylenebisacrylamide in the crosslinking and initiating step is 2% of the total molar amount of the monomers, the amount of the initiator is potassium persulfate, and the amount of the initiator is 2% of the total mass of the monomers.
12. A method for treating low-abundance proteins in plasma for early screening of cancers, which is characterized by comprising the steps of using the temperature-sensitive magnetic beads according to any one of claims 1-8, enabling the temperature-sensitive magnetic beads to be in contact with a diluted plasma sample under the condition of being higher than the low critical dissolution temperature of the temperature-sensitive magnetic beads so as to carry out protein adsorption, and carrying out protein elution on the temperature-sensitive magnetic beads adsorbing low-abundance proteins in the plasma under the condition of being lower than the low critical dissolution temperature so as to enable the proteins to be eluted from the surface of the temperature-sensitive magnetic beads and enter a solution phase; Wherein the adsorption temperature in the protein adsorption process is 37-39 ℃ and/or the elution temperature in the protein elution process is 20-30 ℃.
13. The method according to claim 12, wherein the adsorption temperature is 38 ℃, the elution temperature is 25 ℃ to 30 ℃, and the protein is eluted with urea buffer, dithiothreitol aqueous solution and iodoacetamide aqueous solution.
14. A kit for enriching for low abundance proteins in plasma of a cancer preserver, comprising the temperature sensitive magnetic beads of any one of claims 1-8.

Description

Thermo-sensitive magnetic bead, preparation method thereof and related application thereof in early cancer screening Technical Field The invention relates to the technical fields of biomedical new materials, proteomics and in-vitro diagnosis, in particular to a temperature-sensitive magnetic bead, a preparation method thereof and related 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 systemic changes in protein expression levels, modification states, and interaction networks, which often occur earlier than obvious clinical symptoms. Therefore, the development of a technical means capable of realizing reliable detection in early disease has important significance for improving early diagnosis rate and treatment effect of cancer. The existing tumor detection method mainly comprises imaging examination, 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 evidence, but has the defects of strong invasiveness, limited sampling and difficult repeated implementation, and the traditional serum markers have limited types, insufficient sensitivity and specificity and difficulty in meeting the requirement of early tumor screening. In this context, liquid biopsy is becoming an important development in the field of tumor detection due to its advantages of being minimally invasive, reproducible, and capable of reflecting systemic molecular changes. Among the various analytical subjects of liquid biopsies, circulating proteins are directly involved in and reflect tumor-related biological processes and are considered as a source of biomarkers of great potential application. Mass spectrometry-based proteomics technology can realize large-scale protein identification and quantitative analysis, has the advantages of high flux and high information density, and is widely focused in liquid biopsy research. However, in body fluid systems such as blood, high-abundance proteins occupy most of the total protein mass, severely compress the detection space of low-abundance tumor-related proteins, and become key factors for limiting the sensitivity and coverage depth of mass spectrometry proteomics. In the existing mass spectrometry proteomics liquid biopsy procedure for separating proteins by using magnetic beads, different proteins are simultaneously adsorbed on the surfaces of the magnetic beads and form a complex protein crown structure, and protein molecules are often adsorbed on the solid phase surface in a highly stable manner through a plurality of weak interaction sites. Conventional magnetic beads typically rely on denaturants or surfactants to gradually attenuate protein-solid phase interactions to effect elution, which makes it difficult to achieve simultaneous dissociation of multiple interactions within a limited time, resulting in partial proteins, especially low abundance proteins, that remain at the solid phase interface after elution. This residue can significantly affect the low abundance protein duty cycle, even causing systematic loss of critical low abundance proteins at the detection level, thereby limiting the capture capacity and detection sensitivity of liquid biopsies to early tumor critical signals. In order to break through the limitation of the traditional chemical elution, the intelligent polymer with temperature response characteristic (such as Poly (N-isopropylacrylamide), namely Poly (N-isopropyl acrylamide), PNIPAM for short) provides a new technological opportunity for constructing a controllable adsorption/desorption interface. However, under the condition of low critical solution temperature (Lower Critical Solution Temperature, abbreviated as LCST) about 32 ℃, the application of the conventional PNIPAM to a high-complexity plasma proteomics system has obvious theoretical short plates, namely, on one hand, the phase transition temperature of 32 ℃ is too close to the room temperature operation environment, the adsorption is very easy to be started accidentally when the magnetic beads and the plasma are not uniformly mixed, so that high-abundance proteins are preempted by virtue of concentration advantages, and on the other hand, the strong hydrophobic rigid interface formed after the phase transition of the pure PNIPAM can prevent the dynamic exchange of protein molecules, so that the adsorbed high-abundance impurities are difficult to be replaced by the Vroman effect by the low-abundance proteins with stronger affinity. Therefore, how to improve the interface characteristics of the magnetic bead material, avoid the interference of early adsorption and improve the current situation of difficult protein replacement, and improve t