CN-122017227-A - Choline phosphate modified magnetic ball and preparation method and application thereof

CN122017227ACN 122017227 ACN122017227 ACN 122017227ACN-122017227-A

Abstract

The invention relates to the technical field of exosome separation detection, in particular to a choline phosphate modified magnetic sphere and a preparation method and application thereof. The choline phosphate modified magnetic sphere comprises an Fe 3 O 4 magnetic nanosphere and a choline phosphate polymer coupled to the Fe 3 O 4 magnetic nanosphere, wherein the choline phosphate polymer comprises an azide polymer and choline phosphate grafted on the azide polymer. The choline phosphate modified magnetic sphere can extract exosomes from fecal samples with high efficiency and high purity through intermolecular multivalent coordination of choline phosphate polymer and phosphatidylcholine on the exosome membrane surface.

Inventors

LIU DINGBIN
LI XIAOMIN
DU RUI
GUO ZIFANG
TANG YUJING
LI QIANG
WANG YING
GUO MIN

Assignees

中国石油化工股份有限公司
中石化(北京)化工研究院有限公司
南开大学

Dates

Publication Date: 20260512
Application Date: 20241112

Claims (16)

1. A choline phosphate modified magnetic sphere, wherein the choline phosphate modified magnetic sphere comprises a Fe 3 O 4 magnetic nanosphere and a choline phosphate polymer coupled to the Fe 3 O 4 magnetic nanosphere; Wherein the choline phosphate polymer comprises an azide polymer and choline phosphate grafted onto the azide polymer.
2. The choline phosphate modified magnetic sphere of claim 1, wherein the Fe 3 O 4 magnetic nanospheres are coupled with the choline phosphate polymer by a silane coupling agent.
3. The choline phosphate modified magnetic sphere according to claim 1 or 2, wherein the diameter of the choline phosphate modified magnetic sphere is 10-50nm.
4. A choline phosphate modified magnetic sphere according to any one of claims 1-3, wherein the azide polymer has a degree of polymerization of 25-100.
5. A method for preparing a choline phosphate modified magnetic sphere, comprising the steps of: (1) Reacting a silane coupling agent with the Fe 3 O 4 magnetic nanospheres with surface hydroxyl groups to obtain silanized Fe 3 O 4 magnetic nanospheres; (2) Reacting an azide polymer with a reactive end group with a choline phosphate derivative containing a triple bond to obtain a choline phosphate polymer with a reactive end group; (3) And (3) reacting the silanized Fe 3 O 4 magnetic nanospheres with a choline phosphate polymer with a reactive end group to obtain choline phosphate modified magnetic spheres.
6. The method according to claim 5, wherein in the step (1), the weight ratio of the silane coupling agent to the amount of the Fe 3 O 4 magnetic nanospheres having surface hydroxyl groups is 0.05-20:1; Preferably, the silane coupling agent is a maleimide group-containing silane coupling agent; preferably, the Silane coupling agent is Silane-PEG-Mal.
7. The method according to claim 5, wherein the choline phosphate derivative having a triple bond has a structural formula shown in formula (1); Wherein R 1 is selected from R 2 is selected from methyl,
8. The method of claim 5, wherein the azide polymer having a reactive end group has a degree of polymerization of 25 to 100.
9. The method according to claim 5 or 8, wherein the azide polymer having reactive end groups is an azide compound obtained by RAFT polymerization; Preferably, the structural formula of the azide compound is shown as a formula (2); Wherein m=2 to 8, and m is an integer; Preferably, the chain transfer agent used in the RAFT polymerization reaction is a trithiocarbonate chain transfer agent.
10. The method according to any one of claims 7 to 9, wherein in step (2) the molar ratio of the azide polymer having a reactive end group to the amount of the choline phosphate derivative having a triple bond is 1:1 to 1.2, wherein the azide polymer having a reactive end group is based on azide groups.
11. The method of claim 5, wherein in step (3), the amount of said choline phosphate polymer having reactive end groups is in the range of 1:1-100 with respect to the amount of said silanized Fe 3 O 4 magnetic nanospheres.
12. A choline phosphate modified magnetic ball prepared by the method of any one of claims 5-11.
13. Use of the choline phosphate modified magnetic sphere of any one of claims 1-4 and 12 in exosome detection.
14. A method for separating exosomes in a fecal sample, the method comprising the steps of: Mixing the choline phosphate modified magnetic ball with the fecal sample pretreatment solution, and then carrying out solid-liquid separation; Wherein the choline phosphate modified magnetic ball is the choline phosphate modified magnetic ball of any one of claims 1-4 and 12.
15. The method of claim 14, wherein the preparation of the fecal sample pretreatment solution comprises mixing the fecal sample with PBS buffer, centrifuging and filtering to obtain the fecal sample pretreatment solution.
16. The method of claim 14, wherein the mixing is for a period of 10 to 40 minutes; preferably, the solid-to-liquid ratio of the choline phosphate modified magnetic ball to the fecal sample pretreatment solution is 20-100 μg to 1mL.

Description

Choline phosphate modified magnetic ball and preparation method and application thereof Technical Field The invention relates to the technical field of exosome separation detection, in particular to a choline phosphate modified magnetic sphere and a preparation method and application thereof. Background Colorectal cancer (Colorectal cancer, CRC) is a malignancy originating from colorectal mucosal epithelium, one of the most common malignant tumors worldwide and clinically. Along with social progress and changes of life habits of people, the incidence rate and death rate of colorectal cancer tend to rise year by year, and life health of people is seriously endangered. It has been found that most colorectal cancers take up to 5-10 years to progress from precancerous lesions to invasive adenocarcinoma. However, early colorectal cancer patients have few symptoms, and conventional physical examination generally does not involve intestinal examination, so that once the vast majority of patients find it, they have progressed to mid-to late stages, with five-year survival rates drastically dropping to around 10%. If early discovery, early diagnosis and early treatment can be achieved, the survival rate of patients can be greatly improved. At present, colonoscopy is a 'gold standard' for early colorectal cancer diagnosis, and the method can clearly see the conditions in the intestinal tract, but complicated intestinal tract preparation before colonoscopy is painful, and large-scale screening is difficult to realize. Due to the special association of faeces with the intestinal tract, detection of abnormal indicators in faeces (such as biomarkers for colorectal cancer) has great value in the diagnosis of colorectal cancer. At present, the colorectal cancer is diagnosed by in vitro diagnosis technology (fecal immunochemistry test and multi-target fecal detection), and the method has the advantages of low invasiveness, high sensitivity, simplicity, convenience and rapidness, but clinical results show that the sensitivity of the method to early colorectal cancer is still not ideal, the detection cost is high, and the method is difficult to popularize in a large range. Therefore, we want to establish a new means for early colorectal cancer screening by separating and further detecting exosome molecular information carried in the patient's feces through a functionalized magnetic sphere. Exosomes (exosomes) are phospholipid bilayer vesicles that are secreted by most cells, with diameters of 30-150nm, carrying important molecules such as specific membrane proteins, nucleic acids, lipids, etc. of the parent cells. Since normal intestinal epithelial cells have different molecular biological behaviors than colorectal cancer cells, the exosomes secreted by both have different molecular "fingerprints". The exosomes in the fecal sample of the patient are subjected to large-scale protein analysis, nucleic acid analysis or metabolite analysis by using molecular diagnosis technologies such as immunodetection, nucleic acid detection and the like, and the specific exosome markers of colorectal cancer are screened and identified, so that the colorectal cancer can be effectively screened and prevented. However, the components of the stool sample are extremely complex, and it is difficult to efficiently separate the exosomes enriched therein with high purity. The exosome separation method commonly used at present comprises an ultracentrifugation method, a size-based ultrafiltration method, an immunomagnetic bead method, a polymer precipitation method and the like. Ultracentrifugation is widely regarded as a "gold standard" for exosome separation, relying on differences in size and density between exosomes and other components. However, the yield and purity of the isolated exosomes are affected by a number of factors such as the type of rotor, the centrifugation time and the viscosity of the sample, and repeated ultracentrifugation may reduce the yield of exosomes and negatively affect its quality. Size-based ultrafiltration allows separation of small particles and soluble molecules from the exosomes, but correspondingly, the exosomes can adhere to the filter membrane causing losses. And the exosomes may be deformed or damaged by the addition of additional force to pass the analytical liquid through the filter membrane. The immune magnetic bead method combines the antibody or the aptamer modified on the magnetic particles with specific proteins on the surface of the exosome, and realizes the rapid separation of the exosome under the action of an external magnetic field. However, the magnetic bead-based separation strategy is not suitable for large-scale exosome separation, and the heterogeneity of exosome protein expression results in high cost and low capture rate. Polymer precipitation techniques rely primarily on the use of polymers (typically polyethylene glycol) to precipitate extracellular vesicles before further purification treatme