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CN-121994774-A - Polymer Fc binding peptide Raman probe and preparation method and application thereof

CN121994774ACN 121994774 ACN121994774 ACN 121994774ACN-121994774-A

Abstract

The invention belongs to the technical field of biological probes, and particularly relates to a high-molecular Fc binding peptide Raman probe, and a preparation method and application thereof. Compared with the prior art, the invention synthesizes the macromolecule linker containing the noble metal nanoparticle binding molecules and the Fc binding peptides, efficiently combines the noble metal nanoparticles, the Raman reporter molecules and the antibodies, generates a 'hot spot' effect in a nanogap formed among the noble metal nanoparticles, can remarkably amplify Raman signals, realizes high-sensitivity and specificity detection of low-concentration circulating tumor cells, simultaneously introduces the Fc binding peptides as universal anchor points of the antibodies, improves the suitability of the probes for antibodies corresponding to different tumor types, and enhances the practicability of the detection of the circulating tumor cells in various tumor patients.

Inventors

  • SHEN NA
  • YE XIAOLU
  • TANG CHAOHUI

Assignees

  • 中国科学院长春应用化学研究所

Dates

Publication Date
20260508
Application Date
20260209

Claims (10)

  1. 1. The macromolecular Fc binding peptide Raman probe is characterized by comprising a macromolecular connecting structure, a Raman reporter molecule, an enhanced substrate and an antibody; the macromolecule connecting structure comprises a structure shown in a formula (I): Formula (I); Wherein x, y and z are molar contents of repeating units, 0< x <1,0< y <1,0< x+y <1, z is polymerization degree, and z is an integer of 10-500; r 1 is selected from substituted or unsubstituted C2-C10 alkyl and substituted or unsubstituted C6-C20 aryl, wherein the substituents in the substituted C2-C10 alkyl and the substituted C6-C20 aryl are respectively and independently selected from one or more of C1-C5 alkyl and C6-C10 aryl; R 2 is selected from H or a cation; r 3 is selected from residues of the Fc binding peptide that lose one H; R 4 is selected from alkynyl-containing groups; r 5 is selected from H or acyl of C2-C10; L 1 、L 2 and L 3 are independently selected from C1-C5 alkylene; the reinforcing substrate comprises precious metal nanoparticles; The macromolecule connecting structure is connected with the reinforced substrate through a covalent bond.
  2. 2. The polymeric Fc-binding peptide raman probe according to claim 1, wherein x is 0.8 to 0.98, y is 0.005 to 0.4, and z is an integer of 80 to 240; R 1 is selected from C2-C8 alkyl or C6-C14 aryl; the R 2 is selected from H, a metal cation or an organic cation; said R 3 is selected from the group consisting of residues of Fc-III-4C which lose one H; The R 4 is selected from the group represented by formula (II): formula (II); R 5 is selected from H or acyl of C2-C5; R 6 is selected from C1-C8 alkylene, C6-C14 aryl or a group formed by singly bonding the above groups; L 1 、L 2 and L 3 are each independently selected from C1-C2 alkylene groups.
  3. 3. The high molecular Fc binding peptide raman probe according to claim 2, wherein said R 1 is selected from the group consisting of ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl, or anthracyl; The R 2 is selected from H, sodium ion, potassium ion, ammonium ion or positively charged amino acid ion; Said R 5 is selected from H, formyl, acetyl, propionyl or butyryl; R 6 is selected from alkylene of C1-C5, aryl of C6-C10 or a group formed by singly bonding the above groups.
  4. 4. The polymeric Fc-binding peptide raman probe according to claim 1, wherein the polymeric linking structure has a structure represented by formula (III): formula (III); x is 0.85-0.95, y is 0.005-0.4, z; is an integer of 80 to 240.
  5. 5. The high molecular Fc binding peptide raman probe according to claim 1 wherein said raman reporter is selected from the group consisting of p-mercaptobenzoic acid, p-mercaptobenzonitrile and p-nitrothiophen; the noble metal nanoparticles are selected from gold nanoparticles and/or silver nanoparticles.
  6. 6. The high molecular Fc binding peptide raman probe according to claim 1, wherein said antibody is selected from one or more of PD1、PDL1、OX40、OX40L、CD16、41BB、EGFR、CD3、CD3ε、CD19、CD28、BCMA、MET、CD47、CTLA-4、EpCAM、CD20、TROP2、CD73、CD69、DNAM-1、NKG2D、CSF1R、TIGIT、CD40、CD80 and CD86 antibodies.
  7. 7. A method of preparing a macromolecular Fc binding peptide raman probe according to claim 1 comprising the steps of: And mixing and heating the macromolecule connecting structure shown in the formula (I) with the reinforced substrate to react, thereby obtaining the macromolecule Fc binding peptide Raman probe.
  8. 8. A kit comprising the macromolecular Fc binding peptide raman probe of any one of claims 1-6.
  9. 9. The kit of claim 8, wherein the kit is used to detect the level of antigen expression on tumor cells of a cancer patient.
  10. 10. The kit of claim 9, wherein the cancer comprises one or more of nasal and sinus malignancy, nasopharyngeal carcinoma, oral cancer, laryngeal carcinoma, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, large intestine cancer, sigmoid and rectal cancer, liver cancer, pancreatic and peri-ampullate cancer, biliary tract cancer, renal cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibroblastic cancer, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, ewing's sarcoma, leukemia, lymphoma, and multiple myeloma.

Description

Polymer Fc binding peptide Raman probe and preparation method and application thereof Technical Field The invention belongs to the technical field of biological probes, and particularly relates to a high-molecular Fc binding peptide Raman probe, and a preparation method and application thereof. Background APDL 1A is a core immunotherapy scheme for various solid tumors (such as lung cancer and melanoma) by blocking the interaction between PDL1 on the surface of tumor cells and PD1 on the surface of T cells and restoring the killing function of an immune system to the tumors. However, aPDL has significant individual difference (20% -40%) in treatment response rate, the curative effect of the treatment is closely related to the expression level of the tumor cell PDL1, namely, the treatment response rate of a PDL1 high-expression patient can reach more than 60%, and the treatment response rate of a low-expression patient is less than 10%, so that accurate evaluation of the expression level of the tumor PDL1 is a key for guiding aPDL1 treatment decision and prognosis judgment. The current mainstream method for clinically evaluating the expression of PDL1 still depends on traditional tissue sampling, but has the obvious limitations that ① is invasive and can possibly cause complications such as bleeding, infection and the like, ② tumor heterogeneity leads to insufficient sample representativeness, ③ cannot dynamically monitor the change of PDL1 in the treatment process, and the treatment scheme is difficult to adjust in real time. Liquid biopsy is used as a noninvasive detection technology, tumor assessment is realized by analyzing tumor markers (such as circulating tumor cells and circulating tumor DNA) in body fluid such as blood and the like, and the defect of tissue sampling is overcome. Wherein, the Circulating Tumor Cells (CTCs) are taken as complete cells which directly fall off in tumor tissues, carry real-time biological information of the tumors, can dynamically reflect the state of the tumors in the treatment process, and are ideal markers for evaluating aPDL1 treatment prognosis. However, CTCs have extremely low concentration (only 1-10 per mL) in blood, and complex blood components (containing a large amount of red blood cells, white blood cells and free proteins), and the CTCs can be accurately detected by a high-sensitivity and high-specificity detection technology. Surface Enhanced Raman Spectroscopy (SERS) is based on the principle of raman spectroscopy, with the aid of a mechanism of chemical enhancement and electromagnetic enhancement of noble metal nanoparticles such as gold/silver, etc., resulting in a signal that is 10 2~1014 times stronger than the normal raman spectrum. SERS has the characteristics of narrow spectrum peak of Raman spectrum, strong correspondence between peak position and corresponding chemical structure and rich spectrum peak information, and the ultrasensitive property, so that the SERS can be used as an effective detection means of CTCs. The SERS probe is based on the principle that the noble metal nano particles can enhance Raman signals, and the corresponding nano particles are modified according to the research purpose so as to overcome the problems of insufficient specificity, weak Raman signals of the sample, nanoparticle aggregation caused by the sample and the like in the detection. The basic structure of a SERS probe generally consists of four parts, 1) a SERS substrate, such as gold/silver nanoparticles, that enhances the raman signal intensity of a target substance, 2) a raman reporter, such as p-mercaptobenzoic acid, rhodamine isothiocyanate, bipyridine, etc., as a signal source in an indirect detection mode, 3) a coating, such as a biomolecule, a high molecular polymer, a liposome, etc., that reduces non-specific adsorption and enhances nanoparticle stability, and 4) a targeting molecule, such as an antibody, an aptamer, a biomolecule, etc. According to the structure, the noble metal nano particles are modified step by step, so that corresponding probes suitable for research purposes are constructed, and the sensitivity and specificity requirements in the CTCs detection process can be effectively met by means of the ultrasensitivity of the SERS technology and the specificity provided by the targeting molecule modified by the probes. Improved SERS Nanoparticles for Direct Detection of Circulating Tumor Cells in the Blood（Wu,X.X.;Luo,L.Q.;Yang,S.;Ma,X.H.;Li,Y.L.;Dong,C.;Tian,Y.C.;Zhang,L.;Shen,Z.Y.;Wu,A.G.;ACS Appl. Mater. Interfaces. 2015,7,9965-9971.） It is proposed that detection of CTCs in the blood of cancer patients is critical for early cancer diagnosis, cancer prognosis, assessment of the effect of chemotherapy treatment, and selection of cancer treatment options. This work suggests a new SERS nanoparticle that binds raman reporter molecules and folate receptors for direct detection of CTCs in blood. However, the raman probe designed by the method is a