Polymer Fc binding peptide Raman probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological probes, and particularly relates to a high-molecular Fc binding peptide Raman probe as well as a preparation method and application thereof. Compared with the prior art, the precious metal nanoparticles, the Raman reporter molecules and the antibody are efficiently combined by synthesizing the macromolecular linker containing the precious metal nanoparticle binding molecules and the Fc binding peptide, and nanogaps formed among the precious metal nanoparticles generate a hot spot effect, so that a Raman signal can be remarkably amplified; the high-sensitivity and specific detection on the low-concentration circulating tumor cells is realized; meanwhile, an Fc binding peptide is introduced as a universal anchor point of an antibody, so that the adaptability of the probe to antibodies corresponding to different tumor types is improved, and the practicability of the probe in detection of circulating tumor cells in bodies of patients with various tumors is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biological probe technology, and particularly relates to a polymeric Fc-binding peptide Raman probe, its preparation method and application. Background Technology

[0002] aPDL1, by blocking the interaction between PDL1 on the surface of tumor cells and PD1 on the surface of T cells, restores the immune system's tumor-killing function and has become a core immunotherapy regimen for various solid tumors (such as lung cancer and melanoma). However, the response rate of aPDL1 treatment varies significantly among individuals (20%–40%), and its efficacy is closely related to the expression level of PDL1 on tumor cells—the treatment response rate in patients with high PDL1 expression can reach over 60%, while it is less than 10% in those with low expression. Therefore, accurately assessing the PDL1 expression level in tumors is crucial for guiding aPDL1 treatment decisions and prognostic assessment.

[0003] Currently, the mainstream method for clinical assessment of PDL1 expression still relies on traditional tissue sampling, but this method has significant limitations: ① It is invasive and may cause complications such as bleeding and infection; ② Tumor heterogeneity leads to insufficient sample representativeness; ③ It cannot dynamically monitor changes in PDL1 during treatment, making it difficult to adjust the treatment plan in real time. Liquid biopsy, as a non-invasive detection technology, assesses tumors by analyzing tumor markers (such as circulating tumor cells and circulating tumor DNA) in body fluids such as blood, thus overcoming the shortcomings of tissue sampling. Among them, circulating tumor cells (CTCs), as intact cells directly shed from tumor tissue, carry real-time biological information of the tumor and can dynamically reflect the state of the tumor during treatment, making them an ideal biomarker for assessing the prognosis of aPDL1 treatment. However, the concentration of CTCs in blood is extremely low (only 1-10 cells / mL), and blood composition is complex (containing a large number of red blood cells, white blood cells, and free proteins), requiring highly sensitive and specific detection technologies to accurately detect CTCs.

[0004] Surface-enhanced Raman scattering (SERS) is based on the principles of Raman spectroscopy. It utilizes the chemical and electromagnetic enhancement mechanisms of noble metal nanoparticles such as gold / silver to produce Raman spectra that are 10 times stronger than ordinary Raman spectra. 2 ~10 14The signal is twice that of the target substance. SERS has the characteristics of narrow Raman spectral peaks, strong correspondence between peak positions and corresponding chemical structures, and rich spectral information. In addition, its ultrasensitivity makes it an effective detection method for CTCs. SERS probes are based on the principle that noble metal nanoparticles can enhance Raman signals. The nanoparticles are modified according to the research purpose to overcome the problems encountered in detection, such as insufficient specificity, weak Raman signals of the sample itself, and nanoparticle agglomeration caused by the sample. The basic structure of SERS probes generally consists of four parts: 1) SERS substrate, such as gold / silver nanoparticles, which can enhance the Raman signal intensity of the target substance; 2) Raman reporter molecules, such as p-mercaptobenzoic acid, rhodamine isothiocyanate, bipyridine, etc., which serve as a signal source in an indirect detection method; 3) Coating layer, such as biomolecules, polymers, liposomes, etc., which can reduce non-specific adsorption and enhance the stability of nanoparticles; 4) Target molecules, such as antibodies, aptamers, biomolecules, etc. Based on the above structure, noble metal nanoparticles are modified stepwise to construct corresponding probes suitable for research purposes. With the help of the ultrasensitivity of SERS technology itself and the specificity provided by the target molecules modified by the probe, the sensitivity and specificity requirements in the detection process of CTCs can be effectively met.

[0005] The paper "Improved SERS Nanoparticles for Direct Detection of Circulating Tumor Cells in the Blood" (Wu,XX; Luo,LQ; Yang,S.; Ma,XH; Li,YL; Dong,C.; Tian,YC; Zhang,L.; Shen,ZY; Wu,AG; ACS Appl. Mater. Interfaces. 2015, 7, 9965-9971.) proposes that detecting circulating tumor cells (CTCs) in the blood of cancer patients is crucial for early cancer diagnosis, cancer prognosis, evaluation of chemotherapy drug efficacy, and selection of cancer treatment. This work proposes a novel SERS nanoparticle, combining a Raman reporter molecule and a folic acid receptor, for the direct detection of CTCs in the blood. However, the designed Raman probe uses a single gold nanoparticle as a substrate, resulting in limited SERS enhancement and low sensitivity for detecting the target analyte.

[0006] The paper "PD-L1 targeted iron oxide SERS bioprobe for accurately detecting circulating tumor cells and delineating tumor boundary" (Pan,T; Zhang,DH; You,GM; Wu XX; Zhang,CG; Miao,XY; Ren,WZ; He,YW; He,LL; Gong,YC; Lin,J.; Wu,AG; Shao,GL; Chinese Chemical Letters,2025,36(1):109857-109857.) proposes that an iron oxide (IO)-based SERS bioprobe combined with aPDL1 can be used to detect circulating tumor cells (CTCs) in liquid biopsy. The IO nanoparticles used in this work have a poorer plasmon resonance effect than gold nanoparticles, resulting in a weaker Raman signal enhancement and lower sensitivity for detecting the target analytes. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a polymeric Fc-binding peptide Raman probe with high detection sensitivity and targeting adaptability, as well as its preparation method and application.

[0008] This invention provides a polymeric Fc-binding peptide Raman probe, comprising a polymeric linker structure, a Raman reporter molecule, and an enhancing substrate and antibody;

[0009] The polymer linking structure includes the structure shown in formula (I):

[0010] Formula (I);

[0011] Where x, y, and z are the molar contents of repeating units, 0 < x < 1, 0 < y < 1, 0 < x + y < 1; z is the degree of polymerization, and z is an integer from 10 to 500;

[0012] R1 is selected from substituted or unsubstituted C2-C10 alkyl groups and substituted or unsubstituted C6-C20 aryl groups; the substituents in the substituted C2-C10 alkyl groups and the substituted C6-C20 aryl groups are each independently selected from one or more of C1-C5 alkyl groups and C6-C10 aryl groups.

[0013] R2 is selected from H or a cation;

[0014] R3 is selected from an Fc-binding peptide that has lost an H residue;

[0015] R4 is selected from groups containing an alkyne group;

[0016] R5 is selected from H or C2~C10 acyl groups;

[0017] L1, L2, and L3 are each independently selected from C1 to C5 alkylene groups;

[0018] The enhanced substrate includes noble metal nanoparticles;

[0019] The polymer linking structure is covalently connected to the reinforcing substrate.

[0020] Preferably, x is 0.8~0.98; y is 0.005~0.4; and z is an integer from 80 to 240.

[0021] And / or, R1 is selected from C2-C8 alkyl or C6-C14 aryl;

[0022] The R2 is selected from H, metal cations, or organic cations;

[0023] The R3 is selected from a residue of Fc-III-4C that has lost one H;

[0024] The R4 is selected from the group shown in formula (II):

[0025] Formula (II);

[0026] R6 is selected from C1-C8 alkylene groups, C6-C14 aryl groups, or groups formed by connecting the above groups through single bonds;

[0027] The R5 is selected from H or C2~C5 acyl groups;

[0028] L1, L2 and L3 are each independently selected from C1 to C2 alkylene groups.

[0029] Preferably, R1 is selected from ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl, or anthracene;

[0030] The R2 is selected from H, sodium ions, potassium ions, ammonium ions, or positively charged amino acid ions;

[0031] R6 is selected from C1-C5 alkylene groups, C6-C10 aryl groups, or groups formed by connecting the above groups through single bonds;

[0032] The R5 is selected from H, formyl, acetyl, propionyl or butyryl.

[0033] Preferably, the polymer linking structure has the structure shown in formula (III):

[0034] Formula (III);

[0035] x is 0.85~0.95; y is 0.005~0.4; z is an integer from 80 to 240.

[0036] Preferably, the Raman reporter molecule is selected from p-mercaptobenzoic acid, p-mercaptobenzonitrile, and p-nitrobenzenethiol;

[0037] The noble metal nanoparticles are selected from gold nanoparticles and / or silver nanoparticles.

[0038] Preferably, the antibody is selected from one or more of the following: 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.

[0039] This invention also provides a method for preparing the above-mentioned polymeric Fc-binding peptide Raman probe, comprising the following steps:

[0040] The polymer linking structure shown in formula (I) is mixed with an enhanced substrate and heated to obtain a polymeric Fc-binding peptide Raman probe.

[0041] The present invention also provides a kit comprising the above-described polymeric Fc-binding peptide Raman probe.

[0042] Preferably, the kit is used to detect the antigen expression level on tumor cells of cancer patients.

[0043] Preferably, the cancer includes one or more of the following: malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, malignant tumors of the testis, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, leukemia, lymphoma, and multiple myeloma.

[0044] Compared with existing technologies, this invention synthesizes a polymeric linker containing noble metal nanoparticle binding molecules and Fc binding peptides, which efficiently binds noble metal nanoparticles, Raman reporter molecules and antibodies. The nano-gap formed between the noble metal nanoparticles generates a "hot spot" effect, which can significantly amplify the Raman signal and achieve highly sensitive and specific detection of low concentrations of circulating tumor cells. At the same time, the introduction of Fc binding peptides as universal antibody anchors improves the adaptability of the probe to antibodies corresponding to different tumor types and enhances its practicality in detecting circulating tumor cells in a variety of tumors. Attached Figure Description

[0045] Figure 1 A schematic diagram illustrating the preparation process of the polymeric Fc-binding peptide Raman probe provided by this invention;

[0046] Figure 2 This is a schematic diagram of the process for preparing the polymeric Fc-binding peptide Raman probe in Example 1 of the present invention;

[0047] Figure 3 The structural formula and 1H NMR spectrum of the polymer linking structure pL prepared in Example 1 of this invention;

[0048] Figure 4 The image shows the SERS intensity characterization results of AuNPs-MBA and IgG@AuNPs-MBA / pL prepared in Example 1 of this invention.

[0049] Figure 5 Transmission electron microscopy images of AuNPs-MBA and IgG@AuNPs-MBA / pL prepared in Example 1 of this invention;

[0050] Figure 6 This is a schematic diagram of the CTCs detection process in Embodiment 1 of the present invention;

[0051] Figure 7 This is a graph showing the detection sensitivity of the aPDL1@AuNPs-MBA / pL SERS probe prepared in Example 1 of this invention on 4T1 cells in mouse blood. Detailed Implementation

[0052] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] Terminology Explanation:

[0054] SERS (Surface-Enhanced Raman Scattering): Surface-enhanced Raman scattering refers to the significant enhancement of the Raman signal (10⁻¹⁰) when molecules are adsorbed on the surface of a metal nanostructure. 2 ~10 14 The phenomenon of (times) can be observed, enabling detection at the single-molecule level.

[0055] CTCs (Circulating Tumor Cells): These are tumor cells that detach from the primary tumor or metastatic lesions and enter the peripheral blood circulation. They are key markers of tumor metastasis and recurrence.

[0056] This invention provides a polymeric Fc-binding peptide Raman probe, comprising a polymeric linker structure, a Raman reporter molecule, an enhancing substrate, and an antibody.

[0057] In this invention, the polymer linking structure includes the structure shown in formula (I):

[0058] Formula (I);

[0059] Where x, y, and z are the molar contents of repeating units, 0 < x < 1, 0 < y < 1, 0 < x + y < 1; z is the degree of polymerization, which is an integer from 10 to 500.

[0060] In a specific embodiment of the present invention, x is preferably 0.8 to 0.98; optionally, x is 0.8, 0.82, 0.84, 0.85, 0.86, 0.88, 0.9, 0.92, 0.94, 0.95, 0.96, 0.98 or any two of the above values; y is preferably 0.005 to 0.4; optionally, y is 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 or any two of the above values.

[0061] Furthermore, in a specific embodiment provided by the present invention, x is 0.85~0.95; y is 0.005~0.4.

[0062] Furthermore, in a specific embodiment provided by the present invention, x is 0.9~0.92; y is 0.06~0.09.

[0063] Furthermore, in a specific embodiment provided by the present invention, x is 0.911; y is 0.010.

[0064] In one specific embodiment of the present invention, z can optionally be an integer within the range of 10, 50, 80, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, 380, 400, 420, 450, 480, 500, 520, 550, 580, 600, or any two of the above values.

[0065] Furthermore, in a specific embodiment provided by the present invention, z is preferably an integer from 80 to 240; optionally, z is an integer within the range of 80, 100, 120, 140, 160, 180, 200, 220, 240 or any two of the above values.

[0066] Furthermore, in one specific embodiment provided by the present invention, z is 160.

[0067] R1 is a substituted or unsubstituted C2-C10 alkyl group or a substituted or unsubstituted C6-C20 aryl group; the substituents in the substituted C2-C10 alkyl group and the substituted C6-C20 aryl group are each preferably one or more of C1-C5 alkyl group and C6-C10 aryl group, more preferably one or more of C1-C3 alkyl group and C6-C10 aryl group.

[0068] In one specific embodiment of the present invention, R1 is a substituted or unsubstituted C2-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group; the substituents in the substituted C2-C8 alkyl group and the substituted C6-C14 aryl group are each preferably one or more of C1-C5 alkyl group and C6-C10 aryl group, more preferably one or more of C1-C3 alkyl group and C6-C10 aryl group.

[0069] In a specific embodiment of the present invention, R1 is preferably an alkyl group of C2 to C8 or an aryl group of C6 to C14, more preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl or anthracene.

[0070] R2 is H or a cation, preferably H, a metal cation or an organic cation, more preferably H, a sodium ion, a potassium ion, an ammonium ion or a positively charged amino acid ion.

[0071] R3 is an Fc-binding peptide that has lost an H residue, preferably Fc-III-4C that has lost an H residue.

[0072] R4 is a group containing an alkynyl group, preferably the group shown in formula (II):

[0073] Formula (II);

[0074] Wherein, R6 is a C1-C8 alkylene group, a C6-C14 aryl group, or a group formed by connecting the above groups through a single bond; preferably, R6 is a C1-C8 alkylene group, a C6-C14 aryl group, or a group formed by connecting the above groups through a single bond; more preferably, it is a C1-C5 alkylene group, a C6-C10 aryl group, or a group formed by connecting the above groups through a single bond; even more preferably, it is a C1-C3 alkylene group, a C6-C10 aryl group, or a group formed by connecting the above groups through a single bond; most preferably, it is methylene, ethylene, phenyl, or benzyl.

[0075] R5 is an acyl group of H or C2~C10, preferably an acyl group of H or C2~C8, even more preferably an acyl group of H or C2~C5, and most preferably an acyl group of H, formyl, acetyl, propionyl or butyryl.

[0076] L1, L2 and L3 are each independently C1 to C5 alkylene groups, preferably C1 to C4 alkylene groups, more preferably C1 to C3 alkylene groups, and even more preferably C1 to C2 alkylene groups.

[0077] In one specific embodiment of the present invention, the polymer linking structure has the structure shown in formula (III):

[0078] Formula (III);

[0079] x is 0.85~0.95; y is 0.005~0.4; z is an integer from 80 to 240.

[0080] In this invention, the reinforcing substrate includes noble metal nanoparticles; the noble metal nanoparticles can be any noble metal nanoparticles known to those skilled in the art, and there are no special limitations; in a specific embodiment provided by this invention, the noble metal nanoparticles are preferably gold nanoparticles and / or silver nanoparticles.

[0081] In one specific embodiment of the present invention, the particle size of the noble metal nanoparticles is preferably 10-100 nm; optionally, the particle size of the noble metal nanoparticles is 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm or any two of the above values.

[0082] In another specific embodiment provided by the present invention, the Raman reporter molecule is modified on the surface of noble metal nanoparticles.

[0083] In one specific embodiment of the present invention, the Raman reporter molecule is preferably p-mercaptobenzoic acid, p-mercaptobenzonitrile, and p-nitrobenzenethiol, and more preferably p-mercaptobenzoic acid.

[0084] In this invention, the polymer linking structure and the reinforcing substrate are connected by covalent bonds; specifically, noble metal nanoparticles with surface-modified Raman reporter molecules are covalently linked to the alkyne groups in the polymer linking structure, thereby forming an aggregate of reinforcing substrates and improving the Raman signal; the gap between the reinforcing substrates in the aggregate of reinforcing substrates is 2~3 nm.

[0085] In a specific embodiment of the present invention, the polymeric Fc-binding peptide Raman probe is bound to an antibody; specifically, the polymeric linker structure binds to the antibody via side chain R3, and the R3 specifically binds to the Fc segment of the antibody, thereby achieving targeted recognition of tumor cells; the antibody can be any antibody well known to those skilled in the art, and there are no special limitations. In the present invention, it is preferably one or more of the following antibodies: 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.

[0086] This invention synthesizes a polymeric linker containing noble metal nanoparticle binding molecules and Fc-binding peptides, which efficiently binds noble metal nanoparticles carrying Raman reporter molecules to antibodies. The nano-gap between the nanoparticles generates a "hot spot" effect, which can significantly amplify the Raman signal and achieve highly sensitive and specific detection of low concentrations of circulating tumor cells. At the same time, the introduction of Fc-binding peptides as universal antibody anchors improves the adaptability of the probe to antibodies corresponding to different tumor types and enhances its practicality in detecting circulating tumor cells in a variety of tumors.

[0087] The present invention also provides a method for preparing the above-mentioned polymeric Fc-binding peptide Raman probe, comprising the following steps: mixing the polymeric linking structure shown in formula (I) with an enhanced substrate and heating to react, thereby obtaining the polymeric Fc-binding peptide Raman probe.

[0088] Taking gold nanoparticles as an example of noble metal nanoparticles, the preparation process of the polymer Fc-bound peptide Raman probe is as follows: Figure 1 As shown, M is a Raman reporter molecule and Ab is an antibody.

[0089] In this invention, there are no special restrictions on the source of any raw materials; they can be commercially available.

[0090] In a specific embodiment of the present invention, the polymeric linking structure shown in formula (I) is preferably prepared by the following method: polyglutamic acid (PLG) is activated with a carboxyl activator and then reacted sequentially with an Fc-binding peptide, a compound shown in formula (IV), or a salt of a compound shown in formula (IV) to obtain the polymeric linking structure.

[0091] (IV)

[0092] In a specific embodiment of the present invention, the carboxyl activator can be any carboxyl activator well known to those skilled in the art, and there are no special limitations, including but not limited to carbodiimides and / or activated esters; the carbodiimides include but are not limited to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and dicyclohexylcarbodiimide (DCC); the activated esters include but are not limited to N-hydroxysuccinimide (NHS) and sulfonyl-N-hydroxysuccinimide (Sulfo-NHS).

[0093] In a specific embodiment of the present invention, the activation is preferably carried out in a solvent; the solvent can be any solvent known to those skilled in the art and is not particularly limited, but is preferably N,N-dimethylformamide (DMF) in the present invention; the activation temperature is preferably 25℃~35℃, more preferably 28℃~32℃, and even more preferably 30℃; the activation time is preferably 1~10 h, more preferably 2~8 h, and even more preferably 4~6 h.

[0094] In a specific embodiment of the present invention, the reaction with the Fc-binding peptide after activation is preferably carried out under the conditions of an organic amine; the organic amine is any organic amine well known to those skilled in the art and is not particularly limited, including but not limited to triethylamine; the temperature of the reaction with the Fc-binding peptide after activation is preferably 25°C to 35°C, more preferably 28°C to 32°C, and even more preferably 30°C; the reaction time of the reaction with the Fc-binding peptide after activation is preferably 20 to 30 h.

[0095] In a specific embodiment of the present invention, the reaction temperature of the product after reacting with the Fc-binding peptide and the compound or salt of the compound shown in formula (IV) is preferably 25°C to 35°C, more preferably 28°C to 32°C, and even more preferably 30°C; the reaction time of the product after reacting with the Fc-binding peptide and the compound or salt of the compound shown in formula (IV) is preferably 40 to 50 h.

[0096] In a specific embodiment of the present invention, after the reaction is completed, the polymeric linking structure shown in formula (I) is preferably dialyzed with DMF and pure water in sequence and then freeze-dried.

[0097] In a specific embodiment of the present invention, the reinforcing substrate is preferably carried out according to the following method: Raman reporter molecules and noble metal nanoparticles are mixed and reacted in a solvent to obtain the reinforcing substrate; the Raman reporter molecules and noble metal nanoparticles are as described above, and will not be repeated here; the mixing reaction is preferably carried out at room temperature; the mixing reaction time is preferably 10~60 min; optionally, the mixing reaction time is 10 min, 20 min, 30 min, 40 min, 50 min, 60 min or any two of the above values.

[0098] The polymer linking structure shown in formula (I) is mixed with the reinforcing substrate and heated to react; the temperature of the heating reaction is preferably 40℃~80℃; optionally, the temperature of the heating reaction is 40℃, 50℃, 60℃, 70℃, 80℃ or any two of the above values; the heating reaction time is preferably 1~5 h; optionally, the heating reaction time is 1h, 2h, 3h, 4h, 5h or any two of the above values.

[0099] After heating and reaction, centrifugation is preferred. After washing, the sample is incubated with antibody in buffer to obtain a high molecular weight Fc-binding peptide Raman probe. The buffer can be any buffer known to those skilled in the art and is not particularly limited. In this invention, PBS buffer is preferred. The incubation is preferably carried out at room temperature. The incubation time is preferably 2 to 6 hours. Optionally, the incubation time is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or any two of the above values.

[0100] The present invention also provides a kit comprising the above-described polymeric Fc-binding peptide Raman probe.

[0101] In one specific embodiment of the present invention, the kit is used to detect tumor cells. The tumor cells include isolated tumor cells or tumor cells in vivo; the tumor cells in vivo include cells from tumor tissue and / or circulating tumor cells.

[0102] In one specific embodiment of the present invention, the polymeric Fc-binding peptide Raman probe can be used in clinical scenarios such as tumor diagnosis and prognostic assessment of immunotherapy.

[0103] In one specific embodiment of the present invention, the kit is used to detect the antigen expression level on tumor cells of cancer patients.

[0104] In one specific embodiment of the present invention, the cancer includes one or more of the following: malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, malignant tumors of the testis, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, leukemia, lymphoma, and multiple myeloma.

[0105] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a polymeric Fc-binding peptide Raman probe, its preparation method, and its application.

[0106] All reagents used in the following examples are commercially available.

[0107] Example 1

[0108] See Figure 2 , Figure 2 This is a schematic diagram of the process for preparing the polymeric Fc-binding peptide Raman probe in Example 1 of the present invention.

[0109] Step 1: Preparation of gold nanoparticles (AuNPs) (sodium citrate reduction method)

[0110] Add 200 μL of 10% chloroauric acid (HAuCl4) aqueous solution to 200 mL of ultrapure water and heat to boiling. Dissolve 224.03 mg of sodium citrate dihydrate (Na3C6H5O7·2H2O) in 1 mL of ultrapure water and quickly add it to the boiling solution under vigorous stirring. Continue boiling and stirring for 30 min (the solution changes from pale yellow to wine red). After cooling, 30 nm AuNPs are obtained.

[0111] Step 2: Synthesis of polymer linker (PLG-Alk / Fc-III-4C, pL)

[0112] Under anhydrous conditions, 300 mg of polyglutamic acid (PLG), 77.7 mg of N-hydroxysuccinimide (NHS), and 129.4 mg of 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) were dissolved in 15 mL of anhydrous N,N-dimethylimide (DMF). The mixture was stirred at 30 °C for 4 h, then 104.1 mg of Fc-III-4C and 113.8 mg of triethylamine were added, and the reaction was continued at 30 °C for 24 h. Then, 75.4 mg of (4-alkynylphenyl)methylamine hydrochloride (Alk) was added, and the reaction was continued at 30 °C for another 48 h. The solution was then dialyzed sequentially with DMF and pure water and freeze-dried to obtain product pL. Its structural formula and 1H NMR spectrum are shown below. Figure 3 As shown, the synthesis process allows for precise adjustment of the degree of PLG polymerization, the degree of binding of Fc-III-4C, and Alk.

[0113] Based on elemental analysis and HPLC results, the product pL contains 6.59% Alk and 11.31% Fc-III-4C.

[0114] Step 3: Synthesis of SERS probe (AuNPs-MBA / pL)

[0115] 30 μL of 30 mM MBA solution was added to 12.0 mL of AuNPs dispersion. The mixture was stirred at room temperature for 30 min and then used directly to characterize SERS intensity. Figure 4 The wavelength of the laser was chosen to be 638 nm.

[0116] Dissolve 3 mg pL in 0.5 mL DMF and slowly add it dropwise to AuNPs-MBA (12 mL) solution while stirring vigorously at 60 °C. Continue the reaction for 3 h. Then centrifuge the AuNPs-MBA / pL solution (8000 rpm, 5 min), wash it 3 times with PBS buffer, and disperse it in 2 mL PBS buffer.

[0117] 1.8 μL of mouse IgG (10.01 mg / mL) and 1 mL of AuNPs-MBA / pL solution were mixed and incubated with PBS buffer at 4°C for 4 hours with shaking to prepare IgG@AuNPs-MBA / pL, which was used to characterize SERS intensity. Figure 4 The laser wavelength was chosen to be 638 nm, and TEM was used to characterize the aggregate SERS probe structure. Figure 5 ).

[0118] 1.8 μL of PDL1 antibody (10.01 mg / mL) and 1 mL of AuNPs-MBA / pL solution were mixed and incubated with PBS buffer at 4 °C for 4 hours with shaking to prepare aPDL1@AuNPs-MBA / pL for the detection of CTCs.

[0119] 4.3 CTCs detection methods ( Figure 6 )

[0120] Two mL of PBS buffer containing varying numbers of 4T1 cells was mixed with two mL of fresh mouse whole blood. Lymphocytes and 4T1 cell layers were extracted using mouse peripheral blood separation medium. Cells were washed twice with PBS buffer and then redispersed in 1 mL of PBS buffer. 100 μL of aPDL1@AuNPs-MBA / pL was added to the cell suspension and incubated at 37°C for 30 min. The sample was centrifuged at 1000 rpm for 5 min, washed three times with PBS buffer, and redispersed in 50 μL of PBS buffer. SERS intensity was characterized using a laser wavelength of 638 nm.

[0121] Figure 7 To detect the SERS patterns of different concentrations of 4T1 cells in mouse peripheral blood. From Figure 7 As shown in (a), the SERS signal gradually increases with increasing cell concentration, and a significant SERS signal is still visible at a concentration of 1 cells / mL, indicating that the SERS probe has sufficient sensitivity for cell detection. Figure 7 (c) It can be seen that the constructed SERS probe exhibits good linearity (RL) in the range of 1–10 cells / mL when used to simulate blood sample testing. 2 =0.962), which can be used for the detection of CTCs in clinical settings.

[0122] In summary, through the C-Au bond formed between the alkyne group (-C≡CH) of pL and Au, a 2-3 nm nanoscale gap is generated between AuNPs, forming a "hot spot" of gold nanoparticle aggregates. Its Raman signal is 4 times stronger than that of single-particle probes. The detection limit of this polymeric Fc-binding peptide Raman probe is 1 tumor cell / mL, which meets the requirements for clinical CTC detection. Fc-III-4C mediates universal targeting, is compatible with multiple antibodies, and can be applied to multiple cancer types.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polymeric Fc-binding peptide Raman probe, characterized in that, This includes polymeric linker structures, Raman reporter molecules, and enhancements to the substrate and antibody. The polymer linking structure includes the structure shown in formula (I): Equation (I); Where x, y, and z are the molar contents of repeating units, 0 < x < 1, 0 < y < 1, 0 < x + y < 1; z is the degree of polymerization, and z is an integer from 10 to 500; R1 is selected from substituted or unsubstituted C2-C10 alkyl groups and substituted or unsubstituted C6-C20 aryl groups; the substituents in the substituted C2-C10 alkyl groups and the substituted C6-C20 aryl groups are each independently selected from one or more of C1-C5 alkyl groups and C6-C10 aryl groups. R2 is selected from H or a cation; R3 is selected from an Fc-binding peptide that has lost an H residue; R4 is selected from groups containing an alkyne group; R5 is selected from H or C2~C10 acyl groups; L1, L2, and L3 are each independently selected from C1 to C5 alkylene groups; The enhanced substrate includes noble metal nanoparticles; The polymer linking structure is covalently connected to the reinforcing substrate.

2. The polymeric Fc-binding peptide Raman probe according to claim 1, characterized in that, The x is 0.8~0.98; the y is 0.005~0.4; and the z is an integer from 80 to 240. R1 is selected from C2-C8 alkyl groups or C6-C14 aryl groups; The R2 is selected from H, metal cations, or organic cations; The R3 is selected from a residue of Fc-III-4C that has lost one H; The R4 is selected from the group shown in formula (II): Formula (II); The R5 is selected from H or C2~C5 acyl groups; R6 is selected from C1-C8 alkylene groups, C6-C14 aryl groups, or groups formed by connecting the above groups through single bonds; L1, L2 and L3 are each independently selected from C1 to C2 alkylene groups.

3. The polymeric Fc-binding peptide Raman probe according to claim 2, characterized in that, R1 is selected from ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, phenyl, naphthyl, biphenyl, or anthracene; The R2 is selected from H, sodium ions, potassium ions, ammonium ions, or positively charged amino acid ions; The R5 is selected from H, formyl, acetyl, propionyl, or butyryl; R6 is selected from C1-C5 alkylene groups, C6-C10 aryl groups, or groups formed by connecting the above groups through single bonds.

4. The polymeric Fc-binding peptide Raman probe according to claim 1, characterized in that, The polymer linking structure has the structure shown in formula (III): Formula (III); x is 0.85~0.95; y is 0.005~0.4; z is an integer from 80 to 240.

5. The polymeric Fc-binding peptide Raman probe according to claim 1, characterized in that, The Raman reporter molecule is selected from p-mercaptobenzoic acid, p-mercaptobenzonitrile, and p-nitrobenzyl mercaptan; The noble metal nanoparticles are selected from gold nanoparticles and / or silver nanoparticles.

6. The polymeric Fc-binding peptide Raman probe according to claim 1, characterized in that, The antibody is selected from one or more of the following: 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. A method for preparing the polymeric Fc-binding peptide Raman probe according to claim 1, characterized in that, Includes the following steps: The polymer linking structure shown in formula (I) is mixed with an enhanced substrate and heated to obtain a polymeric Fc-binding peptide Raman probe.

8. A reagent kit, characterized in that, The polymeric Fc-binding peptide Raman probe includes any one of claims 1 to 6.

9. The reagent kit according to claim 8, characterized in that, The kit is used to detect the level of antigen expression on tumor cells of cancer patients.

10. The reagent kit according to claim 9, characterized in that, The cancers mentioned include one or more of the following: malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, malignant tumors of the testis, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, leukemia, lymphoma, and multiple myeloma.

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