Immune raman multicolor imaging kit for liver cancer prediction and evaluation

Abstract

This invention relates to an immunoRaman multicolor imaging kit for the prediction and assessment of liver cancer. The immunoRaman multicolor imaging reagent contains five Raman probes, which are composed of Raman spheres and antibodies attached to the surface of the Raman spheres. The Raman spheres are polymerized from triple-bonded monomers with (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10–100 nm and a Raman shift of 1800–2800 cm⁻¹. ‑1 The antibodies used for the five Raman probes are selected from one of the following: AFP antibody, HBX antibody, GPC3 antibody, GS antibody, and CD34 antibody. The immunoRaman multicolor imaging reagent and kit provided by this invention can achieve one-step labeling and multiplex color development of five liver cancer-related protein markers in the same liver tissue section. Compared with multiplex fluorescence immunohistochemistry, this invention has significant advantages in both efficiency and accuracy. Experiments show that the detection results of this invention are consistent with clinical diagnostic results, demonstrating great application potential.

Description

Technical Field

[0001] This invention relates to the field of precision medical diagnostics, and in particular to an immunoRaman multicolor imaging kit for the prediction and assessment of liver cancer. Background Technology

[0002] In clinical practice, liver cancer is mostly diagnosed at an advanced stage. Its early symptoms are often subtle, its malignancy is high, and its prognosis is poor, hence it is known in the medical community as a "silent killer." Studies show that the early cure rate for liver cancer can exceed 70%, but current early diagnosis still mainly relies on imaging examinations and routine histopathological examinations. However, imaging examinations are easily interfered with by factors such as hemangiomas, non-uniform fatty liver, cirrhosis, and hepatic dysplastic nodules; routine histopathological examinations involve cumbersome and time-consuming procedures such as tissue sectioning and staining, and the diagnostic results often depend on the pathologist's subjective judgment. These uncertain results can easily lead to misdiagnosis, thus affecting the timeliness and effectiveness of treatment plans.

[0003] Globally, there are approximately 257 million people chronically infected with hepatitis B, of whom 30%–40% will develop cirrhosis or liver cancer. In my country, 80% of liver cancer patients have a history of hepatitis B-related cirrhosis, and hepatitis B infection-related liver cancer accounts for as high as 84%. Hepatitis B virus infection is considered a core pathogenic factor for liver cancer. Although antiviral therapy can reduce the risk of liver cancer, it cannot eliminate the risk (the annual incidence of liver cancer still exceeds 1%). Therefore, rapid and accurate detection of the causes of liver cancer is of great significance. Revealing the deep-seated mechanisms of persistent hepatitis B virus-induced carcinogenesis is key to the radical cure of liver cancer. Detection of liver cancer-related biomarkers, through multi-indicator feedback on tumor etiology, can effectively improve the sensitivity and specificity of liver cancer diagnosis, providing strong support for accurate diagnosis of liver cancer. Currently, there is no standardized clinical method for simultaneous detection of multiple molecular biomarkers related to liver cancer. The main difficulties lie in the identification of biomarkers and the signal overlap problem when detecting multiple proteins simultaneously, and existing multiplex imaging is costly. Summary of the Invention

[0004] This invention provides an immunoRaman multicolor imaging kit for the prediction and assessment of liver cancer. This kit simultaneously images and detects five emerging causative proteins of hepatitis B virus-induced liver cancer, as well as multiple proteins in the liver cancer microenvironment during development. These five liver cancer-related proteins are: alpha-fetoprotein (AFP), glutamine synthase (GS), phosphatidylinositol proteoglycan 3 (GPC3), liver susceptibility virus protein (HBX), and a highly glycosylated type I transmembrane glycoprotein (CD34). These proteins are closely related to viral infection status, malignancy, and metastatic progression in primary liver cancer. Simultaneous detection can effectively achieve differential diagnosis of liver cancer etiology and assess the malignancy and progression of liver cancer. This invention achieves innovative "one-step labeling, multiplex color development" detection of key proteins in liver cancer etiology using this kit. Compared with traditional immunohistochemical detection, this invention has significant advantages in accuracy and efficiency, and the experimental results fully meet clinical testing requirements.

[0005] The technical solution provided by this invention is as follows:

[0006] In a first aspect, the present invention provides an immunoRaman multicolor imaging reagent for the prediction and assessment of liver cancer, comprising five Raman probes, wherein the Raman probes are composed of Raman spheres and antibodies attached to the surface of the Raman spheres; wherein:

[0007] The Raman spheres are polymerized from triple-bonded monomers with (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10~100 nm and a Raman shift of 1800~2800 cm⁻¹. -1 The triple-bonded monomers of the five Raman probes are selected from compounds 1 to 7, wherein compounds 1 and 2 are not used simultaneously, and compounds 3 and 4 are used simultaneously. The structural formulas of compounds 1 to 7 are shown below:

[0008] ;

[0009] The antibodies for the five Raman probes were derived from one of the following: AFP antibody, HBX antibody, GPC3 antibody, GS antibody, and CD34 antibody.

[0010] In conjunction with the first aspect of the present invention, in some embodiments: the triple bond monomers in the five Raman probes are compound 2, compound 3, compound 5, compound 6, and compound 7, respectively;

[0011] The characteristic peak of the Raman probe with compound 2 as the triple bond monomer is at 2160 cm⁻¹. -1 ;

[0012] The characteristic peak of the Raman probe with compound 3 as the triple bond monomer is 2186 cm⁻¹. -1 ;

[0013] The characteristic peak of the Raman probe with compound 5 as the triple bond monomer is 2227 cm⁻¹. -1 ;

[0014] The characteristic peak of the Raman probe with compound 6 as the triple bond monomer is 2241 cm⁻¹. -1 ;

[0015] The characteristic peak of the Raman probe with compound 7 as the triple bond monomer is 2260 cm⁻¹. -1 .

[0016] In conjunction with the first aspect of the invention, some embodiments include:

[0017] The antibody on the Raman probe of compound 2, whose triple bond monomer is AFP antibody, is an AFP antibody.

[0018] The antibody on the Raman probe of compound 3, which is a triple bond monomer, is the GPC3 antibody.

[0019] The antibody on the Raman probe of compound 5, which has a triple bond monomer, is the HBX antibody.

[0020] The antibody on the Raman probe of compound 6, whose triple bond monomer is GS antibody, is a GS antibody.

[0021] The antibody on the Raman probe of compound 7, which is a triple-bonded monomer, is a CD34 antibody.

[0022] In conjunction with the first aspect of the invention, some embodiments include:

[0023] The Raman sphere is chemically bonded to the antibody; and / or,

[0024] The Raman spheres have a particle size of 30~70 nm.

[0025] Secondly, the present invention provides an immunoRaman multicolor imaging kit for predicting and evaluating liver cancer, comprising the above-mentioned immunoRaman multicolor imaging reagent; in use, liver tissue sections are co-incubated with the immunoRaman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL.

[0026] In conjunction with a second aspect of the invention, in some embodiments: the immunoRaman multicolor imaging kit further includes PBS, wherein the total volume of the PBS is configured such that, after mixing with all immunoRaman multicolor imaging reagents, the concentration of each Raman probe is not less than 0.5 mg / mL.

[0027] Thirdly, this invention provides a liver cancer prediction and assessment system, including a sample processing module, a collection module, and an acquisition module, wherein the modules are physically or communicatively connected; wherein:

[0028] The sample processing module is configured to incubate a liver tissue slice of the subject with the above-mentioned immunoRaman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL;

[0029] The acquisition module is configured to acquire Raman imaging information of the liver tissue slices;

[0030] The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, liver susceptibility virus protein, phosphatidylinositol proteoglycan 3, glutamine synthase, and highly glycosylated type I transmembrane glycoprotein based on Raman imaging information.

[0031] In conjunction with the third aspect of the present invention, in some embodiments: the liver cancer prediction and assessment system further includes an assessment module:

[0032] The assessment module is configured to assess the subject's risk of developing liver cancer, the cause of liver cancer, the degree of malignancy, and the metastasis based on the expression level.

[0033] In conjunction with the third aspect of the present invention, in some embodiments: the evaluation module is specifically configured to: perform multi-dimensional evaluation based on the differences in expression levels of various biomarkers, using the average level of healthy liver tissue as a reference, including:

[0034] The risk of liver cancer in subjects was assessed based on the level of alpha-fetoprotein expression.

[0035] The level of expression of hepatitis B virus (HBV) proteins in the liver can be used to determine whether liver cancer is caused by HBV.

[0036] The malignancy and risk of progression of liver cancer can be assessed based on the expression level of phosphatidylinositol proteoglycan 3.

[0037] The status of liver cancer metastasis was assessed based on the combined expression levels of glutamine synthase and highly glycosylated type I transmembrane glycoproteins.

[0038] Fourthly, the present invention provides a liver cancer prediction and assessment instrument, including the above-mentioned liver cancer prediction and assessment system.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] 1. The immunoRaman multicolor imaging reagent and kit provided by the present invention can achieve one-step labeling and multiple color development of five liver cancer-related protein markers in the same liver tissue section. It is simple to operate, low in cost, and has high labeling efficiency.

[0041] 2. Compared with existing liver cancer diagnostic methods, the immunoRaman multicolor imaging kit provided by this invention is the first to achieve simultaneous imaging of five liver cancer-related biomarkers: AFP, HBX, GPC3, GS, and CD34. It is also the first to construct a method for predicting liver cancer and assessing its progression based on the combined imaging results. The expression levels of these five biomarkers in the patient's tumor tissue are used for the first time to assess the risk of hepatitis B-related liver cancer, its degree of malignancy, and metastasis. This guides clinicians in providing scientific and personalized treatment methods for liver cancer patients and provides the latest evidence for the prognosis and treatment of patients.

[0042] 3. The reagent kit provided by this invention is simple to prepare, has good stability, long shelf life, and is easy to scale up for production. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 The images show the Raman spectra of monomers 1 through 7.

[0045] Figure 2 The images shown are transmission electron microscope (TEM) images of the nanospheres prepared in Example 2 of this invention. a is a nanosphere polymerized from compound 2; b is a nanosphere polymerized from compound 3; c is a nanosphere polymerized from compound 5; d is a nanosphere polymerized from compound 6; and e is a nanosphere polymerized from compound 7.

[0046] Figure 3 The images shown are Raman spectra of the compounds before and after polymerization in Example 2 of this invention. M1 is the Raman spectrum of compound 2; P1 is the Raman spectrum of nanospheres of compound 2; M2 is the Raman spectrum of compound 3; P2 is the Raman spectrum of nanospheres of compound 3; M3 is the Raman spectrum of compound 5; P3 is the Raman spectrum of nanospheres of compound 5; M4 is the Raman spectrum of compound 6; P4 is the Raman spectrum of nanospheres of compound 6; M5 is the Raman spectrum of compound 7; P5 is the Raman spectrum of nanospheres of compound 7.

[0047] Figure 4 The following are infrared spectra of the compounds before and after polymerization in Example 2 of this invention: M1 is the infrared spectrum of compound 2; P1 is the infrared spectrum of nanospheres of compound 2; M2 is the infrared spectrum of compound 3; P2 is the infrared spectrum of nanospheres of compound 3; M3 is the infrared spectrum of compound 5; P3 is the infrared spectrum of nanospheres of compound 5; M4 is the infrared spectrum of compound 6; P4 is the infrared spectrum of nanospheres of compound 6; M5 is the infrared spectrum of compound 7; P5 is the infrared spectrum of nanospheres of compound 7.

[0048] Figure 5 The flowchart illustrates the preparation and use of the immunoRaman multicolor imaging kit provided by this invention.

[0049] Figures 6a to 6j Raman imaging images of the immunoRaman probe prepared in Example 5 to confirm hepatitis B infection and liver cancer diagnosis; wherein:

[0050] Figure 6a For Raman imaging of AFP protein in Example 5, a 2160 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0051] Figure 6b For example, in Raman imaging of Example 5, the corresponding 2160 cm was selected. -1 Raman spectra of characteristic peaks;

[0052] Figure 6c For Raman imaging of HBX protein in Example 5, a 2227 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0053] Figure 6d For example, in Raman imaging of Example 5, the corresponding 2227 cm was selected. -1 Raman spectra of characteristic peaks;

[0054] Figure 6e For Raman imaging of the GPC3 protein in Example 5, a 2186 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0055] Figure 6f For example, in Raman imaging of Example 5, the corresponding 2186 cm⁻¹ was selected. -1 Raman spectra of characteristic peaks;

[0056] Figure 6g For Raman imaging of the GS protein in Example 5, a 2241 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0057] Figure 6h For example, in Raman imaging of Example 5, the corresponding 2241 cm was selected. -1 Raman spectra of characteristic peaks;

[0058] Figure 6i For Raman imaging of CD34 protein in Example 5, a 2260 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0059] Figure 6jFor example, in Raman imaging of Example 5, the corresponding 2260 cm⁻¹ was selected. -1 The Raman spectrum of the characteristic peaks is shown.

[0060] Figures 7a to 7j The immunoRaman probe prepared for Example 6 is used to demonstrate Raman imaging of liver cancer progression; wherein:

[0061] Figure 7a For Raman imaging of AFP protein in Example 6, a 2160 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0062] Figure 7b For example, in Raman imaging of Example 6, the corresponding 2160 cm⁻¹ was selected. -1 Raman spectra of characteristic peaks;

[0063] Figure 7c For Raman imaging of HBX protein in Example 6, a 2227 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0064] Figure 7d For example, in Raman imaging of Example 6, the corresponding 2227 cm was selected. -1 Raman spectra of characteristic peaks;

[0065] Figure 7e For Raman imaging of the GPC3 protein in Example 6, a 2186 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0066] Figure 7f For example, in Raman imaging of Example 6, the corresponding 2186 cm⁻¹ was selected. -1 Raman spectra of characteristic peaks;

[0067] Figure 7g For Raman imaging of the GS protein in Example 6, a 2241 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0068] Figure 7h For example, in the Raman imaging of Example 6, the corresponding 2241 cm was selected. -1 Raman spectra of characteristic peaks;

[0069] Figure 7i For Raman imaging of CD34 protein in Example 6, a 2260 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0070] Figure 7j For example, in Raman imaging of Example 6, the corresponding 2260 cm⁻¹ was selected.-1 The Raman spectrum of the characteristic peaks is shown.

[0071] Figures 8a to 8j The immunoRaman probes prepared for Example 7 were used to demonstrate Raman imaging of liver cancer progression and metastasis; wherein:

[0072] Figure 8a For Raman imaging of AFP protein in Example 7, a 2160 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0073] Figure 8b For example, in the Raman imaging of Example 7, the corresponding 2160 cm was selected. -1 Raman spectra of characteristic peaks;

[0074] Figure 8c For Raman imaging of HBX protein in Example 7, a 2227 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0075] Figure 8d For example, in the Raman imaging of Example 7, the corresponding 2227 cm was selected. -1 Raman spectra of characteristic peaks;

[0076] Figure 8e For Raman imaging of the GPC3 protein in Example 7, a 2186 cm⁻¹ microscope was used. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0077] Figure 8f For example, in Raman imaging of Example 7, the corresponding 2186 cm⁻¹ was selected. -1 Raman spectra of characteristic peaks;

[0078] Figure 8g For Raman imaging of the GS protein in Example 7, a 2241 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0079] Figure 8h For example, in the Raman imaging of Example 7, the corresponding 2241 cm was selected. -1 Raman spectra of characteristic peaks;

[0080] Figure 8i For Raman imaging of CD34 protein in Example 7, a 2260 cm⁻¹ spectral density was selected. -1 Raman imaging pattern with characteristic peaks of specific Raman signals;

[0081] Figure 8j For example, in Raman imaging of Example 7, the corresponding 2260 cm⁻¹ was selected. -1 The Raman spectrum of the characteristic peaks is shown. Detailed Implementation

[0082] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0083] In this article, the term "immunoRaman multicolor imaging reagent" is defined as a reagent containing five Raman probes, wherein the Raman probes consist of Raman spheres and antibodies attached to their surfaces; the Raman spheres are polymerized from triple-bonded monomers with acrylic acid or styrene and acrylic acid, with a particle size of 10–100 nm and a Raman shift of 1800–2800 cm⁻¹. -1 The antibodies for the five probes are AFP antibody, HBX antibody, GPC3 antibody, GS antibody, and CD34 antibody.

[0084] In this article, the term "Raman probe" is defined as a detection unit consisting of a Raman sphere and a specific antibody attached to its surface. Each probe has a unique Raman characteristic peak and can specifically identify liver cancer-related protein biomarkers.

[0085] In this paper, the term "Raman sphere" is defined as nanoparticles polymerized from triple-bonded monomers with (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10–100 nm and a Raman shift of 1800–2800 cm⁻¹. -1 It is the core structure of the Raman probe.

[0086] In this paper, the term "triple bond monomer" is defined as a compound (compounds 1 to 7) used in the synthesis of Raman spheres, possessing specific Raman characteristic peaks, wherein compounds 2, 3, 5, 6, and 7 correspond to 2160 cm⁻¹, respectively. -1 2186 cm -1 2227 cm -1 2241 cm -1 2260 cm -1 Characteristic peaks.

[0087] In this article, the term "AFP antibody" is defined as an alpha-fetoprotein antibody attached to the surface of a Raman sphere, used to detect alpha-fetoprotein (AFP) expression levels and assess the risk of liver cancer.

[0088] In this article, the term "HBX antibody" is defined as an antibody against liver susceptibility virus protein attached to the surface of a Raman sphere, used to detect hepatitis B virus X protein (HBX) and determine whether liver cancer is caused by hepatitis B virus.

[0089] In this article, the term "GPC3 antibody" is defined as a phosphatidylinositol proteoglycan 3 antibody attached to the surface of a Raman sphere, used to detect GPC3 protein expression levels and assess the malignancy and risk of progression of liver cancer.

[0090] In this article, the term "GS antibody" is defined as a glutamine synthase antibody attached to the surface of a Raman sphere, used to detect GS protein expression levels and, in conjunction with CD34, to assess liver cancer metastasis.

[0091] In this article, the term "CD34 antibody" is defined as a highly glycosylated type I transmembrane glycoprotein antibody attached to the surface of a Raman sphere, used to detect CD34 protein expression levels and in conjunction with GS to assess liver cancer metastasis.

[0092] In this article, the term "immunoRaman multicolor imaging kit" is defined as a detection tool containing immunoRaman multicolor imaging reagents and PBS. When used, liver tissue sections are incubated with the reagents in PBS (each probe concentration is not less than 0.5 mg / mL) to achieve simultaneous detection of five biomarkers.

[0093] In this article, the term "liver cancer prediction and assessment system" is defined as a system that includes modules for sample processing, collection, acquisition, and assessment, used for liver tissue section incubation, Raman imaging acquisition, biomarker expression analysis, and multi-dimensional assessment of liver cancer risk, etiology, malignancy, and metastasis.

[0094] In this article, "positive expression" is defined as an expression level that is significantly higher than the average expression level of healthy liver tissue.

[0095] This invention selects HBX, GS, GPC3, AFP, and CD34 as core biomarkers for hepatocellular carcinoma caused by hepatitis B, based on their specific coverage and complementarity of the hepatitis B carcinogenesis mechanism. HBx, as the hepatitis B virus X protein, is the "causal label" of hepatitis B-related hepatocellular carcinoma; HBx protein can be detected in 85% of hepatitis B-related hepatocellular carcinoma tissues, directly linking to the virus-driven source of carcinogenesis. GPC3 is a hepatocellular carcinoma-specific membrane protein, with a positive rate as high as 82% in hepatitis B-related hepatocellular carcinoma, and is almost not expressed in benign lesions outside the background of hepatitis B cirrhosis, allowing for precise differentiation between hepatitis B-related malignant proliferation and benign hepatocyte regeneration. Although alpha-fetoprotein (AFP) is a classic biomarker for hepatocellular carcinoma, its dynamic changes in hepatitis B-related hepatocellular carcinoma are unique. Elevated AFP levels in patients with chronic hepatitis B are often associated with liver damage, and relying solely on AFP levels can lead to misdiagnosis. Glutamine synthase (GS), a glutamine synthase, is diffusely and strongly expressed in hepatitis B-related hepatocellular carcinoma (HCC) due to abnormal activation of the Wnt / β-catenin pathway, reflecting virus-induced metabolic reprogramming of hepatocytes—a key metabolic characteristic of hepatitis B carcinogenesis. CD34, a marker of tumor vascular endothelium, is also present in HCC with a "completely positive" vascular pattern (staining of sinusoidal vessels throughout the lobules) due to high expression of virus-induced angiogenesis factors (AGF), with a positive rate of 89%, reflecting virus-driven tumor angiogenesis activity and associated with HCC invasiveness. Therefore, these five indicators pinpoint key links in the etiological chain: AFP for HCC early warning, HBx for viral integration, GS for metabolic adaptation, GPC3 / AFP for malignant transformation, and CD34 for microenvironmental changes. This study proposes for the first time a non-overlapping etiological-pathological assessment system for HCC formation based on simultaneous detection of HBx, GS, GPC3, AFP, and CD34.

[0096] This invention mixes five Raman probes with different Raman characteristic peaks to form an immunoRaman multicolor imaging reagent, which is used for immunodetection point labeling of liver tissue sections. Based on the Raman characteristic peaks of the five Raman probes, simultaneous multicolor imaging of tissue is achieved, and the imaging results can be analyzed as a method for diagnosing the etiology of liver cancer.

[0097] The method for preparing the above-mentioned Raman probes according to the present invention includes: polymerizing a triple-bonded monomer with (i) acrylic acid or (ii) styrene and acrylic acid to form Raman spheres with a particle size of 10-100 nm; activating the carboxyl groups on the surface of the Raman spheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and connecting the carboxyl groups on the surface of the Raman spheres to the amino groups of the target molecule using NHS, including: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide to an aqueous dispersion of the Raman spheres at room temperature to activate the carboxyl groups on the surface of the Raman spheres; adding N-hydroxysuccinimide ester to the aqueous dispersion of the Raman spheres, stirring for at least 30 minutes, and then adding the target molecule and reacting for at least 2 hours. When preparing each Raman probe, the added target molecule is selected from one of AFP antibody, HBX antibody, GPC3 antibody, GS antibody, and CD34 antibody.

[0098] In some embodiments, when preparing the Raman probe, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the reaction solution is 10-50 mg / mL, the mass fraction of N-hydroxysuccinimide ester is 10-50 mg / mL, and the mass ratio of Raman sphere to target molecule is 50-100:1.

[0099] During immunodetection labeling, the immunoRaman multicolor imaging reagent was co-incubated with the biological tissue sample for at least 12 hours, followed by washing to remove unbound Raman probes. The labeled biological sample was then imaged using a Raman spectrometer, with samples collected at 2160 cm⁻¹. -1 2186cm -1 2227cm -1 2241cm -1 and 2260cm -1 Raman signal at the location.

[0100] The liver cancer prediction and assessment system provided by this invention includes a sample processing module, a collection module, and an acquisition module, with physical or communication connections between the modules; wherein:

[0101] The sample processing module is configured to incubate a liver tissue slice of the subject with the above-mentioned immunoRaman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL;

[0102] The acquisition module is configured to acquire Raman imaging information of the liver tissue slices;

[0103] The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, liver susceptibility virus protein, phosphatidylinositol proteoglycan 3, glutamine synthase, and highly glycosylated type I transmembrane glycoprotein based on Raman imaging information.

[0104] In some embodiments, the liver cancer prediction and assessment system further includes an assessment module configured to assess the subject's risk of developing liver cancer, its etiology, malignancy, and metastasis based on the expression levels. For example, positive co-expression of AFP and HBX predicts a high likelihood of liver cancer and identifies the primary cause as hepatitis B virus, with existing data indicating a diagnostic accuracy of up to 100%. Simultaneous positivity of AFP, HBX, and GPC3 suggests a higher degree of liver cancer progression, potentially larger tumor size, possible complications such as cirrhosis, and a higher likelihood of metastasis. Simultaneous positivity of AFP, HBX, GPC3, GS, and CD34 indicates a high degree of liver cancer progression and the presence of metastases.

[0105] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0106] The following are embodiments of the present invention. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Compound 6, acrylonitrile, was purchased from Aladdin Reagent Company with a purity of 99%; compounds 1-5 and 7 were synthesized in the laboratory. Antibody information used in the embodiments is shown in Table 1.

[0107] Table 1 Antibody information used in the examples

[0108]

[0109] Clinical Sample Information: The paraffin blocks of liver tissue used in Examples 5-7 of this invention were all provided by Hubei Provincial People's Hospital. The preparation method of the paraffin blocks of liver tissue was as follows: After liver tissue was collected, it was fixed in 4% paraformaldehyde or neutral formalin fixative, followed by dehydration, clearing, paraffin impregnation, and embedding. The paraffin blocks were continuously sliced ​​into 5μm thick sections using a microtome. Subsequently, the prepared tissue sections were dewaxed and hydrated. The patient's clinical information was confirmed by the pathology department and clinicians, as follows:

[0110] Example 5: The patient was diagnosed with hepatitis B-related liver cancer (early stage), and no cirrhosis or distant metastases of the tumor were found.

[0111] Example 6: The patient was diagnosed with hepatitis B and cirrhosis, and the tumor was larger than 10 cm.

[0112] Example 7: The patient was diagnosed with hepatitis B and cirrhosis, and the liver cancer had metastasized to distant sites (bone metastasis).

[0113] Example 1: Synthesis of Monomers

[0114] 1. Synthesis of compound 1

[0115]

[0116] To a solution of methyl 3-bromo-4-iodobenzoate (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL), triisopropylsilylacetylene (218.4 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol), and bis(triphenylphosphine)palladium dichloride (14 mg, 0.2 mmol) were added. The mixture was cooled with liquid nitrogen to freeze the reaction system. 420 μL of dry triethylamine (TEA) was then added, and the mixture was refluxed in an oil bath at 65 °C for 24 h under N2 protection. After cooling to room temperature, the solvent was evaporated under reduced pressure, using a mixture of petroleum ether (PE) and ethyl acetate (EA) (volume ratio 10:1) as the eluent to give 300 mg of a white solid intermediate, with a yield of 94.2%.

[0117] The above-mentioned white solid intermediate (955 mg, 2.5 mmol) was dissolved in 20 mL of toluene, and tributylvinyltin (0.95 mL, 3 mmol) and tetrakis(triphenylphosphine)palladium (57.7 mg, 0.05 mmol) were added. The reaction system was cooled with liquid nitrogen to freeze it, then protected with N2, refluxed in an oil bath, and reacted at 115 °C for 24 h. The solvent was then evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of PE and EA (volume ratio 10:1) as the eluent to obtain a yellow liquid intermediate (0.7 g, 81.8%).

[0118] The yellow liquid intermediate (342 mg, 1 mmol) was dissolved in a mixed solution of methanol (MeOH, 5 mL) and water (1 mL), and lithium hydroxide (LiOH, 224 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, using a mixture of dichloromethane (DCM) and MeOH (10:1 v / v) as the eluent, to give 319 mg of compound 1 as a white solid, with a yield of 97.2%. 1H NMR (400 MHz, CDCl3): δ 8.30 (d, J = 4.0 Hz, 1H), 7.91 (q, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 5.94 (d, J = 16.0 Hz, 1H), 5.54 (d, J = 12.0Hz, 1H), 1.15 (s, 2H).

[0119] 2. Synthesis of compound 4

[0120]

[0121] To a solution of methyl 3-bromo-4-iodobenzoate (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL), 4-pentyn-1-ol (100.94 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol), and bis(triphenylphosphine)palladium dichloride (14 mg, 0.2 mmol) were added. The mixture was cooled with liquid nitrogen to freeze the reaction system. 420 μL of dry TEA was then added, and the mixture was refluxed in an oil bath at 65 °C for 24 h under N2 protection. After cooling to room temperature, the solvent was evaporated under reduced pressure, using a mixture of DCM and MeOH (100:1 v / v) as the eluent to give 300 mg of a pale yellow liquid intermediate, with a yield of 97.6%.

[0122] The above-mentioned pale yellow liquid intermediate (891 mg, 3 mmol) was dissolved in 20 mL of toluene, and tributylvinyltin (1.14 mL, 3.6 mmol) and tetrakis(triphenylphosphine)palladium (69 mg, 0.06 mmol) were added. The reaction system was cooled with liquid nitrogen to freeze it, then protected with N2, refluxed in an oil bath, and reacted at 115 °C for 24 h. The solvent was then evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of DCM and MeOH (100:1 v / v) as the eluent to obtain the yellow liquid intermediate (0.4 g, 54.6%).

[0123] The yellow liquid intermediate (244 mg, 1 mmol) was dissolved in a mixed solution of MeOH (5 mL) and water (1 mL), and LiOH (96 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, using a mixture of DCM and MeOH (10:1 v / v) as the eluent, to give 83 mg of compound 4 as a yellow solid, with a yield of 36.0%. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J = 4.0 Hz, 1H), 7.78 (q, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 20.0 Hz, 12 Hz, 1H), 5.98 (d, J = 20.0 Hz, 1H), 5.46 (d, J = 12.0 Hz, 1H), 3.53 (t, J = 8.0 Hz, 2H), 2.55 (t, J = 8.0 Hz, 2H), 1.72 (m, 2H).

[0124] 3. Synthesize compound 5

[0125]

[0126] 4-Bromobenzonitrile (1820.2 mg, 10 mmol) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 231 mg, 0.2 mmol) were added to a 250 mL three-necked flask. After removing all O2 from the flask using a double-row tube, tributylvinyltin (3804 mg, 12 mmol) was dissolved in 10 mL of toluene and injected into the flask using a syringe. The mixture was cooled with liquid nitrogen, completely frozen, and then O2 was removed three times. The mixture was refluxed and reacted at 120 °C and 600 rpm for 24 h. After quenching with saturated potassium fluoride solution, the reaction solution was extracted with EA and deionized water, washed three times with deionized water, and three times with saturated NaCl solution. The organic phase was dried over anhydrous NaSO4, concentrated by rotary evaporation, and purified by column chromatography using a mixture of PE and EA (20:1 v / v) as the eluent to give 435 mg of compound 5 as a clear liquid, with a yield of 34.0%. 1H NMR (400 MHz, CDCl3): δ 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J =8.0 Hz, 2H), 7.26 (s, 1H), 6.73 (q, J = 16.0 Hz, 1H), 5.88 (d, J = 20.0 Hz, 1H), 5.45 (d, J = 12.0 Hz, 1H), 1.60 (s, 1H).

[0127] 4. Synthesis of compound 2: 2-Methacrylic acid and p-iodobenzyl alcohol undergo esterification to produce p-iodobenzyl 2-methacrylic acid. The p-iodobenzyl 2-methacrylic acid then reacts with trimethylsilylacetylene under the catalysis of cuprous iodide and palladium dichloride to produce compound 2.

[0128] 5. Synthesis of compound 3: 2-Methylacrylic acid and trimethylsilylpropynol undergo esterification to generate compound 3.

[0129] 6. Synthesis of compound 7: 2-methacrylic acid and trimethylsilylpropynol undergo esterification to generate compound 3; compound 3 and propynol are coupled via Sonogashira under Pd / Cu catalysis to obtain compound 7.

[0130] Example 2: Preparation of Raman spheres

[0131] like Figure 1 As shown, the Raman characteristic peak of compound 1 is at 2154 cm⁻¹. -1 The Raman characteristic peak of compound 2 is 2160 cm⁻¹. -1 The Raman characteristic peak of compound 4 is 2214 cm⁻¹. -1 The Raman characteristic peak of compound 5 is 2227 cm⁻¹. -1Significant band overlap was observed. To improve the identification of characteristic peaks, this embodiment specifically selected five monomers for Raman sphere preparation: using a 2160 cm⁻¹... -1 Compound 2 with characteristic peak at 2186 cm⁻¹ -1 Compound 3 with characteristic peaks at 2227 cm⁻¹ -1 Compound 5 with characteristic peaks at 2241 cm⁻¹ -1 Compound 6 with characteristic peaks at 2260 cm⁻¹ -1 Compound 7, with its characteristic peaks, was used to construct Raman spheres with unique Raman fingerprint signals.

[0132] 1. Preparation of Raman spheres of compound 2: 0.06 g of the compound was used to prepare the Raman spheres with a characteristic peak at 2160 cm⁻¹. -1 Compound 2, 0.02 g of acrylic acid, 0.006 g of sodium dodecylbenzenesulfonate, and 5 mL of deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred continuously for 30 min. Then, 0.008 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was completed, unreacted compound 2 and emulsifier were removed by dialysis (dialysis membrane with a pore size of 8-14 kD), yielding a pure compound with a Raman characteristic peak at 2160 cm⁻¹. -1 Aqueous dispersion of compound 2 Raman spheres.

[0133] 2. Preparation of Raman spheres of compound 3: 0.2 g of the Raman sphere with a characteristic peak at 2186 cm⁻¹ was prepared. -1 Compound 3, 0.1 g styrene, 0.05 g acrylic acid, 0.008 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred for 30 min. 0.03 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 4 h. After the reaction was complete, unreacted compound 3 and emulsifier were removed by dialysis (dialysis membrane with a pore size of 8–14 kD), yielding a pure compound with a Raman characteristic peak at 2186 cm⁻¹. -1 Aqueous dispersion of compound 3 Raman spheres.

[0134] 3. Preparation of Raman spheres of compound 5: 0.1 g of the compound with a Raman characteristic peak at 2227 cm⁻¹ was prepared. -1 Compound 5, 0.02 g of acrylic acid, 0.005 g of sodium dodecylbenzenesulfonate, and 5 mL of deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred continuously for 30 min. 0.015 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was complete, unreacted compound 5 and the emulsifier were removed by dialysis (dialysis membrane with a pore size of 8–14 kD), yielding a pure compound with a Raman characteristic peak at 2227 cm⁻¹. -1 An aqueous dispersion of compound 5 Raman spheres.

[0135] 4. Preparation of Raman spheres for compound 6: 0.2 g styrene, 0.15 g acrylic acid, 0.015 g sodium dodecylbenzenesulfonate, and 10 mL deionized water were added to a reaction vessel. After purging with nitrogen gas to remove oxygen, the mixture was heated to 70 °C and stirred continuously for 30 min. Then, 0.45 g of a compound with a Raman characteristic peak at 2241 cm⁻¹ was added. -1 Acrylonitrile (compound 6) and 0.02 g of azobisisobutyronitrile were reacted thoroughly under constant pressure nitrogen protection for 2 h. After the reaction, unreacted compound 6 and emulsifier were removed by dialysis (dialysis membrane pore size 8~14 kD), yielding a pure compound with a Raman characteristic peak at 2241 cm⁻¹. -1 An aqueous dispersion of compound 6 Raman spheres.

[0136] 5. Preparation of Raman spheres of compound 7: 0.05 g of the compound 7 Raman spheres was prepared with a Raman characteristic peak at 2260 cm⁻¹. -1 Compound 7, 0.03 g styrene, 0.02 g acrylic acid, 0.003 g sodium dodecylbenzenesulfonate, and 5 mL deionized water were added to a reaction vessel. After purging with nitrogen to remove oxygen, the mixture was heated to 70 °C and stirred for 30 min. 0.01 g potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 5 h. After the reaction was complete, unreacted compound 7 and emulsifier were removed by dialysis (dialysis membrane with a pore size of 8–14 kD), yielding a pure compound with a Raman characteristic peak at 2260 cm⁻¹. -1 An aqueous dispersion of compound 7 Raman spheres.

[0137] Table 2. Raman spheres obtained in Example 2 and their preparation parameters

[0138]

[0139] Example 3: Synthesis of Raman Probe

[0140] In this embodiment, the target molecules (AFP antibody, HBX antibody, GPC3 antibody, GS antibody, CD34 antibody) are chemically coupled to the surface of the Raman sphere, giving it specific recognition ability.

[0141] Take 1 mL of each of the five Raman sphere aqueous dispersions prepared in Example 2 and treat them separately: add 5 μL of 50 mg / mL EDC aqueous solution and stir for 30 minutes to activate the carboxyl groups on the surface of the Raman spheres; add 5 μL of 50 mg / mL NHS aqueous solution as a coupling agent and continue stirring for 30 minutes; add 5 μL of the targeting molecule and continue stirring at room temperature for 2 hours to allow the targeting molecule to covalently bind to the carboxyl groups on the surface of the Raman spheres through amide bonds, thus obtaining the Raman probe.

[0142] In this embodiment, the surface of the compound 2 Raman sphere was modified with AFP antibody, and the characteristic peak was 2160 cm⁻¹.-1 Compound 3, with its Raman sphere surface modified with GPC3 antibody, exhibits a characteristic peak at 2186 cm⁻¹. -1 Compound 5, with its Raman sphere surface modified with HBX antibody, exhibits a characteristic peak at 2227 cm⁻¹. -1 Compound 6, with its Raman sphere surface modified with GS antibody, exhibits a characteristic peak at 2241 cm⁻¹. -1 Compound 7, with its surface modified with CD34 antibody and Raman spheres, exhibits a characteristic peak at 2260 cm⁻¹. -1 .

[0143] Example 4: Preparation of Immunoraramian Multicolor Imaging Reagent

[0144] The five Raman probes with different Raman characteristic peaks prepared in Example 3 were mixed in a Raman intensity ratio of 1:1:1:1:1 to obtain an immunoRaman multicolor imaging reagent. Note: The concentration of each Raman probe is approximately 1 mg / mL, but actual operation may vary. The experiment mainly aims to prepare a solution with approximately the same Raman intensity.

[0145] Example 5: Application in predicting liver cancer and hepatitis B-related liver cancer

[0146] The following experiment uses the immunoRaman multicolor imaging reagent prepared in Example 4 for the labeling and imaging of five liver cancer markers in liver tissue. The specific steps are as follows:

[0147] 1. Slide preparation: The embedded paraffin blocks are sectioned using a Leica pathology microtome. The sectioned tissue slides are placed in a 40°C water bath and spread out. The slide is tilted and inserted into the water to retrieve the slide, ensuring that the slide adheres to the appropriate position on the slide. The slides are then baked in a 60°C oven for 3 hours.

[0148] 2. Dewaxing of sections: Place the paraffin sections in xylene I (20 min) - xylene II (20 min) - xylene III (20 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% ethanol (5 min) - 90% ethanol (5 min) - 80% ethanol (5 min) - 70% ethanol (5 min) in sequence, and then rinse with distilled water for 5 min.

[0149] 3. Antigen Retrieval: Antigen retrieval is performed using an electric ceramic furnace. Place the dewaxed and hydrated tissue sections on a heat-resistant plastic slide holder in a beaker (or retrieval box). Add an appropriate amount of retrieval solution (0.01 M Tris-EDTA retrieval solution (pH 9.0)) to the beaker, ensuring the solution level covers the tissue sections to a certain height. Initially, heat the electric ceramic furnace on high to bring the solution to a boil. Once boiling, reduce to medium and start timing. The retrieval time is 15 minutes. During this process, do not allow the tissue to dry (ensure sufficient retrieval solution is available). After the time is up, remove the beaker from the microwave and allow it to cool naturally. Once the retrieval solution has cooled to room temperature, remove the slides and rinse them three times with TBST (pH 7.4), 3 minutes each time (do not rinse directly onto the tissue during rinsing to avoid damaging it).

[0150] 4. Add Raman probe: Add diluted immunoRaman multicolor imaging reagent and incubate in a humidified chamber at 4°C for 12 hours.

[0151] 5. Mounting: Wash the slides three times with PBS, wipe the liquid off the slides with absorbent paper, and mount with a cover slide.

[0152] 6. Imaging liver tissue sections using a commercially available ordinary Raman imaging system: The location of the tissue section was captured using a bright-field microscope. After selecting the imaging area, the following experimental conditions were set: laser 532 nm, laser power 50 mW, objective lens 50X, exposure time 1 s, integration times 1, and step size 50 μm.

[0153] 7. Image Processing: After the imaging test is completed, a 2160 cm² image will be selected. -1 2186 cm -1 2227 cm -1 2241 cm -1 2260 cm -1 Raman characteristic peaks impart different colors to tissue imaging regions, and the position of the characteristic peak corresponds to the positive expression position of the biomarker.

[0154] Results and Analysis:

[0155] like Figures 6a to 6j As shown: AFP (2160 cm) -1 ) and HBX (2227 cm) -1 The presence of GPC3 (2186 cm) indicates a significant positive expression, suggesting that the patient is a carrier of the hepatitis B virus and has been preliminarily diagnosed with liver cancer; -1 ), GS (2241 cm) -1 CD34 (2260 cm) -1 The low positive expression indicates that the cancer has not worsened and is currently at a low level of deterioration, which is consistent with the clinical diagnosis of early-stage liver cancer.

[0156] Example 6: Application in predicting the progression of hepatitis B-related liver cancer

[0157] Experimental procedure: Liver tissue sections were processed according to the procedure in Example 5, and Raman imaging was performed after incubation with immunoRaman multicolor imaging reagent for 12 h.

[0158] Results and Analysis:

[0159] like Figures 7a to 7j As shown, AFP, HBX, and GPC3 were clearly positive, confirming that the patient had hepatitis B-related liver cancer and was at risk of worsening (GPC3 grows synchronously with liver cancer cells, and positive expression confirms that liver cancer cells grow rapidly and have the risk of proliferation and spread, thus indicating that the patient's liver cancer may further worsen); GS and CD34 were not highly positive but were expressed to some extent, suggesting that the cancer had not yet progressed to an advanced stage, which is consistent with the clinical assessment of the risk of worsening of "tumor size exceeding 10 cm".

[0160] Example 7: Application in assessing the progression of hepatitis B-related liver cancer

[0161] Experimental procedure: Liver tissue sections were processed according to the procedure in Example 5, and Raman imaging was performed after incubation with immunoRaman multicolor imaging reagent for 12 h.

[0162] Results and Analysis:

[0163] like Figures 8a to 8j As shown: five liver cancer markers, AFP, HBX, GPC3, GS, and CD34, were clearly expressed positively; positive expression of AFP and HBX can clearly identify the patient as a hepatitis B-related liver cancer patient; positive expression of GPC3 can reflect the proliferation of malignant tumors, and its positive expression confirms that the liver cancer is in the growth and evolution stage; GS and CD34 reflect that the entire liver detection area is in a state of high metabolic activity and abnormal blood and energy supply, which can confirm that this sample has hepatitis B tumor metastasis and that the hepatitis B-related liver cancer has seriously deteriorated, consistent with the clinical result of "bone metastasis".

[0164] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. An immunoRaman multicolor imaging reagent for the prediction and assessment of liver cancer, characterized in that, It contains five Raman probes, each consisting of a Raman sphere and an antibody attached to the surface of the Raman sphere; wherein: The Raman spheres are polymerized from a three-key monomer and (i) acrylic acid or (ii) styrene and acrylic acid, the particle size of the Raman spheres is 30-70 nm, and the Raman shift is 1800-2800 cm -1 ; The triple bond monomers of the five Raman probes were selected from compounds 1 to 7, wherein compounds 1 and 2 were not used simultaneously, and compounds 3 and 4 were used simultaneously. The structural formulas of compounds 1 to 7 are shown below: ； The antibodies for the five Raman probes were derived from one of the following: AFP antibody, HBX antibody, GPC3 antibody, GS antibody, and CD34 antibody.

2. The immunoRaman multicolor imaging reagent according to claim 1, characterized in that: The triple bond monomers in the five Raman probes are compound 2, compound 3, compound 5, compound 6, and compound 7, respectively. The characteristic peak of the triple bond monomer for the Raman probe of compound 2 is 2160 cm -1 ; The characteristic peak of the triple bond monomer is 2186 cm -1 ; The characteristic peak of the Raman probe with compound 5 as the triple bond monomer is 2227 cm⁻¹. -1 ; The characteristic peak of the Raman probe with compound 6 as the triple bond monomer is 2241 cm⁻¹. -1 ; The characteristic peak of the Raman probe with compound 7 as the triple bond monomer is 2260 cm⁻¹. -1 .

3. The immunoRaman multicolor imaging reagent according to claim 2, characterized in that: The antibody on the Raman probe of compound 2, whose triple bond monomer is AFP antibody, is an AFP antibody. The antibody on the Raman probe of compound 3, which is a triple bond monomer, is the GPC3 antibody. The antibody on the Raman probe of compound 5, which has a triple bond monomer, is the HBX antibody. The antibody on the Raman probe of compound 6, whose triple bond monomer is GS antibody, is a GS antibody. The antibody on the Raman probe of compound 7, which is a triple-bonded monomer, is a CD34 antibody.

4. The immunoRaman multicolor imaging reagent according to claim 1, characterized in that: The Raman sphere is connected to the antibody via chemical bonds.

5. An immunoRaman multicolor imaging kit for predicting and assessing liver cancer, characterized in that, The reagent comprises the immunoRaman multicolor imaging reagent according to any one of claims 1 to 4; when used, liver tissue sections are incubated with the immunoRaman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL.

6. The immunoRaman multicolor imaging kit for predicting and assessing liver cancer according to claim 5, characterized in that, The immunoRaman multicolor imaging kit also includes PBS, wherein the total volume of the PBS is configured such that, after mixing with all immunoRaman multicolor imaging reagents, the concentration of each Raman probe is not less than 0.5 mg / mL.

7. A liver cancer prediction and assessment system, characterized in that, This includes a sample processing module, a data acquisition module, and an acquisition module, with physical or communication connections between these modules; among which: The sample processing module is configured to incubate a liver tissue slice of the subject with the immunoRaman multicolor imaging reagent as described in any one of claims 1 to 4 in PBS, wherein the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL; The acquisition module is configured to acquire Raman imaging information of the liver tissue slices; The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, liver susceptibility virus protein, phosphatidylinositol proteoglycan 3, glutamine synthase, and highly glycosylated type I transmembrane glycoprotein based on Raman imaging information.

8. The liver cancer prediction and assessment system according to claim 7, characterized in that: The liver cancer prediction and assessment system also includes an assessment module: The assessment module is configured to assess the subject's risk of developing liver cancer, the cause of liver cancer, the degree of malignancy, and the metastasis based on the expression level.

9. The liver cancer prediction and assessment system according to claim 8, characterized in that, The assessment module is specifically configured as follows: using the average level of healthy liver tissue as a reference, it performs multi-dimensional assessment based on the differences in the expression levels of various biomarkers, including: The risk of liver cancer in subjects was assessed based on the level of alpha-fetoprotein expression. The level of expression of hepatitis B virus (HBV) proteins in the liver can be used to determine whether liver cancer is caused by HBV. The malignancy and risk of progression of liver cancer can be assessed based on the expression level of phosphatidylinositol proteoglycan 3. The status of liver cancer metastasis was assessed based on the combined expression levels of glutamine synthase and highly glycosylated type I transmembrane glycoproteins.

10. A liver cancer prediction and assessment instrument, characterized in that: The system includes the liver cancer prediction and assessment system as described in any one of claims 7 to 9.

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