Digestive tumor inflammation pathology dynamic detection and targeted therapy method

Abstract

The invention discloses a digestive tumor inflammation pathology dynamic detection and targeted therapy method, and relates to the technical field of biological sample detection analysis, and the method comprises the following specific steps: 1, collecting tissue samples and blood samples from digestive tumor inflammation patients, non-tumor inflammation patients and healthy people, the method has the advantages that tissue and blood samples are collected from a large number of digestive tumor inflammation patients, non-tumor inflammation patients and healthy people, the specific recognition molecules are specifically combined with the digestive tumor inflammation specific markers in the samples, and the signal intensity is detected through the fluorospectro photometer; according to the method, the content of the marker is obtained through comparison with a standard curve, the illness state is judged according to a threshold value, the whole process is linked with one another, specific recognition and combination of unique biomarkers related to digestive tumor inflammation are achieved from sample collection to result judgment, false positive results are effectively reduced, and the accuracy of clinical diagnosis is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological sample detection and analysis, and particularly to a method for dynamic detection and targeted therapy of digestive tumor inflammation pathology. Background Art

[0002] Digestive tumor inflammation is a class of diseases that seriously threaten human health, covering various types such as gastric cancer, colorectal cancer, liver cancer, etc. Its incidence rate shows an increasing trend year by year. According to the statistical data of the World Health Organization, the number of newly added patients with digestive tumor inflammation is numerous globally every year, and the mortality rate remains high. These diseases not only bring great physical pain to patients, but also cause a heavy economic burden on families and society. The occurrence and development of digestive tumor inflammation is a complex process, involving abnormal changes in multiple genes and signaling pathways. In the early stage of the disease, patients often have no obvious symptoms and are easily ignored, resulting in delayed illness. When obvious symptoms appear, most patients are already in the middle and late stages, the treatment effect is greatly reduced, and the prognosis is poor. Therefore, early and accurate diagnosis and dynamic monitoring of digestive tumor inflammation are crucial for improving the survival rate and quality of life of patients; There are certain defects in the existing technology. First of all, there is a lack of specific markers. Most of the currently known digestive tumor inflammation-related markers lack high specificity and may also show abnormal expression in other non-tumor inflammatory diseases or physiological states. This makes it easy to obtain false positive results when detecting only one or a few markers, interfering with clinical diagnosis. Secondly, the existing technology has deficiencies in aspects such as sample anticoagulation stability, marker mining accuracy, and reagent performance, seriously affecting the diagnostic accuracy of digestive tumor inflammation. For this reason, we propose a method for dynamic detection and targeted therapy of digestive tumor inflammation pathology. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for dynamic detection and targeted therapy of digestive tumor inflammation pathology.

[0004] To solve the problems raised in the above background art, the present invention provides the following technical solution: A method for dynamic detection of digestive tumor inflammation pathology, the detection method includes the following specific steps: Step 1: Collect tissue samples and blood samples from patients with digestive tumor inflammation, non-tumor inflammation patients, and healthy people. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from non-tumor inflammation patients are inflammatory tissues, and the samples collected from healthy people are normal digestive tract tissues. At the same time, collect the blood samples of each person. Add sodium citrate to the collected blood samples, and then divide the collected samples into two parts. Perform proteomics analysis and transcriptomics analysis on half of the samples. After screening out potential markers through data analysis, use immunofluorescence technology for verification to complete the screening and verification of potential specific markers; Step 2: According to the structural data of the screened potential specific markers, perform molecular docking simulation with the help of Schrödinger software to design aptamers, and carry out chemical synthesis of aptamers by the solid-phase phosphoramidite triester method. Using Controlled Pore Glass as the carrier, perform synthesis on an automatic nucleic acid synthesizer. Each nucleotide addition cycle includes deprotection, coupling, capping, and oxidation steps; Step 3: Prepare yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles by the hydrothermal method. Dissolve yttrium chloride, ytterbium chloride, and erbium chloride in water, add oleic acid and octadecene and stir to form a homogeneous solution. Then transfer the solution to a high-pressure reaction kettle for reaction. After the reaction is completed and cooled naturally, the product is washed alternately with ethanol and n-hexane, and then the product is collected by centrifugation. Next, couple the aptamer with the upconversion nanoparticles. Disperse the upconversion nanoparticles in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol for reaction, and then add the aptamer containing a thiol group for reaction. After washing with PBS buffer, redisperse the coupled product in PBS buffer to obtain an aptamer-upconversion nanoparticle complex; Step 4: Use citric acid and urea as raw materials to synthesize functionalized carbon quantum dots by the microwave-assisted method. Add the aptamer-upconversion nanoparticle complex to a solution containing functionalized carbon quantum dots, add polyvinylpyrrolidone as a dispersant and dimethyl sulfoxide, stir evenly and then let stand to complete the preparation of the detection reagent solution; Step 5: Collect blood samples and tissue samples from patients. Centrifuge the blood samples to separate the serum, digest and centrifuge the tissue samples to take the supernatant. Take a certain amount of the sample supernatant and serum and add them to the detection reagent solution, mix well and incubate. After the incubation is completed, use a fluorescence spectrophotometer to detect the upconversion luminescence signal and the fluorescence signal of the carbon quantum dots. Using the blank control as a reference, record the signal intensity of the sample, establish a standard curve with a standard product of known concentration, substitute the sample signal intensity into the standard curve, calculate the marker content, and determine the threshold according to clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

[0005] As a further solution of the present invention: in the above Step 1, collect tissue samples and blood samples from 600 - 700 patients with digestive tumor inflammation, 400 - 500 patients with non-tumor inflammation, and 300 - 400 healthy people. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from patients with non-tumor inflammation are inflammatory tissues, and the samples collected from healthy people are normal digestive tract tissues. The collection amount of each sample is 6g - 8g, and blood samples are collected from each person. The collection amount of the blood sample is 8mL - 12mL. Add sodium citrate with a concentration of 3.0% - 4.0% to the collected blood samples.

[0006] As a further solution of the present invention: in the first step, for proteomics analysis, the isotope-coded relative and absolute quantification technique is adopted. First, the proteins in the sample are extracted, and then the extracted proteins are subjected to a labeling reaction. The labeling reaction lasts for 1.5 h - 2.5 h at 35°C - 38°C. After the labeling is completed, separation is carried out using a liquid chromatograph with a C18 chromatographic column. During the separation process, different elution gradients are set so that different proteins can be separated in the chromatographic column according to their own characteristics, and information on the types, contents, and modification states of the proteins in the sample is obtained. For transcriptomics analysis, the digital gene expression profiling technique is adopted. First, RNA is extracted from the sample, and then the extracted RNA is sequenced and analyzed using the SOLiD sequencing platform. The sequencing depth is maintained at 100X - 150X to obtain gene expression data and understand the gene expression situation in the sample, including information on which genes are up-regulated and which genes are down-regulated. After potential markers are screened out through data analysis, they are verified using immunofluorescence techniques. Another half of the collected sample is used. In the immunofluorescence experiment, the primary antibody is incubated at 3°C - 5°C for 12 h - 18 h, the secondary antibody is incubated at 36°C - 38°C for 1 h - 2 h, and the in situ hybridization reaction temperature is controlled at 40°C - 45°C for 12 h - 20 h.

[0007] As a further solution of the present invention: in the second step, according to the structural data of the screened potential specific markers, molecular docking simulation is carried out with the help of Schrödinger software to design aptamers. During the design process, by flexibly adjusting the nucleotide sequence and spatial conformation of the aptamers, their high specificity and affinity for the markers are ensured. The coupling reaction time is controlled at 3 min - 5 min. After the synthesis is completed, 25% - 30% concentrated ammonia water is used to treat at 50°C - 60°C for 12 h - 18 h to remove the protecting groups and release the aptamers from the carrier, thus completing the design and synthesis of the specific recognition molecules.

[0008] As a further solution of the present invention: in the step three, yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles are prepared by a hydrothermal method. 0.4 mmol - 0.6 mmol of yttrium chloride, 0.4 mmol - 0.5 mmol of ytterbium chloride, and 0.04 mmol - 0.06 mmol of erbium chloride are dissolved in 8 mL - 12 mL of water, 8 mL - 12 mL of oleic acid and 8 mL - 12 mL of octadecene are added, and the mixture is stirred at 110 °C - 130 °C for 0.5 h - 1.5 h to form a homogeneous solution. Subsequently, the solution is transferred to a high-pressure reactor and reacted at 180 °C - 220 °C for 6 h - 10 h. After the reaction is completed and naturally cooled, the product is washed alternately with ethanol and n-hexane for 2 - 4 times, collected by centrifugation at 7000 rpm - 9000 rpm for 8 min - 12 min, and then the aptamer is coupled with the upconversion nanoparticles. The upconversion nanoparticles are dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol, and surface-functionalized by stirring at 35 °C - 39 °C for 5 h - 7 h. Then, a nucleic acid aptamer containing a thiol group is added and reacted at room temperature for 16 h - 20 h. After the reaction is completed, it is separated by centrifugation at 9000 rpm - 11000 rpm for 12 min - 18 min, washed 2 - 4 times with a PBS buffer solution with a pH value of 7.2 - 7.6, and the coupled product is redispersed in the PBS buffer solution to obtain an aptamer-upconversion nanoparticle complex.

[0009] As a further solution of the present invention: in the step four, functionalized carbon quantum dots are synthesized by a microwave-assisted method using citric acid and urea as raw materials. Citric acid and urea are mixed according to a molar ratio of 1:1.5 - 2.5, dissolved in deionized water according to a volume ratio of 1:20 - 30, then transferred to a microwave reaction vessel, and microwave irradiated at a power of 400 W - 600 W for 4 min - 10 min. After the reaction is completed, it is cooled to room temperature, and impurities are removed by filtration through a 0.2 μm - 0.4 μm filter membrane to obtain a solution containing functionalized carbon quantum dots. The aptamer-upconversion nanoparticle complex is added to the solution containing functionalized carbon quantum dots so that the final concentration of the upconversion nanoparticles is maintained at 0.04 mg / mL - 0.12 mg / mL, and the final concentration of the carbon quantum dots is 0.1 mg / mL - 0.4 mg / mL. At the same time, 0.6% - 1.0% of polyvinylpyrrolidone is added as a dispersant, and 0.2% - 0.4% of dimethyl sulfoxide. After stirring evenly, it is left standing at 3 °C - 5 °C for 2 h - 6 h.

[0010] As a further solution of the present invention: in the fifth step, 5 mL - 7 mL of the patient's blood sample and 4 g - 6 g of the tissue sample are collected. After the blood sample is collected, it is centrifuged at 3°C - 5°C and 3000 rpm - 4000 rpm for 15 min - 25 min to separate the serum. After the tissue sample is minced, it is added with a lysis buffer containing proteinase K and incubated at 35°C - 39°C for 1 h - 3 h for digestion. Then, it is centrifuged at 12000 rpm - 14000 rpm for 20 min - 30 min, and the supernatant is taken. 50 μL - 70 μL of the sample supernatant and serum are respectively taken and added to 100 μL - 140 μL of the detection reagent solution. After mixing, it is incubated in a constant temperature shaker at 35°C - 39°C for 1 h - 3 h, and the shaker speed is controlled at 120 rpm - 180 rpm.

[0011] As a further solution of the present invention: after the incubation in the fifth step, the upconversion luminescence signal is detected using a fluorescence spectrophotometer. The excitation wavelength is set at 960 nm - 990 nm, and the emission wavelength range is 510 nm - 690 nm. At the same time, the fluorescence signal of the carbon quantum dots is detected. The excitation wavelength is 350 nm - 370 nm, and the emission wavelength is 440 nm - 460 nm. Using the reagent only without the sample as the blank control as the reference, the signal intensity of the sample is recorded. The standard curve is established by detecting with the standard product of known concentration according to the above steps. The sample signal intensity is substituted into the standard curve to calculate the content of the biomarker. The threshold value is determined based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of the inflammation.

[0012] In addition, the present invention also provides a targeted treatment method for digestive tumor inflammation. After determining the structural data of the potential specific biomarker, a targeting molecule capable of specifically recognizing the biomarker is designed and synthesized. The targeting molecule is conjugated with a drug and a bioactive substance with therapeutic functions to form a targeted treatment complex. The targeted treatment complex is administered to the patient with digestive tumor inflammation, so that the targeted treatment complex can specifically bind to the potential specific biomarker at the tumor inflammation site, thereby realizing the targeted treatment of digestive tumor inflammation.

[0013] Adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention comprehensively analyzes by collecting tissue and blood samples from a large number of patients with digestive tumor inflammation, non-tumor inflammation patients, and healthy people, using proteomics and transcriptomics technologies. Through data processing and verification processes, potential markers with high specific expression in patients with digestive tumor inflammation are screened out to clarify the key targets for subsequent detection. Based on the structures of the screened potential markers, specific recognition molecules, including nucleic acid aptamers, are prepared by computer-aided design and chemical synthesis. These can highly specifically bind to unique biomarkers related to digestive tumor inflammation, ensuring the specificity of detection at the molecular level and laying a foundation for reducing false positive results. Then, a signal amplification system with yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles as the core is constructed and coupled with the specific recognition molecules. This system can significantly enhance the detection signal, enabling accurate detection even when the marker content is extremely low, greatly improving the detection sensitivity, making the detection results more reliable, further enhancing the recognition ability of low-abundance markers, and helping to accurately judge the condition. Next, the aptamer-upconversion nanoparticle complex is mixed with a functionalized carbon quantum dot solution, and the buffer solution formula is optimized and protective agents and stabilizers are added to ensure the stability and activity of the detection reagent under different conditions. This enables the specific recognition molecules to continuously and stably bind to the markers during the detection process, ensuring the reliability and repeatability of the detection results and reducing errors caused by reagent instability. Finally, after collecting and preprocessing the patient samples, they are mixed and incubated with the detection reagent. The specific recognition molecules specifically bind to the digestive tumor inflammation-specific markers in the samples, and the signal intensity is detected by a fluorescence spectrophotometer. By comparing with the standard curve, the marker content is obtained, and the condition is judged based on the threshold. The whole process is interlocked. From sample collection to result judgment, it realizes the specific recognition and binding of unique biomarkers related to digestive tumor inflammation, effectively reduces false positive results, and significantly improves the accuracy of clinical diagnosis; 2. In the sample collection step of the present invention, sodium citrate is used for blood anticoagulation to ensure the stability of the collected blood samples, providing reliable materials for subsequent analysis. Proteomics and transcriptomics analyses can achieve in-depth analysis of the samples to identify potential specific markers. When synthesizing specific recognition molecules, Controlled Pore Glass carriers support the synthesis of nucleic acid aptamers, and the solid-phase phosphoramidite method ensures the accuracy of synthesis. Concentrated ammonia water is used to remove protecting groups to obtain aptamers with high purity and correct structures, enhancing the specific binding ability to the markers. In the construction of the signal amplification system, various metal salts are used to synthesize upconversion nanoparticles, and oleic acid and octadecene assist in forming a homogeneous solution to ensure the performance of the nanoparticles. 5-Amino-1,3,4-thiadiazole-2-thiol functionalizes the surface of the nanoparticles, facilitating coupling with aptamers and significantly enhancing the detection signal intensity. When preparing the detection reagent solution, citric acid and urea are used to synthesize functionalized carbon quantum dots, polyvinylpyrrolidone prevents particle aggregation, dimethyl sulfoxide enhances stability, and the optimized buffer maintains the activity of the composite molecules to ensure the stability and reliability of the detection reagent. These materials work together to comprehensively ensure the accuracy, specificity, and sensitivity of the detection from sample acquisition to final detection, greatly improving the accuracy of the diagnosis of digestive tumor inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic flowchart of the method steps in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following further describes the specific embodiments of the present invention with reference to the drawings. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention.

[0016] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0017] Please refer to the attached Figure 1 , A method for dynamically detecting the pathology of digestive tumor inflammation according to the present invention, the detection method includes the following specific steps: Step 1: Collect tissue samples and blood samples from patients with digestive tumor inflammation, patients with non-tumor inflammation, and healthy people. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from patients with non-tumor inflammation are inflammatory tissues, and the samples collected from healthy people are normal digestive tract tissues. At the same time, collect the blood samples of each person. Add sodium citrate to the collected blood samples, and then divide the collected samples into two parts. Perform proteomics analysis and transcriptomics analysis on half of the samples. After screening for potential markers through data analysis, use immunofluorescence technology for verification to complete the screening and verification of potential specific markers; Step 2: Based on the structural data of the screened potential specific markers, molecular docking simulation is carried out with the help of Schrödinger software to design aptamers. The chemical synthesis of aptamers is carried out by the solid-phase phosphoramidite triester method, using Controlled Pore Glass as the carrier and carried out on an automatic nucleic acid synthesizer. Each nucleotide addition cycle includes deprotection, coupling, capping, and oxidation steps; Step 3: Ytterbium, erbium co-doped sodium fluoride upconversion nanoparticles are prepared by a hydrothermal method. Yttrium chloride, ytterbium chloride, and erbium chloride are dissolved in water, oleic acid and octadecene are added and stirred to form a homogeneous solution. Subsequently, the solution is transferred to a high-pressure reaction kettle for reaction. After the reaction is completed and cooled naturally, the product is washed alternately with ethanol and n-hexane, and then the product is collected by centrifugation. Then, the coupling of the aptamer and the upconversion nanoparticles is carried out. The upconversion nanoparticles are dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol for reaction, and then an aptamer containing a thiol group is added for reaction. After washing with PBS buffer, the coupled product is redispersed in PBS buffer to obtain an aptamer-upconversion nanoparticle complex; Step 4: Using citric acid and urea as raw materials, functionalized carbon quantum dots are synthesized by a microwave-assisted method. The aptamer-upconversion nanoparticle complex is added to a solution containing functionalized carbon quantum dots, polyvinylpyrrolidone is added as a dispersant and dimethyl sulfoxide, and after stirring evenly, it is left standing to complete the preparation of the detection reagent solution; Step 5: Collect blood samples and tissue samples from patients. The blood samples are centrifuged to separate serum, and the tissue samples are digested and centrifuged to take the supernatant. A certain amount of the sample supernatant and serum are respectively added to the detection reagent solution, mixed evenly and incubated. After the incubation is completed, the upconversion luminescence signal and the fluorescence signal of the carbon quantum dots are detected using a fluorescence spectrophotometer. Using the blank control as a reference, record the signal intensity of the sample, establish a standard curve with a standard product of known concentration, substitute the sample signal intensity into the standard curve, calculate the marker content, and determine the threshold based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

[0018] In an embodiment of the present invention: In Step 1, tissue samples and blood samples are collected from 600-700 patients with digestive tumor inflammation, 400-500 patients with non-tumor inflammation, and 300-400 healthy people. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from patients with non-tumor inflammation are inflammatory tissues, and the samples collected from healthy people are normal digestive tract tissues. The collection amount of each sample is 6g-8g, and blood samples are collected from each person. The collection amount of the blood sample is 8mL-12mL, and sodium citrate with a concentration of 3.0%-4.0% is added to the collected blood sample.

[0019] In one embodiment of the present invention: In step one, for proteomics analysis, the isotope-labeled relative and absolute quantification technique is adopted. First, the proteins in the sample are extracted, and then a labeling reaction is carried out on the extracted proteins. The labeling reaction lasts for 1.5 h - 2.5 h at 35°C - 38°C. After the labeling is completed, a liquid chromatography instrument with a C18 chromatographic column is used for separation. During the separation process, different elution gradients are set to enable different proteins to be separated in the chromatographic column according to their own characteristics, so as to obtain information on the types, contents, and modification states of the proteins in the sample. For transcriptomics analysis, the digital gene expression profiling technique is adopted. First, RNA is extracted from the sample, and then the extracted RNA is sequenced and analyzed using the SOLiD sequencing platform. The sequencing depth is maintained at 100X - 150X to obtain gene expression data and understand the gene expression situation in the sample, including information on which genes are up-regulated and which genes are down-regulated. After potential markers are screened out through data analysis, immunofluorescence technology is used for verification. Using the other half of the collected sample, in the immunofluorescence experiment, the primary antibody is incubated at 3°C - 5°C for 12 h - 18 h, the secondary antibody is incubated at 36°C - 38°C for 1 h - 2 h, and the in-situ hybridization reaction temperature is controlled at 40°C - 45°C for 12 h - 20 h.

[0020] In one embodiment of the present invention: In step two, according to the structural data of the screened potential specific markers, molecular docking simulation is carried out with the aid of Schrödinger software to design aptamers. During the design process, by flexibly adjusting the nucleotide sequence and spatial conformation of the aptamer, its high specificity and affinity for the marker are ensured. The coupling reaction time is controlled at 3 min - 5 min. After the synthesis is completed, the aptamer is treated with concentrated ammonia water with a concentration of 25% - 30% at 50°C - 60°C for 12 h - 18 h to remove the protecting groups and release the aptamer from the carrier, thus completing the design and synthesis of the specific recognition molecule.

[0021] In one embodiment of the present invention: In step three, yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles are prepared by a hydrothermal method. 0.4 mmol - 0.6 mmol of yttrium chloride, 0.4 mmol - 0.5 mmol of ytterbium chloride, and 0.04 mmol - 0.06 mmol of erbium chloride are dissolved in 8 mL - 12 mL of water, 8 mL - 12 mL of oleic acid and 8 mL - 12 mL of octadecene are added, and the mixture is stirred at 110 °C - 130 °C for 0.5 h - 1.5 h to form a homogeneous solution. Subsequently, the solution is transferred to a high-pressure reaction kettle and reacted at 180 °C - 220 °C for 6 h - 10 h. After the reaction is completed and naturally cooled, the product is washed alternately with ethanol and n-hexane 2 - 4 times, collected by centrifugation at 7000 rpm - 9000 rpm for 8 min - 12 min, and then the aptamer is coupled with the upconversion nanoparticles. The upconversion nanoparticles are dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol, and surface functionalization is carried out by stirring at 35 °C - 39 °C for 5 h - 7 h. Then, a nucleic acid aptamer containing a thiol group is added and reacted at room temperature for 16 h - 20 h. After the reaction is completed, it is separated by centrifugation at 9000 rpm - 11000 rpm for 12 min - 18 min, washed 2 - 4 times with a PBS buffer solution with a pH value of 7.2 - 7.6, and the coupled product is redispersed in the PBS buffer solution to obtain an aptamer-upconversion nanoparticle complex.

[0022] In one embodiment of the present invention: In step four, functionalized carbon quantum dots are synthesized by a microwave-assisted method using citric acid and urea as raw materials. Citric acid and urea are mixed in a molar ratio of 1:1.5 - 2.5, dissolved in deionized water according to a volume ratio of 1:20 - 30, and then transferred to a microwave reaction vessel. Microwave radiation is carried out at a power of 400 W - 600 W for 4 min - 10 min. After the reaction is completed, it is cooled to room temperature, and impurities are removed by filtration through a 0.2 μm - 0.4 μm filter membrane to obtain a solution containing functionalized carbon quantum dots. The aptamer-upconversion nanoparticle complex is added to the solution containing functionalized carbon quantum dots so that the final concentration of the upconversion nanoparticles is maintained at 0.04 mg / mL - 0.12 mg / mL, and the final concentration of the carbon quantum dots is 0.1 mg / mL - 0.4 mg / mL. At the same time, 0.6% - 1.0% of polyvinylpyrrolidone is added as a dispersant, and 0.2% - 0.4% of dimethyl sulfoxide. After stirring evenly, it is left to stand at 3 °C - 5 °C for 2 h - 6 h.

[0023] In one embodiment of the present invention: In step five, 5 mL - 7 mL of the patient's blood sample and 4 g - 6 g of the tissue sample are collected. After the blood sample is collected, it is centrifuged at 3°C - 5°C and 3000 rpm - 4000 rpm for 15 min - 25 min to separate the serum. After the tissue sample is minced, a lysis buffer containing proteinase K is added, and it is incubated at 35°C - 39°C for 1 h - 3 h for digestion. Then, it is centrifuged at 12000 rpm - 14000 rpm for 20 min - 30 min, and the supernatant is taken. 50 μL - 70 μL of the sample supernatant and serum are respectively taken and added to 100 μL - 140 μL of the detection reagent solution. After mixing, it is incubated in a constant temperature shaker at 35°C - 39°C for 1 h - 3 h, and the shaker speed is controlled at 120 rpm - 180 rpm.

[0024] In one embodiment of the present invention: In step five, after the incubation is completed, an upconversion luminescence signal is detected using a fluorescence spectrophotometer. The excitation wavelength is set at 960 nm - 990 nm, and the emission wavelength range is 510 nm - 690 nm. At the same time, the fluorescence signal of the carbon quantum dots is detected. The excitation wavelength is 350 nm - 370 nm, and the emission wavelength is 440 nm - 460 nm. Using only the reagent without the sample as a blank control as a reference, the signal intensity of the sample is recorded. A standard curve is established by detecting a standard product with a known concentration according to the above steps. The sample signal intensity is substituted into the standard curve to calculate the biomarker content. The threshold is determined based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

[0025] In one embodiment of the present invention: In step one, the specific operation of extracting proteins from the sample is as follows: For the tissue sample, it is first minced and an appropriate amount of RIPA lysis buffer containing a protease inhibitor is added. The RIPA lysis buffer contains components such as Tris-HCl, NaCl, NP-40, sodium deoxycholate, and SDS, and the pH value is maintained at 7.4 - 7.5. Homogenization treatment is carried out on ice to fully break the cells. Then, the homogenate is centrifuged at 4°C at a speed of 12,000 rpm - 15,000 rpm for 15 min - 20 min, and the supernatant is taken to obtain a crude extract containing proteins. For the blood sample, serum or plasma is first separated by centrifugation, and then an equal volume of pre-cooled acetone is added, and it is precipitated at -20°C for 2 h - 4 h. Then, it is centrifuged at a speed of 10,000 rpm - 12,000 rpm for 10 min - 15 min, and the supernatant is discarded. The precipitate is washed 2 - 3 times with pre-cooled 70% ethanol, dried, and resuspended with an appropriate amount of protein lysis buffer to obtain a protein extract.

[0026] In one embodiment of the present invention: In step one, the separation of different proteins according to their own characteristics in the chromatographic column is specifically achieved by setting different elution gradients. During the liquid chromatography separation process, a low-concentration eluent is used in the initial stage, and proteins with relatively weak binding force to the chromatographic column are eluted first with a weak elution ability. As the elution time progresses, the concentration of the eluent is gradually increased to enhance the elution ability, so that proteins with relatively strong binding force to the chromatographic column are eluted in sequence. In this way, different proteins are separated in the C18 chromatographic column according to their own characteristics such as hydrophobicity, charge properties, and molecular size.

[0027] In one embodiment of the present invention: In step one, after the proteins in the sample are separated by the C18 chromatographic column, mass spectrometry analysis technology is used to identify the types, contents, and modification status information of the proteins. Each separated protein component is sequentially introduced into the mass spectrometer. Through the ionization process, protein molecules are converted into charged ions, and then under the action of an electric field and a magnetic field, they are separated and detected according to the mass-to-charge ratio of the ions. By comparing with a known protein database, the types of proteins are determined. Based on information such as the intensity and peak area of the ions, combined with a standard curve, the content of the proteins is quantitatively analyzed. At the same time, using the high resolution and accurate mass determination ability of the mass spectrometer, the mass changes of protein molecules, the presence of specific ions, etc. are analyzed to infer the modification status of the proteins, including modification information such as phosphorylation, glycosylation, and acetylation.

[0028] In one embodiment of the present invention: In step one, the operation of first extracting RNA from the sample is to cut the tissue sample into small pieces, add an appropriate amount of Trizol reagent, and homogenize thoroughly to lyse the cells. Then add chloroform, mix well by shaking, and centrifuge at 12,000 rpm for 15 min at 4°C. At this time, the solution will be layered, and the RNA is present in the upper aqueous phase. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, invert and mix well, and let it stand at -20°C for 10 min - 30 min to precipitate the RNA. Then centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash the precipitate with 75% ethanol 2 - 3 times, dry it, and dissolve the RNA with an appropriate amount of RNase-free water. For blood samples, use a special blood RNA extraction kit and operate according to the steps in the kit instruction manual. First, add an anticoagulant to prevent blood coagulation, then remove impurities such as cell debris by centrifugation, and then add a lysis solution to lyse the cells. Pure RNA is obtained through steps such as column adsorption, washing, and elution.

[0029] In an embodiment of the present invention: In step one, after screening out potential markers through data analysis, the specific data analysis steps are as follows: First, the data obtained from proteomics and transcriptomics analyses are standardized to eliminate experimental errors and batch effects. For proteomics data, the quantitative information of proteins in different samples is normalized to make the data between different samples comparable. For transcriptomics data, the gene expression levels are standardized, and the TPM (Transcripts Per Million) method is used for conversion. Then, statistical methods such as t-tests and analysis of variance are used to compare the data of samples from patients with digestive tumor inflammation, non-tumor inflammation patients, and healthy individuals. In the proteomics data, proteins with significantly higher or lower expression levels in the samples of patients with digestive tumor inflammation compared to those of non-tumor inflammation patients and healthy individuals are identified. In the transcriptomics data, genes with differential expression in patients with digestive tumor inflammation are screened out. Then, correlation analysis is performed on the selected potential protein and gene markers to study their mutual relationships. By constructing a protein-protein interaction network or a gene co-expression network, key markers or combinations of markers are identified.

[0030] In an embodiment of the present invention: In step one, the specific operation for verification using immunofluorescence technology is as follows: The samples for verification are processed. For tissue samples, they are cut into slices with a thickness of 4 μm - 8 μm and attached to polylysine-treated glass slides, fixed with 4% paraformaldehyde for 15 min - 30 min, then rinsed with PBS buffer three times, 5 minutes each time. Then, permeabilization treatment is carried out with a PBS solution containing 0.1% - 0.3% Triton X-100, incubated at room temperature for 10 min - 15 min, and rinsed with PBS three times, 5 min each time. Next, blocking is performed with a PBS solution containing 5% - 10% bovine serum albumin (BSA) or 10% normal goat serum, incubated at room temperature for 1 h - 2 h. The diluted primary antibody is dripped onto the slices and incubated in a humid box at 3°C - 5°C for 12 h - 18 h. Then, the slices are rinsed with PBS three times, 10 min - 15 min each time. Then, the fluorescently labeled secondary antibody is dripped onto the slices and incubated in a humid box at 36°C - 38°C for 1 h - 2 h. Finally, the slices are rinsed with PBS three times, 10 min - 15 min each time, anti-fluorescence quenching mounting medium is dripped, a coverslip is covered, and observation is carried out under a fluorescence microscope. The distribution and expression of potential markers in tissues or cells are judged according to the position and intensity of the fluorescence signals.

[0031] Example 1. Please refer to the appendix Figure 1, tissue samples and blood samples were collected from 700 patients with digestive tumor inflammation, 500 patients with non-tumor inflammation, and 400 healthy individuals. The amount of each tissue sample collected was 8 g, and the amount of blood sample collected was 12 mL. Sodium citrate with a concentration of 4.0% was added to the collected blood samples. Half of the collected samples were taken for proteomics and transcriptomics analysis. For proteomics analysis, isotope-labeled relative and absolute quantification technology was used. The extracted proteins were subjected to a 2.5-h labeling reaction at 38 °C. After labeling, separation was performed using a liquid chromatography instrument with a C18 chromatographic column by setting different elution gradients to obtain protein-related information. For transcriptomics analysis, digital gene expression profiling technology was used. After extracting RNA from the samples, sequencing analysis was performed using the SOLiD sequencing platform at a sequencing depth of 150X to obtain gene expression data. After screening for potential markers through data analysis, the other half of the collected samples were used. In the immunofluorescence experiment, the primary antibody was incubated at 5 °C for 18 h, and the secondary antibody was incubated at 38 °C for 2 h. The in situ hybridization reaction was carried out at 45 °C for 20 h to complete the screening and verification of potential specific markers. According to the structural data of the screened potential specific markers, nucleic acid aptamers were designed using Schrödinger software and synthesized on an automatic nucleic acid synthesizer using the solid-phase phosphoramidite triester method with Controlled Pore Glass as the carrier. The reaction time for each nucleotide addition cycle coupling was controlled at 5 min. After synthesis, it was treated with 30% concentrated ammonia water at 60 °C for 18 h to remove the protecting groups and release the aptamer, completing the design and synthesis of specific recognition molecules. The hydrothermal method was used to prepare yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles. 0.6 mmol of yttrium chloride, 0.5 mmol of ytterbium chloride, and 0.06 mmol of erbium chloride were dissolved in 12 mL of water, and 12 mL of oleic acid and 12 mL of octadecene were added. It was stirred at 130 °C for 1.5 h to form a homogeneous solution, transferred to a high-pressure reaction kettle, and reacted at 220 °C for 10 h. After the reaction ended and cooled naturally, the product was washed alternately with ethanol and n-hexane 4 times and collected by centrifugation at 9000 rpm for 12 min. Then, the upconversion nanoparticles were dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol and stirred at 39 °C for 7 h for surface functionalization. The nucleic acid aptamer containing a thiol group was added and reacted at room temperature for 20 h. After the reaction was completed, it was separated by centrifugation at 11000 rpm for 18 min and washed 4 times with PBS buffer with a pH of 7.6. The coupled product was redispersed in PBS buffer to obtain the aptamer-upconversion nanoparticle complex. Citric acid and urea were dissolved in deionized water at a molar ratio of 1:2.5 and a volume ratio of 1:30, transferred to a microwave reaction vessel, and microwave irradiated at a power of 600 W for 10 min. After the reaction ended and cooled to room temperature, with 0.Filter impurities with a 4-μm filter membrane to obtain a solution containing functionalized carbon quantum dots. Add the aptamer-upconversion nanoparticle complex to it, keeping the final concentration of upconversion nanoparticles at 0.12 mg / mL and the final concentration of carbon quantum dots at 0.4 mg / mL. At the same time, add 1.0% polyvinylpyrrolidone as a dispersant and 0.4% dimethyl sulfoxide. After stirring evenly, let it stand at 5 °C for 6 h to complete the preparation of the detection reagent solution. Collect 7 mL of the patient's blood sample and 6 g of tissue sample. Centrifuge the blood sample at 5 °C and 4000 rpm for 25 min to separate the serum. After cutting the tissue sample into pieces, add lysis buffer containing proteinase K and incubate at 39 °C for 3 h for digestion. Then centrifuge at 14000 rpm for 30 min and take the supernatant. Take 70 μL of the sample supernatant and serum respectively, and add them to 140 μL of the detection reagent solution. After mixing evenly, incubate in a constant temperature shaker at 39 °C for 3 h, and control the shaker speed at 180 rpm. After the incubation, use a fluorescence spectrophotometer to detect. Set the excitation wavelength at 990 nm to detect the upconversion luminescence signal (the emission wavelength range is 510 nm - 690 nm), and set the excitation wavelength at 370 nm to detect the carbon quantum dot fluorescence signal (the emission wavelength is 440 nm - 460 nm). Record the sample signal intensity with only the reagent without the sample as the blank control as the reference. Use the standard product with a known concentration to detect according to the above steps to establish a standard curve. Substitute the sample signal intensity into the standard curve to calculate the biomarker content. Determine the threshold based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

[0032] Example 2. Please refer to the appendix Figure 1, tissue samples and blood samples were collected from 600 patients with digestive tumor inflammation, 400 patients with non-tumor inflammation, and 300 healthy individuals. The collection amount of each tissue sample was 6 g, and the collection amount of blood sample was 8 mL. Sodium citrate with a concentration of 3.0% was added to the collected blood samples. Half of the collected samples were taken for proteomics and transcriptomics analysis. Isotope labeling relative and absolute quantification technology was used for proteomics analysis. The extracted proteins were subjected to a 1.5-h labeling reaction at 35 °C. After the labeling was completed, separation was carried out using a liquid chromatograph with a C18 chromatographic column by setting different elution gradients to obtain protein-related information. Digital gene expression profiling technology was used for transcriptomics analysis. After extracting RNA from the samples, sequencing analysis was performed using the SOLiD sequencing platform at a sequencing depth of 100X to obtain gene expression data. After screening out potential markers through data analysis, the other half of the collected samples were used. In the immunofluorescence experiment, the primary antibody was incubated at 3 °C for 12 h, and the secondary antibody was incubated at 36 °C for 1 h. The in situ hybridization reaction continued at 40 °C for 12 h to complete the screening and verification of potential specific markers. According to the structural data of the screened potential specific markers, nucleic acid aptamers were designed with the help of Schrödinger software and synthesized on an automatic nucleic acid synthesizer using the solid-phase phosphoramidite triester method with Controlled Pore Glass as the carrier. The cycle coupling reaction time for each nucleotide addition was controlled at 3 min. After the synthesis was completed, it was treated with 25% concentrated ammonia water at 50 °C for 12 h to remove the protecting groups and release the aptamer, completing the design and synthesis of specific recognition molecules. The yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles were prepared by the hydrothermal method. 0.4 mmol of yttrium chloride, 0.4 mmol of ytterbium chloride, and 0.04 mmol of erbium chloride were dissolved in 8 mL of water, 8 mL of oleic acid and 8 mL of octadecene were added, and the mixture was stirred at 110 °C for 0.5 h to form a homogeneous solution. Then it was transferred to a high-pressure reaction kettle and reacted at 180 °C for 6 h. After the reaction ended and cooled naturally, the product was washed alternately with ethanol and n-hexane 2 times and collected by centrifugation at 7000 rpm for 8 min. Then the upconversion nanoparticles were dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol and stirred at 35 °C for 5 h for surface functionalization. The thiol-containing nucleic acid aptamer was added and reacted at room temperature for 16 h. After the reaction was completed, it was separated by centrifugation at 9000 rpm for 12 min and washed 2 times with PBS buffer with a pH value of 7.2. The coupling product was redispersed in PBS buffer to obtain the aptamer-upconversion nanoparticle complex. Citric acid and urea were dissolved in deionized water at a molar ratio of 1:1.5 and a volume ratio of 1:20, transferred to a microwave reaction vessel, and microwave irradiated at a power of 400 W for 4 min. After the reaction ended and cooled to room temperature, impurities were removed by filtering through a 0.2-μm filter membrane to obtain a solution containing functionalized carbon quantum dots. The aptamer-upconversion nanoparticle complex was added to it so that the final concentration of the upconversion nanoparticles remained at 0.The final concentration of the carbon quantum dots is 0.1 mg / mL in a solution with a concentration of 0.4 mg / mL. Meanwhile, 0.6% polyvinylpyrrolidone is added as a dispersant and 0.2% dimethyl sulfoxide. After stirring evenly, the mixture is left to stand at 3°C for 2 h to complete the preparation of the detection reagent solution. 5 mL of the patient's blood sample and 4 g of the tissue sample are collected. The blood sample is centrifuged at 3000 rpm for 15 min at 3°C to separate the serum. After the tissue sample is minced, it is added to the lysis buffer containing proteinase K and incubated at 35°C for 1 h for digestion. Then, it is centrifuged at 12,000 rpm for 20 min to obtain the supernatant. 50 μL of the sample supernatant and serum are respectively added to 100 μL of the detection reagent solution. After mixing evenly, the mixture is incubated in a constant temperature shaker at 35°C for 1 h, and the shaker speed is controlled at 120 rpm. After the incubation, a fluorescence spectrophotometer is used for detection. The excitation wavelength is set at 960 nm to detect the upconversion luminescence signal (the emission wavelength range is 510 nm - 690 nm), and the excitation wavelength is 350 nm to detect the fluorescence signal of the carbon quantum dots (the emission wavelength is 440 nm - 460 nm). The sample signal intensity is recorded with only the reagent without the sample as the blank control as the reference. A standard curve is established by detecting the known concentration standards according to the above steps. The sample signal intensity is substituted into the standard curve to calculate the biomarker content. The threshold is determined based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

[0033] It can be concluded from the above two groups of embodiment contents that by collecting tissue and blood samples from a large number of different populations, comprehensively analyzing and screening using proteomics and transcriptomics technologies, then designing and synthesizing specific recognition molecules based on the structure of potential biomarkers, constructing a signal amplification system, and preparing a stable detection reagent solution, and finally collecting patient samples for detection and other series of closely linked operations, the specific recognition and binding of unique biomarkers related to digestive tumor inflammation are achieved, effectively reducing false positive results, significantly improving the accuracy of clinical diagnosis, and demonstrating the significant technical advantages and broad application prospects of the process of the present invention in the field of digestive tumor inflammation detection.

[0034] Although the present invention is disclosed above in a preferred embodiment, it is not used to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, all modifications, equivalent changes, and decorations made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for dynamically detecting the inflammation pathology of digestive tumors, characterized in that, The detection method includes the following specific steps: Step 1: Collect tissue samples and blood samples from patients with digestive tumor inflammation, patients with non-tumor inflammation, and healthy individuals. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from patients with non-tumor inflammation are inflammatory tissues, and the samples collected from healthy individuals are normal digestive tract tissues. At the same time, collect the blood samples of each person. Add sodium citrate to the collected blood samples, and then divide the collected samples into two parts. Perform proteomics analysis and transcriptomics analysis on half of the samples. After screening out potential markers through data analysis, use immunofluorescence technology for verification to complete the screening and verification of potential specific markers; Step 2: According to the structural data of the screened potential specific markers, use Schrödinger software for molecular docking simulation to design aptamers, and use the solid-phase phosphoramidite triester method for the chemical synthesis of aptamers. Using Controlled Pore Glass as the carrier, perform synthesis on an automatic nucleic acid synthesizer. Each nucleotide addition cycle includes deprotection, coupling, capping, and oxidation steps; Step 3: Prepare yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles by the hydrothermal method. Dissolve yttrium chloride, ytterbium chloride, and erbium chloride in water, add oleic acid and octadecene and stir to form a homogeneous solution. Then transfer the solution to a high-pressure reaction kettle for reaction. After the reaction is completed and naturally cooled, the product is washed alternately with ethanol and n-hexane, and then the product is collected by centrifugation. Then perform the coupling of the aptamer and the upconversion nanoparticles. Disperse the upconversion nanoparticles in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol for reaction, and then add the aptamer containing a thiol group for reaction. After washing with PBS buffer, redisperse the coupling product in PBS buffer to obtain an aptamer-upconversion nanoparticle complex; Step 4: Use citric acid and urea as raw materials and apply the microwave-assisted method to synthesize functionalized carbon quantum dots. Add the aptamer-upconversion nanoparticle complex to the solution containing functionalized carbon quantum dots, add polyvinylpyrrolidone as a dispersant and dimethyl sulfoxide, stir evenly and then let it stand to complete the preparation of the detection reagent solution; Step 5: Collect the patient's blood samples and tissue samples. Centrifuge the blood samples to separate the serum, and digest and centrifuge the tissue samples to obtain the supernatant. Take a certain amount of the sample supernatant and serum and add them to the detection reagent solution, mix well and incubate. After the incubation is completed, use a fluorescence spectrophotometer to detect the upconversion luminescence signal and the fluorescence signal of the carbon quantum dots. Using the blank control as a reference, record the signal intensity of the sample, establish a standard curve with a standard product of known concentration, substitute the sample signal intensity into the standard curve, calculate the marker content, and determine the threshold based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

2. The dynamic detection method for digestive tumor inflammation pathology according to claim 1, characterized in that: In the first step, tissue samples and blood samples are collected from 600 - 700 patients with digestive tumor inflammation, 400 - 500 patients with non-tumor inflammation, and 300 - 400 healthy individuals. The samples collected from patients with digestive tumor inflammation are tumor tissues, the samples collected from patients with non-tumor inflammation are inflammatory tissues, and the samples collected from healthy individuals are normal digestive tract tissues. The collection amount of each sample is 6g - 8g, and blood samples are collected from each person with a collection amount of 8mL - 12mL. Sodium citrate with a concentration of 3.0% - 4.0% is added to the collected blood samples.

3. The dynamic detection method for digestive tumor inflammation pathology according to claim 1, wherein: In the first step, for proteomics analysis, the isotope-labeled relative and absolute quantification technique is adopted. First, the proteins in the samples are extracted, and then the extracted proteins are subjected to a labeling reaction. The labeling reaction lasts for 1.5h - 2.5h at 35℃ - 38℃. After the labeling is completed, a liquid chromatograph with a C18 chromatographic column is used for separation. During the separation process, different elution gradients are set to enable different proteins to be separated in the chromatographic column according to their own characteristics, and information on the types, contents, and modification states of the proteins in the samples is obtained. For transcriptomics analysis, the digital gene expression profile technique is adopted. First, RNA is extracted from the samples, and then the extracted RNA is sequenced and analyzed using the SOLiD sequencing platform. The sequencing depth is maintained at 100X - 150X to obtain gene expression data and understand the gene expression conditions in the samples, including information on which genes are up-regulated and which genes are down-regulated. After potential markers are screened out through data analysis, immunofluorescence technology is used for verification. The other half of the collected samples are used. In the immunofluorescence experiment, the primary antibody is incubated at 3℃ - 5℃ for 12h - 18h, the secondary antibody is incubated at 36℃ - 38℃ for 1h - 2h, and the in-situ hybridization reaction temperature is controlled at 40℃ - 45℃ for 12h - 20h.

4. A method for dynamically detecting the inflammation pathology of digestive tumors according to claim 1, characterized in that: In the second step, according to the structural data of the screened potential specific markers, molecular docking simulation is carried out with the aid of Schrödinger software to design aptamers. During the design process, by flexibly adjusting the nucleotide sequences and spatial conformations of the aptamers, their high specificity and affinity for the markers are ensured. The coupling reaction time is controlled at 3min - 5min. After the synthesis is completed, the aptamers are treated with concentrated ammonia water with a concentration of 25% - 30% at 50℃ - 60℃ for 12h - 18h to remove the protecting groups and release the aptamers from the carriers, thus completing the design and synthesis of the specific recognition molecules.

5. A method for dynamically detecting the inflammation pathology of digestive tumors according to claim 1, characterized in that: In the third step, yttrium ytterbium erbium co-doped sodium fluoride upconversion nanoparticles are prepared by a hydrothermal method. 0.4 mmol - 0.6 mmol of yttrium chloride, 0.4 mmol - 0.5 mmol of ytterbium chloride, and 0.04 mmol - 0.06 mmol of erbium chloride are dissolved in 8 mL - 12 mL of water, 8 mL - 12 mL of oleic acid and 8 mL - 12 mL of octadecene are added, and the mixture is stirred at 110 °C - 130 °C for 0.5 h - 1.5 h to form a homogeneous solution. Subsequently, the solution is transferred to a high-pressure reaction kettle and reacted at 180 °C - 220 °C for 6 h - 10 h. After the reaction is completed and cooled naturally, the product is washed alternately with ethanol and n-hexane 2 - 4 times, collected by centrifugation at 7000 rpm - 9000 rpm for 8 min - 12 min, and then the coupling of the aptamer and the upconversion nanoparticles is carried out. The upconversion nanoparticles are dispersed in a solution containing 5-amino-1,3,4-thiadiazole-2-thiol, and surface functionalization is carried out by stirring at 35 °C - 39 °C for 5 h - 7 h. Then, a nucleic acid aptamer containing a thiol group is added, and the reaction is carried out at room temperature for 16 h - 20 h. After the reaction is completed, it is separated by centrifugation at 9000 rpm - 11000 rpm for 12 min - 18 min, washed 2 - 4 times with PBS buffer solution with a pH value of 7.2 - 7.6, and the coupling product is redispersed in PBS buffer solution to obtain an aptamer-upconversion nanoparticle complex.

6. The dynamic detection method for digestive tumor inflammation pathology according to claim 1, characterized in that: In the fourth step, functionalized carbon quantum dots are synthesized by a microwave-assisted method using citric acid and urea as raw materials. Citric acid and urea are mixed according to a molar ratio of 1:1.5 - 2.5, dissolved in deionized water according to a volume ratio of 1:20 - 30, and then transferred to a microwave reaction vessel. Microwave radiation is carried out at a power of 400 W - 600 W for 4 min - 10 min. After the reaction is completed, it is cooled to room temperature, and impurities are removed by filtration through a 0.2 μm - 0.4 μm filter membrane to obtain a solution containing functionalized carbon quantum dots. The aptamer-upconversion nanoparticle complex is added to the solution containing functionalized carbon quantum dots so that the final concentration of the upconversion nanoparticles is maintained at 0.04 mg / mL - 0.12 mg / mL, and the final concentration of the carbon quantum dots is 0.1 mg / mL - 0.4 mg / mL. At the same time, 0.6% - 1.0% of polyvinylpyrrolidone is added as a dispersant, and 0.2% - 0.4% of dimethyl sulfoxide. After stirring evenly, it is left to stand at 3 °C - 5 °C for 2 h - 6 h.

7. A dynamic detection method for digestive tumor inflammation pathology according to claim 1, characterized in that: In step 5, 5 mL - 7 mL of the patient's blood sample and 4 g - 6 g of the tissue sample are collected. After the blood sample is collected, it is centrifuged at 3°C - 5°C and 3000 rpm - 4000 rpm for 15 min - 25 min to separate the serum. After the tissue sample is minced, a lysis buffer containing proteinase K is added, and it is incubated at 35°C - 39°C for 1 h - 3 h for digestion. Then, it is centrifuged at 12000 rpm - 14000 rpm for 20 min - 30 min, and the supernatant is taken. 50 μL - 70 μL of the sample supernatant and serum are respectively added to 100 μL - 140 μL of the detection reagent solution. After mixing, it is incubated in a constant temperature shaker at 35°C - 39°C for 1 h - 3 h, and the shaker speed is controlled at 120 rpm - 180 rpm.

8. A method for dynamically detecting the inflammation pathology of digestive tumors according to claim 1, characterized in that: In step 5, after the incubation is completed, the upconversion luminescence signal is detected using a fluorescence spectrophotometer. The excitation wavelength is set at 960 nm - 990 nm, and the emission wavelength range is 510 nm - 690 nm. At the same time, the fluorescence signal of the carbon quantum dots is detected. The excitation wavelength is 350 nm - 370 nm, and the emission wavelength is 440 nm - 460 nm. Using only the reagent without the sample as a blank control as a reference, the signal intensity of the sample is recorded. The standard curve is established by detecting the known concentration standard product according to the above steps. The sample signal intensity is substituted into the standard curve to calculate the marker content. The threshold is determined based on clinical verification to judge whether the patient has digestive tumor inflammation and the degree of inflammation.

9. A digestive tumor inflammation targeted therapy method applicable to the digestive tumor inflammation pathological dynamic detection method according to any one of claims 1-8, characterized in that: After determining the structural data of the potential specific marker, the described treatment method designs and synthesizes a targeting molecule that can specifically recognize the marker, couples the targeting molecule with a drug and a bioactive substance with therapeutic functions to form a targeted therapeutic complex, and administers the targeted therapeutic complex to the patient with digestive tumor inflammation, so that the targeted therapeutic complex can specifically bind to the potential specific marker at the tumor inflammation site, thereby achieving targeted treatment of digestive tumor inflammation.

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