Detection method of hematologic tumor biomarker

Abstract

The invention belongs to the field of biomarker detection, and particularly relates to a detection method of a blood tumor biomarker. The hematologic tumor biomarker is circRNA, and particularly relates to circ0035559, circ0001857 and the like, an SERS probe is prepared for detection and is composed of an SERS base material and competitive double-chain molecules, the SERS base material is obtained by modifying Ag NP (at) 4MBN (at) SiO2 NP through poly 4-sodium p-styrenesulfonate, the competitive double-chain molecules are an S1 chain and an S2 chain designed by circRNA to be detected, the S1 chain is connected with the SERS base material through an SH group, the S2 chain contains Raman signal molecules, and the SERS base material is connected with the SERS base material through an SH group. And the RNA competes for the S1 chain together with circRNA to be detected. The SERS probe disclosed by the invention can be used for well detecting a blood tumor biomarker and is relatively high in precision.

Description

Technical Field

[0001] The present invention belongs to the field of biomarker detection, and specifically relates to a method for detecting a blood tumor biomarker. Background Art

[0002] Hematological malignancies (HM) refer to malignant diseases originating from hematopoietic cells. They are heterogeneous diseases that can affect the bone marrow, blood, and various organs and tissues throughout the body. Their morbidity and mortality rates are among the highest, and they are major diseases that seriously endanger human health. Generally speaking, hematological malignancies are divided into 1) Leukemia: a large number of cancer cells spread, affecting the bone marrow or blood, and the ability of the circulatory system to produce blood is severely impaired; 2) Lymphoma: the cancerous formation that affects lymphocytes is called lymphoma; 3) Myeloma: cancerous formation of plasma cells, which can help the body fight infection by identifying and attacking bacteria. When myeloma causes cancer cells to accumulate in the bone marrow, it will crowd out healthy blood cells. The causes of these blood tumors are not fully understood at present. As leukemia and myeloma grow in the bone marrow, they interfere with the bone marrow's ability to produce normal blood cells, including white blood cells, red blood cells, and platelets, which can lead to frequent infections, anemia, and easy bruising and bleeding. Lymphoma usually manifests as swollen lymph nodes, and may also interfere with the body's ability to fight infection. In addition, myeloma produces substances that weaken bones and produce abnormal proteins, which can lead to symptoms such as multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage.

[0003] The classification of blood tumors is complex, and diagnosis and treatment vary greatly from person to person. Early detection, diagnosis, and treatment are important measures to improve the survival rate of blood tumor patients. Therefore, the development of highly sensitive and specific tumor cell detection and identification methods will help implement timely and effective treatment methods, and accurate and rapid identification will also help with drug screening.

[0004] Hematological tumor biomarkers are not only specific cell molecules, but also include various nucleic acids, secreted proteins and small molecule metabolites. Common hematological tumor biomarkers are as follows: 1) DNA, mainly including overexpression of oncogenes / tumor suppressor genes, site mutations, chromosome aberrations, DNA methylation, etc. For example, BCR / ABL fusion gene is one of the most important biomarkers in leukemia, and C-myc gene is often the research focus of malignant lymphoma mechanism; 2) RNA, miR-15 / 16 has been confirmed to be downregulated in chronic lymphocytic leukemia, and miR-21 and miR-155 have been found to be significantly elevated in the serum of B-cell lymphoma patients; 3) Proteins, such as CD20, are 35-47kDa non-glycosylated tetraspanin proteins expressed in most B-cell lymphomas.

[0005] Molecular biology, immunohistochemistry, etc. provide a variety of biomarker detection methods, but there is still a lot of room for further development of new detection methods that are efficient, accurate, rapid, simple, and low-cost. Summary of the invention

[0006] Based on the above problems, the present invention provides a method for detecting a blood tumor biomarker, the blood tumor biomarker is circRNA, specifically involving the detection of circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776, the present invention prepares a SERS probe for detection, the SERS probe is composed of a SERS substrate and a competitive double-stranded molecule, the SERS substrate is obtained by modifying Ag NP@4MBN@SiO2NP with poly-4-styrene sulfonate sodium, the competitive double-stranded molecule is the S1 chain and S2 chain designed for the circRNA to be tested, the S1 chain is connected to the SERS substrate through the SH group, and the S2 chain contains a Raman signal molecule, which competes with the circRNA to be tested for the S1 chain. The SERS probe of the present invention can better detect blood tumor biomarkers with high accuracy.

[0007] The present invention provides a method for detecting a blood tumor biomarker, comprising a SERS probe, wherein the SERS probe is composed of a SERS substrate and a competitive double-stranded molecule, the SERS substrate is obtained by modifying Ag NP@4MBN@SiO2NP with sodium poly-4-styrene sulfonate, and the preparation method of the SERS substrate is as follows: L1. Weigh ultrapure water, anhydrous ethanol and ammonium hydroxide solution according to a volume ratio of 2-2.5:1.5-1.8:1, stir at a speed of 100-180rpm for 20-30min in a 30-40℃ water bath environment to obtain solution 1, weigh ultrapure water and tetraethyl silicate according to a volume ratio of 8-10:1, stir until uniformly dispersed, obtain solution 2, mix solution 1 and solution 2 according to a volume ratio of 1:1-1.2, heat to 40-50℃ and treat at a speed of 200-250rpm for 1-1.5h, wash with a volume fraction of 50% ethanol solution and resuspend, centrifuge at a centrifugal force of 3000-4000g for 20-30min, precipitate and dry to obtain nano-silica; L2. Weigh the nano-silica and 4-mercaptobenzonitrile solution obtained in step L1 according to a mass-to-volume ratio of 20-30:1, perform ultrasonic treatment at a power of 200-250 W for 20-30 min, centrifuge at a centrifugal force of 6000-8000 g for 15-20 min, and resuspend the precipitate in ultrapure water to obtain solution 3; L3. Sodium citrate, silver nitrate, sodium chloride and ultrapure water were weighed in a mass volume ratio of 10-15 mg: 2-3 mg: 1.2-1.5 mg: 5-8 mL, and stirred at a speed of 150-200 rpm for 3-4 min at room temperature to obtain solution 4, and solution 4, ascorbic acid solution and ultrapure water were weighed in a volume ratio of 25-30: 1: 600-610, and the ultrapure water was heated to 45-50 ° C, and then solution 4 and ascorbic acid solution were quickly added, and heated to reflux at a speed of 150-200 rpm for 90-120 min. After the end, stirring was continued until it was cooled to room temperature to obtain a nanosilver sol; L4. The solution 3 obtained in step L2 and the nanosilver sol obtained in step L3 were weighed in a volume ratio of 1:1.2-1.5, stirred at a speed of 2000-2500 rpm for 10-15 min, and then centrifuged at a centrifugal force of 3000-4000 g for 20-30 min. After the end, the precipitate was vacuum dried to obtain substance 1; L5. Disperse the substance 1 obtained in step L4 in a poly (4-styrene sodium sulfonate) solution having a mass percentage concentration of 3-5%, and allow to stand at room temperature for 30-45 minutes. Centrifuge at a centrifugal force of 2800-3000 g for 10-15 minutes, and vacuum dry the precipitate to obtain a SERS substrate.

[0008] Preferably, the concentration of the 4-mercaptobenzonitrile solution in step L2 is 3-4 mM, and the mass volume concentration of the precipitate in ultrapure water is 5-8 g / L.

[0009] Preferably, the mass volume concentration of the ascorbic acid solution in step L3 is 18 g / L.

[0010] The competitive double-stranded molecule comprises the S1 chain and the S2 chain of circRNA, and the circRNA involves circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776.

[0011] Among them, the S1 chain and S2 chain sequences of circ_0035559 are: 5'-TCATCCACACCTTCATGGAAGCTTTTT-SH-3'; 5′-X-GGGGGTCCATGAAGGTGT-3′.

[0012] Among them, the S1 chain and S2 chain sequences of circ_0001857 are: 5'-CTGATTCACTCCTCCATGTCCTTCGTCTCTCTTTTTT-SH-3'; 5′-X-GGGGGAGAGACGAAGGA-3′.

[0013] Among them, the S1 chain and S2 chain sequences of circ_0000745 are: 5'-TTAGTTGTTGTAAAGGCCCCTTGGCCAGCAGACTTTTT-SH-3'; 5'-X-GGGGGTGCTGGCCAAGGCCA-3'.

[0014] Among them, the S1 chain and S2 chain sequences of circ_0132266 are: 5'-AGGATTACATGACATCTGACCTGACAGGCAGACTTTTTT-SH-3'; 5'-X-GGGGGTCTGCCTGTCAGGT-3'.

[0015] Among them, the S1 chain and S2 chain sequences of circ_0000707 are: 5'-ACAAAAGCCAGTTACTGCCAGCAGCTGTGAAACTCTTTTT-SH-3'; 5′-X-GGGGGTTTCACAGCTGCT-3′.

[0016] Among them, the S1 chain and S2 chain sequences of circ_0007099 are: 5'-AACAAACCTAAAAGTCTTTGGTCAGAAGAGGACAGTTTTT-SH-3'; 5'-X-GGGGGTCCTCTTCTGACCAA-3'.

[0017] Among them, the S1 chain and S2 chain sequences of circ_0000190 are: 5'-TCTGGAATTACCCACTATAAAGATACATGAAGATTTTT-SH-3'; 5'-X-GGGGGTCATGTATCTTTATA-3'.

[0018] Among them, the S1 chain and S2 chain sequences of circ_0087776 are: 5'-AGACTCCCTCACATTTAATTCTCCTTCTCCATTTTT-SH-3'; 5'-X-GGGGGAGAAGGAGAATTAA-3'.

[0019] Preferably, X at the 5' end of the S2 chain of each circRNA represents a Raman signal molecule, and the Raman signal molecule is selected from one or more of p-mercaptobenzoic acid (MBA), p-nitrothiophenol (NT), 3,3' diethylthioaldehyde tricarbocyanine iodide (DTTC), rhodamine 6G (R6G), crystal violet (CV), 4-aminobenzenethiol (4-ATP), 4-aminothiophenol (4-ABT), 4-hydroxythiophenol (4-HBT), and 4-mercaptopyridine (4-MPy).

[0020] The preparation method of the SERS probe is as follows: S1. Dissolve the competitive double-stranded molecule in 1×TE buffer to obtain S1 chain solution and S2 chain solution with a concentration of 15-20 μM, mix the S1 chain solution and the S2 chain solution at a volume ratio of 1:1-1.2 to obtain mixed solution 1, add 5-8 times the volume of Tris-HCl buffer solution of mixed solution 1, heat to 90-96°C and keep for 8-10 minutes to obtain mixed solution 2, add 0.02-0.03 times the volume of TCEP solution of mixed solution 2, continue to keep for 30-45 minutes, and obtain mixed solution 3 after the end; S2. Add the SERS substrate to the mixed solution 3, let it stand in a 37°C water bath for 8-10 hours, and then centrifuge it at 5000-6000g for 10-15 minutes. Resuspend the precipitate in ultrapure water, centrifuge it again, and resuspend the precipitate in ultrapure water to obtain a SERS probe with a mass volume concentration of 8-10g / L.

[0021] Preferably, in step S1, the concentration of the Tris-HCl buffer solution is 25 mM, and the concentration of the TCEP solution is 40 μM.

[0022] Preferably, in step S2, the mass volume concentration of the SERS substrate in the mixed solution 3 is 20-30 g / L.

[0023] Preferably, in the preparation of the above-mentioned SERS probe, the amount of each substance used corresponds to the competitive double-stranded molecule involving a single circRNA.

[0024] The beneficial effects of the present invention are as follows: The present invention detects blood tumor biomarkers. The biomarker to be detected is circRNA. circRNA has a covalent closed-loop structure and lacks a polyadenylated tail. This structure makes it stable and resistant to exonuclease degradation. It is not only a key regulatory molecule in cells, but also studies have shown that it plays an important role in the progression of blood tumors. Traditional detection technology is difficult to detect low-abundance circRNA. The present invention designs a SERS probe, which consists of a SERS substrate and a competitive double-chain molecule. The SERS substrate is obtained by modifying Ag NP@4MBN@SiO2NP with sodium poly (4-styrene sulfonate). The SERS substrate has good stability. 4MBN is embedded between two nanomaterials as an internal standard molecule to continuously monitor and calibrate Raman signals without being affected by the external environment. Nanosilver sol is used as an outer layer to generate a strong electromagnetic field enhancement effect by virtue of its hot spot effect, thereby amplifying the detection signal of the Raman signal molecule. The modified sodium poly (4-styrene sulfonate) can protect Ag NP@4MBN@SiO2NP (substance 1) to prevent the nanosilver from being oxidized or contaminated, and at the same time enhance the use effect of the entire SERS substrate. The present invention also provides a competitive double-stranded molecule, which is designed according to the circRNA to be detected. The S1 chain contains an SH group and can be connected to a SERS substrate after being treated with a TCEP reducing agent. The S2 chain contains a Raman signal molecule and competes with the circRNA to be detected for the S1 chain. When the content of the circRNA to be detected is low, the S2 chain is tightly bound to the S1 chain and the Raman signal is strong. When the content of the circRNA to be detected is high, the circRNA to be detected is tightly bound to the S1 chain and the Raman signal is weak.

[0025] The blood tumor biomarkers selected by the present invention, namely circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776, have been studied relatively extensively and have a close relationship with various blood tumor diseases. For example, circ_0035559 is believed to be useful for the prediction / monitoring of acute myeloid leukemia. The SERS probe of the present invention can better detect blood tumor biomarkers with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1The fitting curves are for standard circ_0035559, circ_0001857, circ_0000745, and circ_0132266 samples; Figure 2 The fitting curves are for standard circ_0000707, circ_0007099, circ_0000190, and circ_0087776 samples; Figure 3 The figure shows the detection results of circRNA concentration differences in different cell lines. DETAILED DESCRIPTION

[0027] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0028] Example 1: This example provides a method for preparing a SERS substrate, and the specific steps are as follows: L1. Weigh ultrapure water, anhydrous ethanol and ammonium hydroxide solution in a volume ratio of 2:1.5:1, stir at a speed of 100 rpm for 20 minutes in a 30°C water bath environment to obtain solution 1, weigh ultrapure water and tetraethyl silicate in a volume ratio of 8:1, stir until uniformly dispersed to obtain solution 2, mix solution 1 and solution 2 in a volume ratio of 1:1, heat to 40°C and treat at a speed of 200 rpm for 1 hour, wash with a volume fraction of 50% ethanol solution and resuspend, centrifuge at a centrifugal force of 3000g for 20 minutes, and precipitate and dry to obtain nano-silica; L2. The nano-silica and 4-mercaptobenzonitrile solution obtained in step L1 were weighed at a mass-volume ratio of 20:1, the concentration of the 4-mercaptobenzonitrile solution was 3 mM, and ultrasonic treatment was performed at a power of 200 W for 20 min. After the treatment, the solution was centrifuged at a centrifugal force of 6000 g for 15 min, and the precipitate was resuspended in ultrapure water to obtain solution 3. The mass-volume concentration of the precipitate in ultrapure water was 5 g / L. L3. Sodium citrate, silver nitrate, sodium chloride and ultrapure water were weighed in a mass volume ratio of 10 mg: 2 mg: 1.2 mg: 5 mL, and stirred at 150 rpm for 3 min at room temperature to obtain solution 4. Solution 4, ascorbic acid solution and ultrapure water were weighed in a volume ratio of 25: 1: 600, and the mass volume concentration of the ascorbic acid solution was 18 g / L. After the ultrapure water was heated to 45° C., solution 4 and ascorbic acid solution were quickly added, and heated to reflux at 150 rpm for 90 min. After the end, stirring was continued until it was cooled to room temperature to obtain a nanosilver sol; L4. Weigh the solution 3 obtained in step L2 and the nanosilver sol obtained in step L3 in a volume ratio of 1:1.2, stir at a speed of 2000 rpm for 10 min, centrifuge at a centrifugal force of 3000 g for 20 min, and then vacuum dry the precipitate to obtain substance 1; L5. The substance 1 obtained in step L4 is dispersed in a poly (4-styrene sodium sulfonate) solution having a mass percentage concentration of 3%, and allowed to stand at room temperature for 30 minutes. After the dispersion, the solution is centrifuged at a centrifugal force of 2800 g for 10 minutes, and the precipitate is vacuum dried to obtain a SERS substrate.

[0029] Example 2: This example provides a method for preparing a SERS substrate, and the specific steps are as follows: L1. Weigh ultrapure water, anhydrous ethanol and ammonium hydroxide solution in a volume ratio of 2.1:1.6:1, stir at a speed of 150 rpm for 26 minutes in a 35°C water bath environment to obtain solution 1, weigh ultrapure water and tetraethyl silicate in a volume ratio of 9:1, stir until uniformly dispersed to obtain solution 2, mix solution 1 and solution 2 in a volume ratio of 1:1.1, heat to 45°C and treat at a speed of 220 rpm for 1.2 hours, wash with a volume fraction of 50% ethanol solution and resuspend, centrifuge at a centrifugal force of 3500g for 25 minutes, and precipitate and dry to obtain nano-silica; L2. The nano-silica and 4-mercaptobenzonitrile solution obtained in step L1 were weighed at a mass-volume ratio of 25:1, the concentration of the 4-mercaptobenzonitrile solution was 3.5 mM, and ultrasonic treatment was performed at a power of 230 W for 24 min. After the treatment, the solution was centrifuged at a centrifugal force of 7000 g for 18 min, and the precipitate was resuspended in ultrapure water to obtain solution 3. The mass-volume concentration of the precipitate in ultrapure water was 6 g / L. L3. Sodium citrate, silver nitrate, sodium chloride and ultrapure water were weighed in a mass volume ratio of 12 mg: 2.5 mg: 1.4 mg: 6 mL, and stirred at 180 rpm for 3.5 min at room temperature to obtain solution 4. Solution 4, ascorbic acid solution and ultrapure water were weighed in a volume ratio of 28: 1: 605, and the mass volume concentration of the ascorbic acid solution was 18 g / L. After the ultrapure water was heated to 48° C., solution 4 and ascorbic acid solution were quickly added, and heated to reflux at 180 rpm for 110 min. After the end, stirring was continued until it was cooled to room temperature to obtain nanosilver sol; L4. Weigh the solution 3 obtained in step L2 and the nanosilver sol obtained in step L3 in a volume ratio of 1:1.4, stir at a speed of 2300 rpm for 12 min, centrifuge at a centrifugal force of 3500 g for 24 min, and then vacuum dry the precipitate to obtain substance 1; L5. The substance 1 obtained in step L4 is dispersed in a poly (4-styrene sodium sulfonate) solution having a mass percentage concentration of 4%, and allowed to stand at room temperature for 38 minutes. After the dispersion is completed, the solution is centrifuged at a centrifugal force of 2900 g for 12 minutes. The precipitate is vacuum dried to obtain a SERS substrate.

[0030] Example 3: This example provides a method for preparing a SERS substrate, and the specific steps are as follows: L1. Weigh ultrapure water, anhydrous ethanol and ammonium hydroxide solution in a volume ratio of 2.5:1.8:1, stir at a speed of 180 rpm for 30 min in a 40°C water bath environment to obtain solution 1, weigh ultrapure water and tetraethyl silicate in a volume ratio of 10:1, stir until uniformly dispersed to obtain solution 2, mix solution 1 and solution 2 in a volume ratio of 1:1.2, heat to 50°C and treat at a speed of 250 rpm for 1.5 h, wash with a volume fraction of 50% ethanol solution and resuspend, centrifuge at a centrifugal force of 4000g for 30 min, precipitate and dry to obtain nano-silica; L2. The nano-silica and 4-mercaptobenzonitrile solution obtained in step L1 were weighed at a mass-volume ratio of 30:1, the concentration of the 4-mercaptobenzonitrile solution was 4 mM, and ultrasonic treatment was performed at a power of 250 W for 30 min. After the treatment, the solution was centrifuged at a centrifugal force of 8000 g for 20 min, and the precipitate was resuspended in ultrapure water to obtain solution 3. The mass-volume concentration of the precipitate in ultrapure water was 8 g / L. L3. Sodium citrate, silver nitrate, sodium chloride and ultrapure water were weighed in a mass volume ratio of 15 mg: 3 mg: 1.5 mg: 8 mL, and stirred at 200 rpm for 4 min at room temperature to obtain solution 4. Solution 4, ascorbic acid solution and ultrapure water were weighed in a volume ratio of 30: 1: 610, and the mass volume concentration of the ascorbic acid solution was 18 g / L. After the ultrapure water was heated to 50° C., solution 4 and ascorbic acid solution were quickly added, and heated to reflux at 200 rpm for 120 min. After the end, stirring was continued until it was cooled to room temperature to obtain a nanosilver sol; L4. Weigh the solution 3 obtained in step L2 and the nanosilver sol obtained in step L3 in a volume ratio of 1:1.5, stir at a speed of 2500 rpm for 15 min, centrifuge at a centrifugal force of 4000 g for 30 min, and then vacuum dry the precipitate to obtain substance 1; L5. The substance 1 obtained in step L4 is dispersed in a poly (4-styrene sodium sulfonate) solution having a mass percentage concentration of 5%, and allowed to stand at room temperature for 45 minutes. After the dispersion, the solution is centrifuged at a centrifugal force of 3000 g for 15 minutes, and the precipitate is vacuum dried to obtain a SERS substrate.

[0031] The chemical reagents involved in Examples 1-3 were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and the product number of the ammonium hydroxide solution was 221228.

[0032] Example 4: This example provides a composition of a competitive double-stranded molecule, the specific contents are as follows: The competitive double-stranded molecules of this example involve the S1 and S2 chains of 8 circRNAs, and the 8 circRNAs are specifically circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776; Among them, the S1 chain and S2 chain sequences of circ_0035559 are: 5'-TCATCCACACCTTCATGGAAGCTTTTT-SH-3'; 5'-MBA-GGGGGTCCATGAAGGTGT-3'; Among them, the S1 chain and S2 chain sequences of circ_0001857 are: 5'-CTGATTCACTCCTCCATGTCCTTCGTCTCTCTTTTTT-SH-3'; 5'-MBA-GGGGGAGACGAAGGA-3'; Among them, the S1 chain and S2 chain sequences of circ_0000745 are: 5'-TTAGTTGTTGTAAAGGCCCCTTGGCCAGCAGACTTTTT-SH-3'; 5'-MBA-GGGGGTGCTGGCCAAGGCCA-3'; Among them, the S1 chain and S2 chain sequences of circ_0132266 are: 5'-AGGATTACATGACATCTGACCTGACAGGCAGACTTTTTT-SH-3'; 5'-MBA-GGGGGTCTGCCTGTCAGGT-3'; Among them, the S1 chain and S2 chain sequences of circ_0000707 are: 5'-ACAAAAGCCAGTTACTGCCAGCAGCTGTGAAACTCTTTTT-SH-3'; 5'-MBA-GGGGGGTTTCACAGCTGCT-3'; Among them, the S1 chain and S2 chain sequences of circ_0007099 are: 5'-AACAAACCTAAAAGTCTTTGGTCAGAAGAGGACAGTTTTT-SH-3'; 5'-MBA-GGGGGTCCTCTTCTGACCAA-3'; Among them, the S1 chain and S2 chain sequences of circ_0000190 are: 5'-TCTGGAATTACCCACTATAAAGATACATGAAGATTTTT-SH-3'; 5'-MBA-GGGGGTCATGTATCTTTATA-3'; Among them, the S1 chain and S2 chain sequences of circ_0087776 are: 5'-AGACTCCCTCACATTTAATTCTCCTTCTCCATTTTT-SH-3'; 5'-MBA-GGGGGAGAAGGAGAATTAA-3'.

[0033] Example 5: This example provides a composition of a competitive double-stranded molecule, the specific contents are as follows: The difference between this embodiment and embodiment 4 is that the Raman signal molecule at the 5' end of the S2 chain of each circRNA is replaced by 3,3' diethylthiocarbocyanine iodide (DTTC) instead of p-mercaptobenzoic acid (MBA). The other contents are consistent with embodiment 4.

[0034] Example 6: This example provides a composition of a competitive double-stranded molecule, the specific contents are as follows: The difference between this embodiment and embodiment 4 is that the Raman signal molecule at the 5' end of the S2 chain of each circRNA is replaced by 4-mercaptopyridine (4-MPy) instead of p-mercaptobenzoic acid (MBA), and the other contents are consistent with embodiment 4.

[0035] Example 7: This example provides a composition of a competitive double-stranded molecule, the specific contents are as follows: The difference between this example and Example 4 is that this example involves the S1 chain and the S2 chain of two circRNAs, and the two circRNAs are specifically circ_0035559 and circ_0001857; Among them, the S1 chain and S2 chain sequences of circ_0035559 are: 5'-TCATCCACACCTTCATGGAAGCTTTTT-SH-3'; 5'-NT-GGGGGTCCATGAAGGTGT-3'; Among them, the S1 chain and S2 chain sequences of circ_0001857 are: 5'-CTGATTCACTCCTCCATGTCCTTCGTCTCTCTTTTTT-SH-3'; 5'-4-ATP-GGGGGAGAGACGAAGGA-3'.

[0036] Example 8: This example provides a composition of a competitive double-stranded molecule, the specific contents are as follows: The difference between this example and Example 4 is that this example involves the S1 chain and the S2 chain of two circRNAs, and the two circRNAs are specifically circ_0000745 and circ_0000707; Among them, the S1 chain and S2 chain sequences of circ_0000745 are: 5'-TTAGTTGTTGTAAAGGCCCCTTGGCCAGCAGACTTTTT-SH-3'; 5'-R6G-GGGGGTGCTGGCCAAGGCCA-3'; Among them, the S1 chain and S2 chain sequences of circ_0000707 are: 5'-ACAAAAGCCAGTTACTGCCAGCAGCTGTGAAACTCTTTTT-SH-3'; 5'-4-HBT-GGGGGGTTTCACAGCTGCT-3'.

[0037] Example 9: This example provides a method for preparing a SERS probe, the specific contents are as follows: S1. The competitive double-stranded molecule was dissolved in 1×TE buffer to obtain S1 chain solution and S2 chain solution with a concentration of 15 μM, and the S1 chain solution and the S2 chain solution were mixed at a volume ratio of 1:1 to obtain mixed solution 1, and a Tris-HCl buffer solution with a volume of 5 times that of the mixed solution 1 was added, and the concentration of the Tris-HCl buffer solution was 25 mM, and the solution was heated to 90°C and maintained for 8 min to obtain mixed solution 2, and a TCEP solution with a volume of 0.02 times that of the mixed solution 2 was added, and the concentration of the TCEP solution was 40 μM, and the solution was maintained for 30 min. After the end, a mixed solution 3 was obtained; S2. Add the SERS substrate into the mixed solution 3, the mass volume concentration of the SERS substrate in the mixed solution 3 is 20g / L, and let it stand in a 37°C water bath for 8h. After that, centrifuge it at 5000g for 10min. Resuspend the precipitate with ultrapure water, centrifuge it again, and resuspend the precipitate in ultrapure water to obtain a SERS probe with a mass volume concentration of 8g / L.

[0038] Embodiment 10: This embodiment provides a method for preparing a SERS probe, the specific contents are as follows: S1. The competitive double-stranded molecule was dissolved in 1×TE buffer to obtain S1 chain solution and S2 chain solution with a concentration of 18 μM, and the S1 chain solution and the S2 chain solution were mixed at a volume ratio of 1:1.1 to obtain mixed solution 1, and a Tris-HCl buffer solution with a volume of 7 times that of the mixed solution 1 was added, and the concentration of the Tris-HCl buffer solution was 25 mM. The solution was heated to 94°C and maintained for 9 min to obtain mixed solution 2, and a TCEP solution with a volume of 0.025 times that of the mixed solution 2 was added, and the concentration of the TCEP solution was 40 μM. The solution was maintained for 38 min, and after the end, a mixed solution 3 was obtained; S2. Add the SERS substrate into the mixed solution 3, the mass volume concentration of the SERS substrate in the mixed solution 3 is 25g / L, and let it stand in a 37°C water bath for 9 hours. After that, centrifuge it at 5400g for 12 minutes. Resuspend the precipitate with ultrapure water, centrifuge it again, and resuspend the precipitate in ultrapure water to obtain a SERS probe with a mass volume concentration of 9g / L.

[0039] Example 11: This example provides a method for preparing a SERS probe, the specific contents are as follows: S1. The competitive double-stranded molecule was dissolved in 1×TE buffer to obtain S1 chain solution and S2 chain solution with a concentration of 20 μM, and the S1 chain solution and S2 chain solution were mixed at a volume ratio of 1:1.2 to obtain mixed solution 1, and a Tris-HCl buffer solution with a volume of 8 times that of the mixed solution 1 was added, and the concentration of the Tris-HCl buffer solution was 25 mM. The solution was heated to 96°C and maintained for 10 min to obtain mixed solution 2, and a TCEP solution with a volume of 0.03 times that of the mixed solution 2 was added, and the concentration of the TCEP solution was 40 μM. The solution was maintained for 45 min, and mixed solution 3 was obtained after the end. S2. Add the SERS substrate into the mixed solution 3, the mass volume concentration of the SERS substrate in the mixed solution 3 is 30g / L, and let it stand in a 37°C water bath for 10 hours. After that, centrifuge it at 6000g for 15 minutes. Resuspend the precipitate with ultrapure water, centrifuge it again, and resuspend the precipitate in ultrapure water to obtain a SERS probe with a mass volume concentration of 10g / L.

[0040] Experimental test: The cell lines involved in the experimental test are as follows: SKNO-1 human acute myeloid leukemia cells, K562 human chronic myeloid leukemia cells, CEM / C1 human acute lymphoblastic leukemia cells, Hs 505.T human chronic lymphocytic leukemia cells, DS human diffuse large B-cell lymphoma cells, IM-9 human multiple myeloma cells, SUP-T1T human lymphoblastic lymphoma cells, Mino human mantle cell lymphoma cells, A549 human lung cancer cells, HCT-8 human colorectal cancer cells, Bcap-37 human breast cancer cells, KATO III human gastric cancer cells, HL-7702 human normal liver cells. The above cell lines are from the Chinese Academy of Sciences Cell Bank, the American ATCC Cell Bank, and Wuhan Pronocell Life Science Technology Co., Ltd.

[0041] Cell culture and acquisition of circRNA: The cell lines were activated, digested with trypsin, and inoculated on high-purity quartz plates with an inoculation volume of 2000 cells / quartz plate. They were placed in an incubator at 37°C and 5% CO2 for 24 hours, taken out, and 2 mL of fresh complete medium and 300 μL SERS probe were added. They were placed in the incubator for 24 hours, taken out, and fully washed with pH=7.0 PBS buffer, fixed with paraformaldehyde solution, washed again with PBS buffer, resuspended in sterile PBS buffer, and stored at 4°C. CircRNA was further extracted as follows: AG RNAex Pro RNA extraction reagent (Cat. No. AG21101) purchased from Hunan Aikerui Bioengineering Co., Ltd. was used to extract the cell lines to obtain Total RNA, and then Evo M-MLV Reverse Transcription Kit II (Cat. No. AG11711) was used for reverse transcription, and SYBR Green Pro Taq HS Premixed qPCR Kit was used for qPCR. Finally, the samples to be tested for each cell line were obtained. The specific experimental steps were carried out according to the instructions.

[0042] Raman spectroscopy measurements: The excitation light wavelength was 785 nm, the excitation power was 12 mW, the integration time was 10 s, the number of scans was 3 times, and the detection was performed under a 20× objective lens.

[0043] Testing of standard samples: Table 1 Sequences of standard circRNA samples

[0044] The standard circRNA samples shown in Table 1 were synthesized at Beijing Nosai Genome Research Center Co., Ltd. Taking circ_0035559 as an example, different concentrations of circ_0035559 standard samples were set for Raman spectroscopy detection. The fitting curves of each standard circRNA sample were obtained based on the relationship between concentration and intensity ratio. The intensity ratio = I x / I 2223 , where x represents the Raman peaks of different Raman signal molecules.

[0045] A SERS probe was obtained by combining Example 1+Example 4+Example+Example 9, and a fitting curve was obtained according to the above steps. The fitting curve is shown in FIG. Figure 1 , Figure 2 As shown, the standard circRNA samples are between 1 and 1×10 9 In the range of pM, they all showed a good linear relationship. The fitting curve equation is shown in Table 2. As can be seen from Table 2, the R of the fitting curve equation corresponding to any standard circRNA sample is 2All of them were >0.96, which further proved that the SERS probe obtained in the present application can detect the eight circRNAs, circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776, with high quality.

[0046] Table 2 Fitting curve equations of standard circRNA samples

[0047] Note: The fitting curve equation can be described as y=a×lgC+b, where a is the slope, b is the intercept, and lgC is the logarithmic value of the standard circRNA sample concentration.

[0048] Cell line samples to be tested: A SERS probe was obtained by combining Example 2+Example 5+Example+Example 10, and the fitting curve equation of each standard circRNA sample was obtained as described above. The cell line sample to be tested was subjected to Raman spectroscopy detection to obtain an intensity ratio, which was further substituted into the fitting curve equation of the standard circRNA sample to obtain the circRNA concentration value of each cell line sample to be tested.

[0049] The results are as follows Figure 3 As shown by Figure 3 It can be seen that the concentrations of biomarkers circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776 in cell lines of blood tumor-related diseases are significantly higher than those in other cancers or normal liver cells, and the difference is even more than two orders of magnitude. The content of circ_0035559 in SKNO-1 cell line is significantly higher than that of other circRNAs, proving that circ_0035559 has certain advantages as a predictive / monitoring biomarker for acute myeloid leukemia. The same situation can also be seen in the relationship between other cell lines and circRNAs, but the situation of SUP-T1T cell line and Mino cell line is slightly different, especially Mino cell line. The reason may be that the circRNA to be detected does not contain circRNAs with obvious directivity to mantle cell lymphoma. However, the above 8 circRNAs can still be used as biomarkers for blood tumors. By detecting their concentrations, they can, to a certain extent, warn of the occurrence of adverse events such as early prediction / prognosis of blood tumors.

[0050] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting blood tumor biomarkers, characterized in that: The invention comprises a SERS probe, wherein the SERS probe is composed of a SERS substrate and a competitive double-stranded molecule, and the SERS substrate is obtained by modifying Ag NP@4MBN@SiO2NP with sodium poly-4-styrene sulfonate; The competitive double-stranded molecule comprises the S1 chain and the S2 chain of circRNA, and the circRNA involves circ_0035559, circ_0001857, circ_0000745, circ_0132266, circ_0000707, circ_0007099, circ_0000190 and circ_0087776.

2. The method for detecting blood tumor biomarkers according to claim 1, characterized in that: The preparation method of the SERS substrate is as follows: L1. Weigh ultrapure water, anhydrous ethanol and ammonium hydroxide solution in a volume ratio of 2-2.5:1.5-1.8:1, heat and stir in a water bath to obtain solution 1, weigh ultrapure water and tetraethyl silicate in a volume ratio of 8-10:1, stir until uniformly dispersed to obtain solution 2, mix solution 1 and solution 2 in a volume ratio of 1:1-1.2, heat and stir, wash and resuspend with 50% ethanol solution, centrifuge, precipitate and dry to obtain nano-silica; L2. Weigh the nano-silica and 4-mercaptobenzonitrile solution obtained in step L1 according to a mass-to-volume ratio of 20-30:1, perform ultrasonic treatment, centrifuge after the end, and resuspend the precipitate in ultrapure water to obtain solution 3; L3. Sodium citrate, silver nitrate, sodium chloride and ultrapure water were weighed in a mass volume ratio of 10-15mg: 2-3mg: 1.2-1.5mg: 5-8mL, and stirred at room temperature to obtain a solution 4. Solution 4, ascorbic acid solution and ultrapure water were weighed in a volume ratio of 25-30: 1: 600-610. After the ultrapure water was heated, solution 4 and ascorbic acid solution were quickly added, heated to reflux, and continued to stir until cooled to room temperature to obtain a nanosilver sol; L4. Weigh the solution 3 obtained in step L2 and the nanosilver sol obtained in step L3 according to a volume ratio of 1:1.2-1.5, stir, centrifuge after completion, and vacuum dry the precipitate to obtain substance 1; L5. Disperse the substance 1 obtained in step L4 in a poly (4-styrene sodium sulfonate) solution having a mass percentage concentration of 3-5%, let it stand at room temperature, centrifuge it after completion, and vacuum dry the precipitate to obtain a SERS substrate.

3. The method for detecting blood tumor biomarkers according to claim 2, characterized in that: The concentration of the 4-mercaptobenzonitrile solution in step L2 is 3-4 mM, and the mass volume concentration of the precipitate in ultrapure water is 5-8 g / L.

4. The method for detecting blood tumor biomarkers according to claim 3, characterized in that: The mass volume concentration of the ascorbic acid solution in step L3 is 18 g / L.

5. The method for detecting blood tumor biomarkers according to claim 4, characterized in that: The S1 and S2 sequences of circ_0035559 are: 5'-TCATCCACACCTTCATGGAAGCTTTTT-SH-3'; 5′-X-GGGGGTCCATGAAGGTGT-3′; The S1 and S2 sequences of circ_0001857 are: 5'-CTGATTCACTCCTCCATGTCCTTCGTCTCTCTTTTTT-SH-3'; 5′-X-GGGGGAGAGACGAAGGA-3′; The S1 and S2 sequences of circ_0000745 are: 5'-TTAGTTGTTGTAAAGGCCCCTTGGCCAGCAGACTTTTT-SH-3'; 5'-X-GGGGGTGCTGGCCAAGGCCA-3'; The S1 and S2 sequences of circ_0132266 are: 5'-AGGATTACATGACATCTGACCTGACAGGCAGACTTTTTT-SH-3'; 5'-X-GGGGGTCTGCCTGTCAGGT-3'; The S1 and S2 sequences of circ_0000707 are: 5'-ACAAAAGCCAGTTACTGCCAGCAGCTGTGAAACTCTTTTT-SH-3'; 5′-X-GGGGGTTTCACAGCTGCT-3′; The S1 and S2 sequences of circ_0007099 are: 5'-AACAAACCTAAAAGTCTTTGGTCAGAAGAGGACAGTTTTT-SH-3'; 5'-X-GGGGGTCCTCTTCTGACCAA-3'; The S1 and S2 sequences of circ_0000190 are: 5'-TCTGGAATTACCCACTATAAAGATACATGAAGATTTTT-SH-3'; 5'-X-GGGGGTCATGTATCTTTATA-3'; The S1 and S2 sequences of circ_0087776 are: 5'-AGACTCCCTCACATTTAATTCTCCTTCTCCATTTTT-SH-3'; 5'-X-GGGGGAGAAGGAGAATTAA-3'.

6. The method for detecting blood tumor biomarkers according to claim 5, characterized in that: The X at the 5' end of the S2 chain of each circRNA represents a Raman signal molecule, and the Raman signal molecule is selected from one or more of p-mercaptobenzoic acid, p-nitrothiophenol, 3,3' diethylthioaldehyde tricarbocyanine iodide, rhodamine 6G, crystal violet, 4-aminobenzenethiol, 4-aminothiophenol, 4-hydroxythiophenol, and 4-mercaptopyridine.

7. The method for detecting blood tumor biomarkers according to claim 6, characterized in that: The preparation method of the SERS probe is as follows: S1. Dissolve the competitive double-stranded molecule in 1×TE buffer to obtain S1 chain solution and S2 chain solution with a concentration of 15-20 μM, mix the S1 chain solution and the S2 chain solution at a volume ratio of 1:1-1.2 to obtain mixed solution 1, add 5-8 times the volume of Tris-HCl buffer solution of mixed solution 1, heat and maintain to obtain mixed solution 2, add 0.02-0.03 times the volume of TCEP solution of mixed solution 2 to obtain mixed solution 3; S2. Add the SERS substrate to the mixed solution 3, let it stand in a 37°C water bath, centrifuge it after the end, resuspend the precipitate with ultrapure water, centrifuge it again, and resuspend the precipitate in ultrapure water to obtain a SERS probe with a mass volume concentration of 8-10g / L.

8. The method for detecting blood tumor biomarkers according to claim 7, characterized in that: In step S1, the concentration of the Tris-HCl buffer solution is 25 mM, and the concentration of the TCEP solution is 40 μM.

9. The method for detecting blood tumor biomarkers according to claim 8, characterized in that: In step S2, the mass volume concentration of the SERS substrate in the mixed solution 3 is 20-30 g / L.

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