Exosome surface protein NELISA detection method based on monatomic catalyst activity regulation

Abstract

The invention discloses an exosome surface protein NELISA (nuclease-linked immunosorbent assay) detection method based on monatomic catalyst activity regulation, and belongs to the technical field of biomedical detection. Compared with a commercial ELISA kit, the exosome surface protein NELISA detection method based on monatomic catalyst activity regulation has the advantages that the linear range of exosome surface protein detection is wider, the exosome detection requirements from low concentration to high concentration can be met, and the method is suitable for more diversified sample types and clinical application scenes; the detection limit is lower, exosomes with lower concentration can be detected, and the kit is suitable for early disease diagnosis and detection of exosome low-abundance samples; the aptamer and the nano material are used for replacing an expensive primary antibody and an enzyme-labeled secondary antibody, so that the reagent cost is remarkably reduced, the method is suitable for popularization and application in areas with limited resources, the economic threshold of exosome detection is reduced, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to a method for detecting exosomal surface protein NELISA based on the activity regulation of single-atom catalysts. Background Art

[0002] Exosomes are a type of extracellular vesicles with a diameter of approximately 30 - 150 nanometers, which are produced by cells through endocytosis and released into the extracellular environment. Exosomes carry a variety of biomolecules, including proteins, lipids, RNA, and DNA, and these molecules reflect the physiological and pathological states of their source cells. Exosomes play important roles in processes such as intercellular communication, immune regulation, and the formation of the tumor microenvironment. Due to the molecular information carried by exosomes being closely related to their source cells, exosomes are considered potential biomarkers. Exosomes can be obtained non-invasively through body fluids (such as blood, urine, saliva, etc.), and thus have broad application prospects in clinical diagnosis. For example: 1) Cancer diagnosis: Specific proteins and RNA in exosomes can be used as early diagnostic markers for cancer; 2) Disease monitoring: Exosomes can be used to monitor the progression and treatment effects of diseases; 3) Personalized treatment: By analyzing the molecular information in exosomes, personalized treatment plans can be provided for patients.

[0003] Lee et al. (Enhanced paper-based ELISA for simultaneous EVs / exosomeisolation and detection using streptavidin agarose-based immobilization[J].Analyst,2019,145.) described a method for detecting exosomes (EVs / exosomes) based on paper-based enzyme-linked immunosorbent assay (p-ELISA). Duijvesz et al. (Immuno-based detection of extracellular vesiclesin urine as diagnostic marker for prostate cancer[J].International JournalofCancer=:Journal International du Cancer,2015,137(12):2869-2878.) described a method for detecting exosomes based on time-resolved fluorescence immunoassay (TR-FIA) for detecting exosomes in the urine of patients with prostate cancer (PCa).

[0004] However, the paper-based ELISA (p-ELISA) has low sensitivity, complex preparation steps, and poor repeatability. The time-resolved fluorescence immunoassay (TR-FIA) method has high costs and requires the use of Eu-labeled antibodies and specialized fluorescence detection equipment.

[0005] Therefore, it is of great research value to develop a method for exosome detection with a wide linear range, low detection limit, low cost, simple operation, no need for expensive instruments and equipment, and simple preparation. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide a method for detecting exosome surface protein NELISA based on the activity regulation of single-atom catalysts.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] The present invention provides a kit for detecting exosome surface proteins, and the kit includes the following components: immobilized capture antibodies, aptamers specifically recognizing exosome surface proteins, nucleic acid amplification reagents, catalysts with peroxidase activity, and a color development system.

[0009] Further, the catalyst with peroxidase activity is a single-atom catalyst.

[0010] Further, the single-atom catalyst is nickel hydroxide loaded with single-atom Ru sites.

[0011] Further, the loading amount of Ru is 0.6 wt%.

[0012] Further, the nucleic acid amplification reagents include DNA polymerase, DNA polymerase buffer, and deoxyribonucleoside triphosphate.

[0013] Further, the DNA polymerase is Phi29 DNA polymerase.

[0014] Further, the color development system includes 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide.

[0015] Further, the kit also includes a ligation system for specific recognition, and the ligation system includes DNA ligase and DNA ligase buffer.

[0016] Further, the DNA ligase is T4 DNA ligase.

[0017] Further, the exosome surface proteins are Alix, TSG101, SNARE, RAB GTPase, CD9, CD63, or CD81.

[0018] Furthermore, the exosome surface protein is CD63; the aptamer is the sequence after annealing of the aptamer- primer and the linear template. The nucleotide sequence of the aptamer- primer is shown in SEQ ID No:1, and the nucleotide sequence of the linear template is shown in SEQ ID No:2.

[0019] The present invention also provides a method for detecting exosome surface proteins. The method uses the above- mentioned kit for testing, and the method includes the following steps:

[0020] (1) Mix the immobilized capture antibody with the sample containing exosomes to enable the capture antibody to specifically capture exosomes;

[0021] (2) Add the aptamer to enable it to specifically recognize the exosome surface protein;

[0022] (3) Amplify the nucleic acid signal using nucleic acid amplification technology;

[0023] (4) Add a catalyst with peroxidase activity, incubate, then add a chromogenic system, react, and read the corresponding chromogenic result.

[0024] Furthermore, the sample in step (1) is serum from which cells and large vesicles have been removed.

[0025] The present invention also provides the use of the above- mentioned kit in the preparation of preparations for diagnosing and / or assisting in screening and / or monitoring the treatment of cancer.

[0026] The present invention has achieved the following beneficial effects:

[0027] The present invention provides a method for detecting exosome surface proteins by nucleic acid enzyme- linked immunosorbent assay (NELISA) based on the regulation of the activity of single- atom catalysts. Compared with commercial ELISA kits, the method of the present invention has a wider linear range for detecting exosome surface proteins, can cover the detection requirements of exosomes from low concentration to high concentration, and is applicable to more diverse sample types and clinical application scenarios; has a lower detection limit, can detect exosomes at lower concentrations, and is applicable to the detection of early disease diagnosis and exosome low- abundance samples; the present invention uses aptamers and nanomaterials to replace expensive primary antibodies and enzyme- labeled secondary antibodies, significantly reducing the reagent cost, suitable for popularization and application in areas with limited resources, and reducing the economic threshold for exosome detection; the operation is simple, suitable for non- professionals to use, reducing the operation difficulty and training cost; does not require expensive instruments and equipment, reducing the hardware cost of detection, suitable for popularization and application in areas with limited resources; the preparation steps are simple, improving the repeatability and stability of the experiment, suitable for large- scale production and application, and having good application prospects.

[0028] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0029] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0030] Figure 1 It is a schematic diagram of the principle of the NELISA of the present invention for detecting exosome surface proteins.

[0031] Figure 2 It is the real-time absorbance value of the peroxidation product of the substrate TMB under different concentrations of 0.60-SA-Ru / Ni(OH)x and H2O2 catalytic systems.

[0032] Figure 3 It is the linear range of the NELISA of the present invention and commercial ELISA for detecting exosome surface proteins (n = 3 samples for each concentration of exosomes; ΔAbsorbance = OD450 - background OD′).

[0033] Figure 4 It is the precision of the NELISA of the present invention for detecting human sera with different exosome contents. Detailed Description of the Invention

[0034] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0035] In the following experiments, if no description of the temperature is made, the reaction is carried out under normal temperature conditions. Normal temperature means room temperature, which is 25 ± 5°C.

[0036] The present invention constructs an NELISA (nuclease-linked immunosorbent assay) platform based on the single-atom catalyst 0.60-SA-Ru / Ni(OH)x for highly sensitive detection of exosome surface proteins. The basic principle of this platform is as Figure 1 shown: First, the capture antibody (anti-CD63) immobilized in the microplate is used to specifically capture exosomes; subsequently, the specific protein on the exosome surface is recognized by the aptamer, converting the detection of the target protein into the detection of nucleic acid sequences; finally, the rolling circle amplification (RCA) technology is used to efficiently amplify the nucleic acid signal.

[0037] During the research process, the present invention found that nucleic acid sequences can significantly inhibit the peroxidase activity of single-atom catalysts.

[0038] The synthesis of the single-atom catalyst 0.60-SA-Ru / Ni(OH)x refers to the method of Tao et al. (Tao Y, Guan J, Zhang J, et al. Ruthenium Single Atomic Sites Surrounding the Support Pit with Exceptional Photocatalytic Activity[J]. Angewandte Chemie, 2024, 136(21)). The single-atom catalyst 0.60-SA-Ru / Ni(OH)x is nickel hydroxide loaded with single-atom Ru sites, and the loading amount of Ru is preferably 0.6 wt%.

[0039] Identification of the peroxidase activity of 0.60-SA-Ru / Ni(OH)x.

[0040] Catalytic system: 850 μL sodium acetate buffer (0.2 M, pH 4.5) + 50 μL H2O2 (2 M) + 50 μL TMB (10 mM) + 50 μL single-atom catalyst 0.60-SA-Ru / Ni(OH)x at different concentrations, monitored for 10 minutes. The concentrations of 0.60-SA-Ru / Ni(OH)x are 0.015 mg / mL, 0.03 mg / mL, 0.06 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, respectively.

[0041] The results are as Figure 2 shown. There is a positive correlation between the rate of formation of the TMB peroxidation product and the concentration of 0.60-SA-Ru / Ni(OH)x, indicating that 0.60-SA-Ru / Ni(OH)x exhibits concentration-dependent peroxidase-like activity.

[0042] Example 1: NELISA detection based on single-atom catalysts

[0043] I. Capturing the target

[0044] 1. Add 200 μL of PBS-T buffer (0.05%, v / v, Tween-20) to each well of a forward 96-well plate (i.e., microplate) and wash it once before use.

[0045] 2. Dilute the biotin-labeled CD63 antibody (abcam, ab59479) to 0.5 μg / mL with PBS (0.01 M, pH 7.4) and place it on ice for later use.

[0046] 3. Capture antibody immobilization: Add 100 μL of the biotinylated CD63 antibody with a concentration of 0.5 μg / mL obtained in step 2 to each well of a streptavidin-coated 96-well plate (ACROBiosystems, SP-11), and incubate with shaking at room temperature for 1 h.

[0047] Washing: Aspirate the liquid in the wells as much as possible, and wash 3 times with PBS to obtain a microplate with immobilized capture antibodies.

[0048] 4. Pretreatment of clinical samples: The clinical specimens (serum from breast cancer patients) are centrifuged at 2000×g for 20 min, and the precipitate is discarded to obtain cell-free serum. Then, it is centrifuged at 10000×g for 20 min at 4°C, and the precipitate is discarded to remove large vesicles. The serum sample is obtained, filtered through a 0.22 μm filter, and stored at -80°C for later use.

[0049] 5. Dilution of serum samples: Dilute the serum samples obtained in step 4 1:100 with PBS.

[0050] 6. Target capture: Add 100 μL of negative control (PBS) and the diluted serum samples to each well of the microplate with immobilized capture antibodies obtained in step 3, and place the microplate in an incubator at 37°C for 1 h.

[0051] Washing: Aspirate the liquid in the wells as much as possible, and wash 3 times with PBS.

[0052] II. Binding aptamer

[0053] 1. Annealing: Add 3 μL of aptamer - primer (50 μM) and 3 μL of linear template (50 μM) to 4 μL of deionized water, mix well and then anneal.

[0054] The nucleotide sequences of the aptamer - primer and the linear template are shown in Table 1.

[0055] Table 1 Nucleotide sequences of aptamer - primer and linear template

[0056]

[0057]

[0058] The above annealing procedure is to incubate in a water bath at 95°C for 5 min, then turn off the power and wait for the temperature to naturally drop to room temperature to obtain the annealed sequence.

[0059] Take the annealed sequence (5 μM, 2 μL) and add it to 100 μL of binding buffer (PBS buffer containing 0.5% BSA and 5 μM Mg 2+ to obtain the diluted annealed sequence.

[0060] 2. Aptamer binding to the target: Add 100 μL of the diluted annealed sequence obtained in Step 1 to the wells of the microplate that captures the target obtained in Step 1, and place the microplate in an incubator at 37 °C for 40 min.

[0061] Washing: Aspirate the liquid in the wells as much as possible and wash once with PBS.

[0062] 3. Prepare a 40 μL ligation system according to the following content, mix gently, place on ice for later use.

[0063] Reagent 40 μL Ligation System 2X Fast T4 DNA Ligase Buffer 20 μL Fast T4 DNA Ligase 2 μL Nuclease-free water 18 μL

[0064] T4 ligation: Add 40 μL of the ligation system to the wells and incubate at room temperature for 10 min.

[0065] Washing: Aspirate the liquid in the wells as much as possible and wash once with PBS to obtain the microplate with the target captured by the aptamer bound.

[0066] III. NELISA detection

[0067] 1. RCA amplification: Prepare a 20 μL amplification system according to the following content, mix gently, place on ice for later use.

[0068] Reagent 20 μL Amplification System phi29-X Reaction Buffer (5X) 4 μL dNTPs (10 mM) 1 μL phi29-XT DNA Polymerase 1 μL Nuclease-free Water 14 μL

[0069] RCA amplification: Add 20 μL of the amplification system to the wells and incubate at 37 °C for 60 min.

[0070] Removing the supernatant: Gently place the microplate on a horizontal tabletop and slowly aspirate the supernatant at the bottom of the wells with a 10 μL pipette tip.

[0071] 2. Blocking the active sites of the catalyst: Add 50 μL of the single-atom catalyst 0.60-SA-Ru / Ni(OH)x (0.1 mg / mL) to the wells and let stand at room temperature for 10 min.

[0072] Prepare a 150 μL color development system according to the following content, mix, and wait for the reagent temperature to return to room temperature. Keep away from light and prepare it immediately before use.

[0073] Reagent 150 μL Color Development System Sodium Acetate Buffer (0.2 M, pH 4.5) 130 μL TMB (10 mM) 10 μL <![CDATA[H2O2(2M)]]> 10 μL

[0074] Color development: Add 150 μL of the color development system to the wells and incubate at room temperature for 5 min. Read the corresponding color development results on an ELISA reader (652 nm).

[0075] 3. Performance analysis of the NELISA of the present invention

[0076] Refer to the above steps to analyze the performance of the NELISA of the present invention for testing exosomal surface proteins. The results are as Figure 3As shown, the linear range of the NELISA method of the present invention for detecting the surface protein (PD-L1) of exosomes is 10 1 -10 6 particles / μL, and the detection limit (the average background signal generated by the matrix blank plus 3 times the mean standard deviation) is 3.45 particles / μL; while the linear range of the commercial ELISA kit (AMEKO) for detecting the surface protein (PD-L1) of exosomes is 10 2 -10 5 particles / μL, and the detection limit (3 times the signal-to-noise ratio) is 15.8 particles / μL.

[0077] Therefore, the NELISA method of the present invention has a wider linear range and a lower detection limit, and has better detection performance than commercial ELISA kits.

[0078] IV. Evaluation of the accuracy and precision of the NELISA detection of the present invention

[0079] Accuracy evaluation: Exosomes with concentrations of 10 2 , 10 4 , 10 6 particles / μL were added to exosome-free human serum as test samples, resulting in low-value, medium-value, and high-value samples. The concentration of the exosomes was measured by Zhongke Baice Co., Ltd., and the instrument used was a nanoparticle tracking analyzer (ZetaView PMX110, Particle Metrix, Germany). The low-value, medium-value, and high-value samples were detected respectively, and each sample was detected 3 times. The average concentration was calculated and the relative deviation was calculated. The results are shown in Table 2. The relative deviations of the human serum samples with different exosome contents detected by the NELISA method of the present invention are all within the range of ±20.0%.

[0080] Table 2 Accuracy of the NELISA detection of the present invention for human serum with different exosome contents

[0081]

[0082] Precision evaluation: Exosomes with concentrations of 10 2 , 10 4 , 10 6 particles / μL were added to exosome-free human serum as test samples, resulting in low-value, medium-value, and high-value samples. The concentration of the exosomes was measured by Zhongke Baice Co., Ltd., and the instrument used was a nanoparticle tracking analyzer (ZetaView PMX110, Particle Metrix, Germany). The low-value, medium-value, and high-value samples were detected respectively, and each sample was detected 10 times. The coefficient of variation (CV) of each concentration measurement value was calculated respectively. The results are as followsFigure 4 As shown in Table 3, the precision of the present invention was measured by the coefficient of variation (CV). The CV of the NELISA of the present invention for detecting serum samples with different exosome contents was not greater than 15.0%.

[0083] Table 3 Precision of the NELISA of the present invention for detecting human sera with different exosome contents

[0084] Sample Exosome Content Coefficient of Variation (CV) Low <![CDATA[10 2 particles / μL]]> 8.37％ Medium <![CDATA[10 4 particles / μL]]> 1.71％ High <![CDATA[10 6 particles / μL]]> 2.34％

[0085] V. Comparison of the NELISA of the present invention with other exosome detection methods

[0086] The advantages of the NELISA detection method of the present invention include: the signal amplification ability of RCA amplification endows it with excellent sensitivity; it can directly detect exosomes in serum without the exosome extraction process. The comparison with other exosome detection methods is shown in Table 4.

[0087] Table 4 Comparison of the NELISA of the present invention with other exosome detection methods

[0088]

[0089] The references involved in Table 4 are as follows:

[0090] [1] Liu C, Zhao J, Tian F, et al. Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers [J]. Nature biomedical engineering, 2019, 3(3): 183-193.

[0091] [2] Zhang J, Shi J, Zhang H, et al. Localized fluorescent imaging of multiple proteins on individual extracellular vesicles using rolling circle amplification for cancer diagnosis [J]. Journal of extracellular vesicles, 2020, 10(1): e12025.

[0092] [3]Chen X,Deng Y,Niu R,et al.Cancer-derived small extracellular vesicles PICKER[J].Analytical Chemistry,2022,94(38):13019-13027.

[0093] [4]Feng Y,Yang Y,**ao Y,et al.Multi-parameter inputted logic-gating on aptamer-encoded extracellular vesicles for colorectal cancer diagnosis[J].Analytical Chemistry,2022,95(2):1132-1139.

[0094] [5]Zhou J,Lin Q,Huang Z,et al.Aptamer-initiated catalytic hairpin assembly fluorescence assay for universal,sensitive exosome detection[J].Analytical Chemistry,2022,94(15):5723-5728.

[0095] [6]Yu Y,Guo Q,Jiang W,et al.Dual-aptamer-assisted and logic gate for cyclic enzymatic signal amplification electrochemical detection of tumor-derived small extracellular vesicles[J].Analytical Chemistry,2021,93(32):11298-11304.

[0096] In summary, the present invention provides a method for detecting exosomal surface proteins by NELISA based on the activity regulation of single-atom catalysts. Compared with commercial ELISA kits, the method of the present invention for detecting exosomal surface proteins has a wider linear range, can cover the detection requirements of exosomes from low concentration to high concentration, and is applicable to more diverse sample types and clinical application scenarios; has a lower detection limit, can detect exosomes at lower concentrations, and is applicable to the detection of early disease diagnosis and exosomal low-abundance samples; the present invention uses aptamers and nanomaterials to replace expensive primary antibodies and enzyme-labeled secondary antibodies, significantly reducing the reagent cost, being suitable for popularization and application in areas with limited resources, and reducing the economic threshold for exosome detection; is simple to operate, suitable for use by non-professionals, reducing the operation difficulty and training cost; does not require expensive instruments and equipment, reducing the hardware cost of detection, and being suitable for popularization and application in areas with limited resources; has simple preparation steps, improving the repeatability and stability of experiments, being suitable for large-scale production and application, and having good application prospects.

Claims

1. A kit for detecting exosome surface proteins, characterized in that: The kit comprises the following components: an immobilized capture antibody, an aptamer that specifically recognizes exosome surface proteins, a nucleic acid amplification reagent, a catalyst with peroxidase activity, and a color development system.

2. The kit according to claim 1, characterized in that: The catalyst having peroxidase activity is a single-atom catalyst, and the single-atom catalyst is preferably nickel hydroxide loaded with a single-atom Ru site; the loading amount of Ru is preferably 0.6 wt %.

3. The kit according to claim 1, characterized in that: The nucleic acid amplification reagent comprises DNA polymerase, DNA polymerase buffer and deoxyribonucleoside triphosphates; preferably, the DNA polymerase is Phi29 DNA polymerase.

4. The kit according to claim 1, characterized in that: The color development system comprises 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide.

5. The kit according to claim 1, characterized in that: The kit further comprises a ligation system for specific identification, wherein the ligation system comprises DNA ligase and DNA ligase buffer; preferably, the DNA ligase is T4 DNA ligase.

6. The kit according to claim 1, characterized in that: The exosome surface protein is Alix, TSG101, SNARE, RAB GTPase, CD9, CD63 or CD81.

7. The kit according to claim 1, characterized in that: The exosome surface protein is CD63; the aptamer is a sequence after annealing the aptamer-primer and the linear template, the nucleotide sequence of the aptamer-primer is shown in SEQ ID No: 1, and the nucleotide sequence of the linear template is shown in SEQ ID No:

2.

8. A method for detecting exosome surface proteins, characterized in that: The method is to use the kit according to any one of claims 1 to 7 for testing, and the method comprises the following steps: (1) mixing the immobilized capture antibody with a sample containing exosomes so that the capture antibody specifically captures the exosomes; (2) adding aptamers to enable them to specifically recognize exosome surface proteins; (3) Amplifying nucleic acid signals using nucleic acid amplification technology; (4) adding a catalyst having peroxidase activity, incubating, then adding a color development system, reacting, and reading the corresponding color development result.

9. The method according to claim 8, characterized in that: The sample in step (1) is serum from which cells and large vesicles have been removed.

10. Use of the kit according to any one of claims 1 to 7 in the preparation of a preparation for diagnosis and / or auxiliary screening and / or treatment monitoring of cancer.

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