Colorimetric method for quantitatively detecting exosome

Abstract

The invention discloses a colorimetric method for quantitative detection of exosomes, and belongs to the technical field of non-protein-dependent exosome quantitative methods.The colorimetric method comprises the following steps that firstly, a sample to be detected is diluted with a solution B to obtain the diluted sample to be detected; 2, uniformly mixing a diluted sample to be detected with the suspension A, incubating at room temperature, and performing magnetic separation; and step 3, transferring the supernate obtained by magnetic separation in the step 2 into a 96 hole, adding the solution C and the solution D, reacting in a dark place, and measuring the absorbance in a microplate reader. According to the colorimetric method for quantitatively detecting the exosome, based on the property that the exosome and a magnetic graphene oxide-horse radish peroxidase (MGO-HRP) compound are competitively combined to release HRP, the HRP is utilized to catalyze substrate color development to realize the quantitative detection of the non-protein-dependent exosome.

Description

Technical Field

[0001] This invention relates to the field of non-protein-dependent exosome quantification methods, and in particular to a colorimetric method for quantitative detection of exosomes. Background Technology

[0002] Exosomes are extracellular vesicles, 30-150 nm in diameter, actively secreted by cells. Rich in bioactive components such as mRNA, miRNA, proteins, and lipids, they are widely present in bodily fluids like blood and urine and can mediate intercellular signal transduction. As biomarkers for cell biopsies, they can carry pathological information from parental cells, enabling non-invasive and minimally invasive diagnosis and disease monitoring of tumors and neurodegenerative diseases, avoiding the invasiveness of traditional tissue biopsies. Furthermore, as drug carriers, their good biocompatibility, low immunogenicity, and targeting properties allow for efficient loading of small molecule and nucleic acid drugs, making them suitable for drug delivery. Plant-derived exosomes contain natural active ingredients such as polyphenols and flavonoids, possessing anti-inflammatory and antioxidant medicinal value, providing direction for new drug development. Whether as potential disease diagnostic markers, drug delivery carriers, or potential therapeutic agents, accurate quantification of exosomes is a key technology for all these applications.

[0003] Currently, the main methods for quantifying exosomes include nanoparticle tracking analysis (NTA), flow cytometry, enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB). Among these, NTA is the most widely used because it can simultaneously obtain concentration and particle size distribution. However, NTA has significant drawbacks: it is easily interfered with by impurities such as protein aggregates, leading to overly high results; and it has a high detection limit (approximately 10). 6 The exosome quantification method (particles / mL) cannot accurately quantify low-concentration samples. The equipment is expensive and not readily available for ordinary experiments. Therefore, developing a simple and convenient method for exosome quantification is of great significance.

[0004] Graphene oxide (GO), as an oxidized derivative of graphene, is rich in oxygen-containing groups such as hydroxyl (-OH), epoxy (-O-), and carboxyl (-COOH) groups on its surface and edges. These groups not only give GO good hydrophilicity but also allow it to be stably dispersed in aqueous solutions. In addition, GO exhibits good biocompatibility, showing low toxicity to biological systems at suitable concentrations, thus possessing the basic conditions for developing bio-related applications. GO, as a typical two-dimensional nanomaterial, is characterized by its large specific surface area. Previous literature has reported that horseradish peroxidase (HRP) can be adsorbed onto the GO surface through non-covalent interactions, forming a GO-HRP complex. During their research on the interaction between the GO-HRP complex and liposomes, the inventors discovered a key phenomenon: when liposomes adsorb onto the surface of the GO-HRP complex, the HRP bound to the complex is released. Given the high similarity between liposomes and exosomes in composition (both contain a lipid bilayer structure) and morphology (both are vesicle-like), our research group has preliminarily developed a chemiluminescence method based on the GO-HRP complex for exosome quantification, enabling rapid detection of exosomes. However, this method requires centrifugation for separation during the assay, limiting its ease of operation. Summary of the Invention

[0005] The purpose of this invention is to provide a colorimetric method for the quantitative detection of exosomes. Magnetic graphene oxide (MGO) was prepared, and the property of HRP to catalyze the oxidation of the chromogenic substrate tetramethylbenzidine (TMB) was utilized to construct a colorimetric method based on the MGO-HRP complex for the rapid quantification of exosomes.

[0006] To achieve the above objectives, the present invention provides a colorimetric method for quantitative detection of exosomes, comprising the following steps: Step 1: Dilute the sample to be tested with solution B to obtain the diluted sample to be tested; Step 2: Mix the diluted sample with suspension A until homogeneous, incubate at room temperature, and then perform magnetic separation. Step 3: Transfer the supernatant obtained from magnetic separation in Step 2 to 96 wells, add solution C and solution D, react in the dark, and then measure the absorbance using an ELISA reader.

[0007] Preferably, in step one, solution B is a 0.1-0.2 mol / L PBS solution with a pH of 7.4.

[0008] Preferably, in step two, the volume of the diluted sample to be tested is 50-100 μL, and the volume of suspension A is 20-70 μL.

[0009] Preferably, in step two, suspension A is an MGO-HRP complex dispersion, the concentration of suspension A is 1-1.5 mg / mL, and the mass ratio of MGO to HRP in suspension A is (400-2000):1.

[0010] Preferably, the preparation method of suspension A is as follows: S1. Preparation of MGO: 40 mg of graphene oxide was dispersed in 4 mL of ethylene glycol and 4 mL of diethylene glycol solution and sonicated for 4 h to obtain a suspension. Then, 0.6 g of sodium acrylate, 0.6 g of sodium acetate and 0.216 g of ferric chloride were uniformly dispersed in the above suspension, transferred to a reaction vessel and heated at 200 °C for 10 h. After the reaction was completed, the product was washed with anhydrous ethanol and water and dried under vacuum at 60 °C to obtain MGO. S2. Preparation of the MGO-HRP complex: S2. Preparation of the MGO-HRP complex: 2 mg MGO was dispersed in 1 mL of water and ultrasonically dispersed until uniform. 1 mL of 1-5 μg HRP was added, and the mixture was incubated at room temperature for 30 min. The supernatant was discarded by magnetic separation, and the mixture was resuspended in 2 mL of 0.1 mol / L PBS solution at pH 7.4 to prepare suspension A.

[0011] Preferably, in step two, the incubation is carried out at room temperature for 5-10 minutes.

[0012] Preferably, in step three, the amount of supernatant used is 10-20 μL, the amount of solution C used is 50-60 μL, and the amount of solution D used is 50-60 μL.

[0013] Preferably, in step three, solution C is a TMB solution. The TMB solution is prepared by dissolving TMB in 0.2 mol / L HAc-KAc buffer to obtain a TMB solution with a concentration of 0.5-2 mmol / L. The pH of the HAc-KAc buffer is 4.5.

[0014] Preferably, in step three, solution D is a hydrogen peroxide solution with a concentration of 1-3 mmol / L.

[0015] Preferably, in step three, the reaction time in the dark is 10-20 min, and the absorbance is measured at 652 nm.

[0016] Therefore, the colorimetric method for quantitative detection of exosomes described above has the following beneficial effects: (1) Compared with ELISA, WB and exosome-based targeted detection methods based on immune capture, the present invention is a non-protein-dependent exosome quantification method, which can avoid the problem of inaccurate quantification caused by the absence or unevenness of exosome surface characteristic proteins; (2) Compared with the commonly used NTA, the experimental results of the present invention can be observed by the naked eye or quantitatively measured by ultraviolet-visible spectrophotometer or enzyme-linked immunosorbent assay (ELISA) reader. Both of these devices are experimental instruments available in ordinary laboratories, providing a simple and convenient method for quantitative analysis of exosomes for the development of diagnostic or therapeutic drugs based on exosomes in ordinary experiments. (3) The present invention is simple and convenient, and the exosome content information in the sample can be obtained within 20 minutes.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a test result diagram of Embodiment 1 of the present invention; Figure 2 This is a test result diagram of Embodiment 2 of the present invention; Figure 3 This is a test result diagram of Embodiment 3 of the present invention; Figure 4 This is a test result diagram of Embodiment 4 of the present invention; Figure 5 This is a test result diagram of Embodiment 5 of the present invention; Figure 6 This is a test result diagram of Embodiment 6 of the present invention; Figure 7 This is a test result diagram of Embodiment 7 of the present invention; Figure 8 This is a test result diagram of Embodiment 8 of the present invention; Figure 9 This is a test result diagram of Embodiment 9 of the present invention; Figure 10 This is a test result diagram of Embodiment 10 of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0022] Example 1 Suspension A is an MGO-HRP complex dispersion (1 mg / mL, m MGO :m HRP =1000:1), and its preparation method is as follows: S1. Preparation of MGO: 40 mg of graphene oxide was dispersed in 4 mL of ethylene glycol and 4 mL of diethylene glycol solution and sonicated for 4 h to obtain a suspension. Then, 0.6 g of sodium acrylate, 0.6 g of sodium acetate and 0.216 g of ferric chloride were uniformly dispersed in the above suspension, transferred to a reaction vessel and heated at 200 °C for 10 h. After the reaction was completed, the product was washed with anhydrous ethanol and water and dried under vacuum at 60 °C to obtain MGO. S2. Preparation of the MGO-HRP complex: S2. Preparation of the MGO-HRP complex: 2 mg MGO was dispersed in 1 mL of water and ultrasonically dispersed until uniform. 1 mL of 3 μg HRP was added, and the mixture was incubated at room temperature for 30 min. The supernatant was discarded by magnetic separation, and the mixture was resuspended in 2 mL of 0.1 mol / L PBS solution at pH 7.4 to prepare suspension A.

[0023] Solution B sample dilution (0.1 mol / L PBS, pH 7.4); Solution C is a TMB solution (1.6 mmol / L, 0.2 mol / L HAc-KAc buffer, pH 4.5). Solution D is hydrogen peroxide (2 mmol / L, water).

[0024] The sample to be tested was exosomes. The exosomes were diluted with solution B, and the concentrations of the diluted exosomes were 0 and 9.2 × 10⁻⁶. 2 2.3×10 3 4.6×10 3 9.2×10 3 1.8×10 4 3.7×10 4 4.6×10 4 particles μL -1 .

[0025] This invention provides a colorimetric method for quantitative detection of exosomes, comprising the following steps: Take 80 μL of diluted exosomes and mix thoroughly with 20 μL of suspension A. Incubate at room temperature for 5 min, then magnetically separate and transfer 10 μL of the supernatant into 96 wells. Add 50 μL of solution C and 50 μL of solution D, and react in the dark for 15 min. Measure the absorbance at 652 nm using a microplate reader. The experimental results are as follows: Figure 1 As shown.

[0026] Depend on Figure 1 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and 20 μL of suspension A is used, the standard curve for detecting exosomes using this method has a detection limit of 9.2 × 10⁻⁶. 2 particles μL -1 The linear range is 9.2 × 10⁻⁶. 2 particles μL -1 -4.6×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 98%.

[0027] Example 2 The only difference between this embodiment and Example 1 is that the amount of suspension A used is 30 μL; all other conditions are the same. The experimental results are as follows: Figure 2 As shown.

[0028] Depend on Figure 2 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and the suspension A is 30 μL, the standard curve for detecting exosomes using this method has a detection limit of 2.3 × 10⁻⁶. 3 particles μL -1 The linear range is 2.3 × 10⁻⁶. 3 particles μL -1 -3.7×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 98%.

[0029] Example 3 The only difference between this embodiment and Example 1 is that the amount of suspension A used is 40 μL; all other conditions are the same. The experimental results are as follows: Figure 3 As shown.

[0030] Depend on Figure 3 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and the suspension A is 40 μL, the standard curve for detecting exosomes using this method has a detection limit of 9.2 × 10⁻⁶. 2 particles μL-1 The linear range is 2.3 × 10⁻⁶. 3 particles μL -1 -3.7×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 104%.

[0031] Example 4 The only difference between this embodiment and Example 1 is that the amount of suspension A used is 50 μL; all other conditions are the same. The experimental results are as follows: Figure 4 As shown.

[0032] Depend on Figure 4 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and the suspension A is 50 μL, the standard curve for detecting exosomes using this method has a detection limit of 2.3 × 10⁻⁶. 3 particles μL -1 The linear range is 4.6 × 10⁻⁶. 3 particles μL -1 -3.7×10 4 particles μL -1 When the concentration of exosomes is 9.2 × 10⁻⁶ 3 particles μL -1 At that time, the recovery rate was 101%.

[0033] Example 5 The only difference between this embodiment and Example 1 is that the amount of suspension A used is 60 μL; all other conditions are the same. The experimental results are as follows: Figure 5 As shown.

[0034] Depend on Figure 5 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and the suspension A is 60 μL, the standard curve for detecting exosomes using this method has a detection limit of 4.6 × 10⁻⁶. 3 particles μL -1 The linear range is 4.6 × 10⁻⁶. 3 particles μL -1 -7.4×10 4 particles μL -1 When the concentration of exosomes is 9.2 × 10⁻⁶ 3 particles μL -1At that time, the recovery rate was 97%.

[0035] Example 6 The only difference between this embodiment and Example 1 is that the amount of suspension A used is 70 μL; all other conditions are the same. The experimental results are as follows: Figure 6 As shown.

[0036] Depend on Figure 6 It can be seen that when m MGO :m HRP When the ratio is 1000:1 and the suspension A is 70 μL, the standard curve for detecting exosomes using this method has a detection limit of 4.6 × 10⁻⁶. 3 particles μL -1 The linear range is 9.2 × 10⁻⁶. 3 particles μL -1 -7.4×10 4 particles μL -1 When the concentration of exosomes is 1.8 × 10⁻⁶ 4 particles μL -1 At that time, the recovery rate was 95%.

[0037] Example 7 The only difference between this embodiment and Embodiment 3 is that: in suspension A, m MGO :m HRP With a ratio of 2000:1 and all other conditions remaining the same, the experimental results are as follows: Figure 7 As shown.

[0038] Depend on Figure 7 It can be seen that when m MGO :m HRP When the ratio is 2000:1 and the suspension A is 40 μL, the standard curve for detecting exosomes using this method has a detection limit of 2.3 × 10⁻⁶. 3 particles μL -1 The linear range is 4.6 × 10⁻⁶. 3 particles μL -1 -3.7×10 4 particles μL -1 When the concentration of exosomes is 9.2 × 10⁻⁶ 3 particles μL -1 At that time, the recovery rate was 97%.

[0039] Example 8 The only difference between this embodiment and Embodiment 3 is that: in suspension A, m MGO :m HRP With a ratio of 700:1 and all other conditions remaining the same, the experimental results are as follows: Figure 8 As shown.

[0040] Depend on Figure 8 It can be seen that when m MGO :m HRP When the ratio is 700:1 and the suspension A is 40 μL, the standard curve for detecting exosomes using this method has a detection limit of 2.3 × 10⁻⁶. 3 particles μL -1 The linear range is 2.3 × 10⁻⁶. 3 particles μL -1 -3.7×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 102%.

[0041] Example 9 The only difference between this embodiment and Embodiment 3 is that: in suspension A, m MGO :m HRP With a ratio of 500:1 and all other conditions remaining the same, the experimental results are as follows: Figure 9 As shown.

[0042] Depend on Figure 9 It can be seen that when m MGO :m HRP When the ratio is 500:1 and the suspension A is 40 μL, the standard curve for detecting exosomes using this method has a detection limit of 9.2 × 10⁻⁶. 2 particles μL -1 The linear range is 9.2 × 10⁻⁶. 2 particles μL -1 -4.6×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 102%.

[0043] Example 10 The only difference between this embodiment and Embodiment 3 is that: in suspension A, m MGO :m HRP With a ratio of 400:1 and all other conditions remaining the same, the experimental results are as follows: Figure 10 As shown.

[0044] Depend on Figure 10 It can be seen that when m MGO :m HRPWhen the ratio is 400:1 and the suspension A is 40 μL, the standard curve for detecting exosomes using this method has a detection limit of 9.2 × 10⁻⁶. 2 particles μL -1 The linear range is 9.2 × 10⁻⁶. 2 particles μL -1 -4.6×10 4 particles μL -1 When the concentration of exosomes is 4.6 × 10 3 particles μL -1 At that time, the recovery rate was 97%.

[0045] Therefore, the present invention employs the above-mentioned colorimetric method for quantitative detection of exosomes, which is based on the property that exosomes can release HRP through competitive binding with magnetic graphene oxide-horseradish peroxidase (MGO-HRP) complex, and utilizes HRP to catalyze substrate color development to achieve non-protein-dependent quantitative detection of exosomes.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A colorimetric method for quantitative detection of exosomes, characterized in that: Includes the following steps: Step 1: Dilute the sample to be tested with solution B to obtain the diluted sample to be tested; Step 2: Mix the diluted sample with suspension A until homogeneous, incubate at room temperature, and then perform magnetic separation. Step 3: Transfer the supernatant obtained from magnetic separation in Step 2 to 96 wells, add solution C and solution D, react in the dark, and then measure the absorbance using an ELISA reader.

2. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step one, solution B is a 0.1-0.2 mol / L PBS solution with a pH of 7.

4.

3. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step two, the volume of the diluted sample to be tested is 50-100 μL, and the volume of suspension A is 20-70 μL.

4. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step two, suspension A is an MGO-HRP complex dispersion with a concentration of 1-1.5 mg / mL and a mass ratio of MGO to HRP of (400-2000):

1.

5. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: The preparation method of suspension A is as follows: S1. Preparation of MGO: 40 mg of graphene oxide was dispersed in 4 mL of ethylene glycol and 4 mL of diethylene glycol solution and sonicated for 4 h to obtain a suspension. Then, 0.6 g of sodium acrylate, 0.6 g of sodium acetate and 0.216 g of ferric chloride were uniformly dispersed in the above suspension, transferred to a reaction vessel and heated at 200 °C for 10 h. After the reaction was completed, the product was washed with anhydrous ethanol and water and dried under vacuum at 60 °C to obtain MGO. S2. Preparation of the MGO-HRP complex: 2 mg MGO was dispersed in 1 mL of water and ultrasonically dispersed until uniform. 1 mL of 1-5 μg HRP was added, and the mixture was incubated at room temperature for 30 min. The supernatant was discarded by magnetic separation, and the mixture was resuspended in 2 mL of 0.1 mol / L PBS solution at pH 7.4 to prepare suspension A.

6. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step two, incubate at room temperature for 5-10 minutes.

7. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step three, the volume of supernatant used is 10-20 μL, the volume of solution C is 50-60 μL, and the volume of solution D is 50-60 μL.

8. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step three, solution C is a TMB solution. The TMB solution is prepared by dissolving TMB in 0.2 mol / L HAc-KAc buffer to obtain a TMB solution with a concentration of 0.5-2 mmol / L. The pH of the HAc-KAc buffer is 4.

5.

9. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step three, solution D is a hydrogen peroxide solution with a concentration of 1-3 mmol / L.

10. The colorimetric method for quantitative detection of exosomes according to claim 1, characterized in that: In step three, the reaction time is 10-20 min in the dark, and the absorbance is measured at 652 nm.

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