Exosome visualized detection biosensor and detection method

Abstract

The application discloses an exosome visual detection biosensor and a detection method, wherein the visual detection biosensor comprises a recognition probe one modified magnetic bead for being combined with an exosome to generate a magnetic bead-exosome compound, and further comprises a gold platinum nanometer enzyme modified engineering bacterium for being combined with a recognition probe two to generate a recognition probe two modified engineering bacterium; the recognition probe two modified surface mineralization engineering bacterium is combined with the magnetic bead-exosome compound, and after magnetic separation, a magnetic bead-exosome-surface mineralization engineering bacterium compound is generated; and the corresponding exosome concentration is visually detected through nanometer enzyme catalytic oxidation of the compound on the color developing solution. Gold platinum nanometer enzymes are modified on the surface of escherichia coli through a biological mineralization synthesis method, compared with single gold platinum nanometer enzymes, since the specific surface area of the escherichia coli is large, more gold platinum nanometer enzymes can be accumulated and synthesized on the surface of a single escherichia coli, thereby playing a cascade amplification role, and realizing visual quantitative detection of tumor exosomes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an exosome visual detection biosensor and a detection method. BACKGROUND

[0002] The current gold standard for exosome marker detection is still ELISA, flow cytometry and mass spectrometry, which lack the feasibility of early screening for cancer, such as low sensitivity, the need for expensive instruments and professional operation. Therefore, it is difficult to achieve early screening of cancer in resource-limited areas. Based on the above problems, it is essential to develop a low-cost, simple-to-operate, and high-sensitivity exosome biosensor and detection method for the clinical application of exosomes.

[0003] As the applicant proposed in the Chinese invention patent application with publication number CN118688452A, specifically an exosome detection biosensor based on engineered bacteria and a detection method, which includes: a recognition probe modified magnetic bead for binding with exosomes to generate a magnetic bead-exosome complex; a recognition probe modified engineered bacteria for binding with the magnetic bead-exosome complex to generate engineered bacteria combined with the magnetic bead-exosome, and the non-target adsorbed engineered bacteria are removed by washing, and the engineered bacteria combined with the magnetic bead-exosome are uniformly coated on the corresponding culture medium for culture, and the concentration of the corresponding exosomes is calculated by observing the number of colonies. However, this method is relatively complicated to operate, and the detection time is long. When detecting by the number of colonies, there are many interference items, which are easily affected by external factors, and the detection data cannot be analyzed by instruments, so that the samples cannot be compared intuitively.

[0004] In recent years, due to the progress of synthetic enzyme-based assays and the integration of various types of nanoscale enzymes, colorimetric exosome biosensors with visual readouts have undergone rapid development. These synthetic nanomaterials exhibit unique physicochemical properties and catalytic capabilities, making it possible to construct exosome colorimetric biosensors with new principles. SUMMARY

[0005] The purpose of the present application is to provide an exosome visual detection biosensor and a detection method to solve the problems raised in the background art.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] An exosome visual detection biosensor includes a recognition probe modified magnetic bead for binding with exosomes to generate a magnetic bead-exosome complex, and further includes:

[0008] The surface mineralization modified gold platinum nanoscale enzyme engineering bacteria are used for catalyzing oxidation of the color developing liquid, and the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria are used for being combined with the recognition probe two to generate the recognition probe two modified surface mineralization engineering bacteria;

[0009] The recognition probe two modified surface mineralization engineering bacteria are used for being combined with the magnetic bead-exosome compound, and after magnetic separation, the magnetic bead-exosome-surface mineralization engineering bacteria compound is generated, the gold platinum nanoscale enzyme on the compound is used for catalyzing oxidation of the color developing liquid, and the corresponding exosome concentration is visually detected;

[0010] At least one of the recognition probe one and the recognition probe two is a specific target recognition probe.

[0011] As a further scheme of the present application, the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria preparation method is as follows:

[0012] F1, the E. coli is proliferated in the LB liquid culture medium, and when reaching a predetermined concentration, the E. coli is collected; first, the E. coli bacterial liquid is centrifuged at a centrifugal force of 5000g for 5min, then the precipitate is collected, and the buffer is used for multiple washing to obtain the concentrated E. coli bacterial liquid.

[0013] F2, the E. coli bacterial liquid is added into the anaerobic M9 low-salt culture medium, resuspended, and free oxygen is removed by blowing nitrogen gas, then chloroauric acid and sodium borohydride are injected, and the E. coli bacterial liquid is cultured at 37 DEG C and a rotation speed of 200rpm for 3h, then the E. coli bacterial liquid is centrifuged at a centrifugal force of 5000g for 5min, the supernatant is removed, and the physiological saline with a concentration of 5% is added for washing, and the washing is repeated for 2-3 times, then pure water is added to obtain the surface modification gold nanoparticle engineering bacteria.

[0014] F3, the above synthesized surface modification gold platinum nanoscale enzyme engineering bacteria are added into pure water, resuspended, and chloroplatinic acid, sodium borohydride and sodium citrate are injected, and the stirring treatment is carried out at 1500rpm / min, until the engineering bacteria become black, indicating that the platinum nanoparticles are successfully modified on the surface of the engineering bacteria, then the engineering bacteria are centrifuged at a centrifugal force of 5000g for 5min, the supernatant is removed, the physiological saline with a concentration of 5% is added for washing, and the washing is repeated for 2-3 times, then pure water is added to obtain the surface mineralization modified gold platinum nanoparticle engineering bacteria.

[0015] The gold platinum nanoparticle is modified on the engineering bacteria by a biomimetic synthesis method, the preparation process is simple and environmentally friendly, and the obtained gold platinum nanoparticle has a clean surface, a large specific surface area and good colloidal stability, and can exhibit high catalytic activity. In the present application, the gold platinum nanoenzyme is modified on the surface of E. coli by a biomimetic synthesis method, and compared with a single gold platinum nanoenzyme, the surface of a single E. coli can accumulate and synthesize more gold platinum nanoenzymes due to the large surface area of E. coli, thereby playing a cascade amplification role. Even if the sample (such as serum) to be detected contains a low concentration of tumor exosomes, the signal of the exosomes can be amplified by a small amount of biomimetic synthesis of the engineering bacteria, so that quantitative detection can be realized.

[0016] As a further scheme of the present application, the preparation method of the recognition probe two modified surface mineralized engineering bacteria is as follows: incubate 1%-10% of the thiol modified recognition probe two with 1%-10% of tris(2-carboxyethyl) phosphine at room temperature for 10-60 min;

[0017] Then, the incubation product is added to the engineering bacteria solution of the surface mineralized gold platinum nanoparticle, and co-incubated at room temperature for 10-60 min to obtain the recognition probe two modified surface mineralized engineering bacteria.

[0018] As a further scheme of the present application, the chromogenic solution is specifically 3,3',5,5'-tetramethylbenzidine (TMB) and the like.

[0019] As a further scheme of the present application, the recognition probe one uses a common target aptamer or an antibody and the like as a recognition molecule; and the recognition probe two uses a specific target aptamer or an antibody and the like as a recognition molecule.

[0020] As a further scheme of the present application, the common target aptamer is specifically a CD63 protein aptamer, a CD81 protein aptamer or a CD9 protein aptamer and the like.

[0021] As a further scheme of the present application, the specific target aptamer is specifically a nucleolin protein aptamer, a PD-L1 protein aptamer, a CD44 protein aptamer, a HER2 protein aptamer, an EpCAM protein aptamer or a MUC1 protein aptamer and the like.

[0022] In the present application, the exosomes to be detected can be exosomes derived from different kinds of tumors, such as early, medium and late stage exosomes of breast cancer cell exosomes, lung cancer cell exosomes, liver cancer cell exosomes, leukemia cell exosomes and the like. It can also be exosomes derived from other diseases, such as Alzheimer's disease corresponding cell exosomes, diabetes corresponding cell exosomes, Parkinson's disease corresponding cell exosomes and the like.

[0023] An exosome visual detection method based on the foregoing exosome visual detection biosensor, comprising the following steps:

[0024] S1, the recognition probe one modified magnetic bead is washed with PBS buffer for several times, then the exosome sample is added, and the mixture is incubated at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the PBS buffer is washed to obtain a magnetic bead-exosome complex;

[0025] S2, the magnetic bead-exosome complex is incubated with the recognition probe two modified surface mineralization engineering bacteria at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the PBS buffer is washed to remove the non-target adsorbed engineering bacteria, to obtain a magnetic bead-exosome-surface mineralization engineering bacteria;

[0026] S3, visual detection of exosomes: the magnetic bead-exosome-surface mineralization engineering bacteria are added to 3,3',5,5'-tetramethylbenzidine (TMB), and the catalytically active nanoparticles on the surface of the engineering bacteria are used to catalyze the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H + The color of the solution changes obviously, and a visible signal is generated, and the absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] 1. The gold platinum nanoscale enzyme is modified on the engineering bacteria by the biological mineralization synthesis method, and the preparation process is simple, environmental protection, and the obtained gold platinum nanoscale enzyme has high catalytic activity due to the clean surface, large specific surface area and good colloidal stability;

[0029] 2. The gold platinum nanoscale enzyme is modified on the surface of E. coli by the biological mineralization synthesis method, and compared with the single gold platinum nanoscale enzyme, the E. coli has a rod-shaped 3D structure and a large surface-to-volume ratio, and the surface of a single E. coli can accumulate and synthesize more gold platinum nanoscale enzymes, providing a large number of target points for probe combination, i.e. the large surface area of these nanoparticles can capture a large amount of probes and fix them on the surface of the nanoparticles, thereby playing a cascade amplification role, so that even if the tumor exosome in the sample (such as serum) to be detected has a low concentration, the signal of the tumor exosome can be amplified by the less biological mineralization synthesis of the engineering bacteria, thereby realizing quantitative detection;

[0030] 3、The detection method of the present application is simple to operate, does not require expensive instruments and relatively cumbersome operation, can detect tumor exosomes using a simple method, improves the sensitivity, convenience, reaction time and cost of early tumor exosome detection, and compared with the prior art, the detection result can be visualized, is more intuitive, and is conducive to promoting the application of exosome-based liquid biopsy in early tumor screening. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 A flowchart of the visual detection of tumor cell exosomes according to the present application.

[0032] Figure 2 Characterization of tumor exosomes, wherein, Figure 2 A: TEM imaging of exosomes, Figure 2 B: NTA analysis of exosomes, Figure 2 C: WB characterization of exosomes.

[0033] Figure 3 Characterization of E.coli-Au-Pt, wherein, Figure 3 A: bright field image of E.coli-Au / Pt, Figure 3 B: TEM imaging of E.coli, Figure 3 C: TEM imaging of E.coli-Au, Figure 3 D: TEM imaging of E.coli-Au-Pt, Figure 3 E: DLS measurement of the particle size of E.coli-Au-Pt, Figure 3 F: potential of E.coli, E.coli-Au and E.coli-Au-Pt alone.

[0034] Figure 4 Feasibility verification and reaction condition optimization, Figure 4 A: effect of aptamer and exosome on catalytic efficiency, Figure 4 B: combination of E.coli and different concentrations of DNA (μM), Figure 4 C: optimization of exosome and magnetic bead binding time, Figure 4 D: exosome and E.coli binding time.

[0035] Figure 5 Clinical sample detection, 14 groups of healthy patient serum and 14 groups of tumor patient serum samples were detected by the method of the present application, and the bright field photos and ultraviolet absorption images are shown. DETAILED DESCRIPTION

[0036] Clearly and completely, the technical solutions in the embodiments of the present application will be described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0037] An exosome visual detection biosensor comprises a recognition probe I modified magnetic bead and an engineered bacterium of surface mineralization modified gold platinum nanoszyme, wherein the recognition probe I modified magnetic bead is used for binding with the exosome to generate a magnetic bead-exosome complex;

[0038] In the engineered bacterium of surface mineralization modified gold platinum nanoszyme, the gold platinum nanoszyme is used for catalyzing oxidation of a color developing solution, and the engineered bacterium of surface mineralization modified catalytically active nanoparticle is used for binding with the recognition probe II to generate a recognition probe II modified surface mineralization engineered bacterium;

[0039] The recognition probe II modified surface mineralization engineered bacterium is used for binding with the magnetic bead-exosome complex, and after magnetic separation, a magnetic bead-exosome-surface mineralization engineered bacterium is generated, and the corresponding exosome concentration is visually detected through catalytic oxidation of the color developing solution by the magnetic bead-exosome-surface mineralization engineered bacterium;

[0040] The color developing solution is specifically 3,3',5,5'-tetramethylbenzidine (TMB), and at least one of the recognition probe I and the recognition probe II is a specific target recognition probe.

[0041] For example, the recognition probe I is a common target aptamer, specifically a CD63 protein aptamer, a CD81 protein aptamer or a CD9 protein aptamer;

[0042] In order to screen exosomes without normal cells and facilitate accurate detection of the concentration of tumor cell exosomes, the recognition probe II is a specific target aptamer, specifically a nucleolin protein aptamer, a PD-L1 protein aptamer, a CD44 protein aptamer, a HER2 protein aptamer, an EpCAM protein aptamer or a MUC1 protein aptamer.

[0043] Of course, as other solutions, the recognition probe I can also be a specific target aptamer, and the corresponding recognition probe II can be a common target aptamer.

[0044] In the present application, the exosomes to be detected can be early and middle stage exosomes of different kinds of tumor sources, such as breast cancer cell exosomes, lung cancer cell exosomes, liver cancer cell exosomes, leukemia cell exosomes, etc. They can also be exosomes of other disease sources, such as Alzheimer's disease corresponding cell exosomes, diabetes corresponding cell exosomes, Parkinson's disease corresponding cell exosomes, etc.

[0045] In the present application, the preparation method of the recognition probe one modified magnetic beads is as follows: incubate the streptavidin modified magnetic beads with a concentration of 10 mg / mL and the biotin labeled recognition probe one with a concentration of 10 uM in PBS buffer for 10-60 min, wash the incubation product with PBS buffer for multiple times to remove free recognition probe one, and then block the incubation product with 10% bovine serum albumin at room temperature for 1-2 h to prevent the adsorption of non-targets, thereby obtaining the recognition probe one modified magnetic beads.

[0046] In the present application, the specific preparation method of the surface mineralization modified catalytically active nanoparticle engineering bacteria is as follows:

[0047] F1, proliferate the E. coli in the LB liquid medium, and collect the E. coli when the bacterial OD value reaches 0.9; first, centrifuge the E. coli bacterial solution at a centrifugal force of 5000 g for 5 min, collect the precipitate, and wash it with a buffer for multiple times to obtain a concentrated E. coli bacterial solution.

[0048] F2, add the E. coli bacterial solution to the anaerobic M9 low-salt culture medium, resuspend, remove free oxygen by blowing nitrogen, and then inject 50 uL of chloroauric acid and 100 uL of sodium borohydride, and shake culture at 37°C and a rotation speed of 200 rpm for 3 h; then, centrifuge the E. coli bacterial solution at a centrifugal force of 5000 g for 5 min, remove the supernatant, wash with 5% physiological saline, repeat the washing for 2-3 times, and then add pure water to obtain the surface modification gold nanoparticle engineering bacteria.

[0049] F3, add the above synthesized surface modification gold nanoparticle engineering bacteria to pure water, resuspend, inject 25 uL of chloroplatinic acid, 100 uL of sodium borohydride, and 300 uL of sodium citrate, and stir at a stirring speed of 1500 rpm / min; when the engineering bacteria become black, it indicates that platinum nanoparticles are successfully modified to the surface of the engineering bacteria; then, centrifuge the engineering bacteria at a centrifugal force of 5000 g for 5 min, remove the supernatant, wash with 5% physiological saline, repeat the washing for 2-3 times, and then add pure water to obtain the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria.

[0050] Further, the preparation method of the recognition probe two modified surface mineralization engineering bacteria is as follows: incubate the thiol modified recognition probe two with a concentration of 1%-10% and the tris(2-carboxyethyl) phosphine with a concentration of 1%-10% at room temperature for 10-60 min;

[0051] Then, add the incubation product to the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria solution, and co-incubate at room temperature for 10-60 min to obtain the recognition probe two modified surface mineralization engineering bacteria.

[0052] An exosome visual detection method based on the foregoing exosome visual detection biosensor, comprising the following steps:

[0053] S1, the recognition probe one modified magnetic beads after blocking treatment are washed with PBS buffer for multiple times, then the exosome sample is added, and they are co-incubated at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the magnetic beads-exosome complex is obtained by washing with PBS buffer;

[0054] S2, the magnetic beads-exosome complex is incubated with the surface mineralization engineering bacteria modified by the recognition probe two at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the non-target adsorbed engineering bacteria are removed by washing with 1XPBS buffer with a pH value of 7.0-8.6 and a concentration of 10-100 nm / mL, to obtain the magnetic beads-exosome-surface mineralization engineering bacteria;

[0055] S3, visual detection of exosomes: the magnetic beads-exosome-surface mineralization engineering bacteria are added to 3,3',5,5'-tetramethylbenzidine (TMB), and the gold platinum nanoscale enzyme modified by the surface mineralization of the engineering bacteria catalyzes the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H + , so that the color of the solution changes obviously, a visible signal is generated, and the absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0056] Example 1

[0057] An exosome visual detection method, please refer to Figure 1 , comprising the following steps:

[0058] S1, the CD63 protein aptamer modified magnetic beads after blocking treatment are washed with PBS buffer for multiple times, then the exosome sample is added, and they are co-incubated at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the magnetic beads-exosome complex is obtained by washing with PBS buffer;

[0059] S2, the magnetic beads-exosome complex is incubated with the surface mineralization engineering bacteria modified by the nucleolin protein aptamer at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the non-target adsorbed engineering bacteria are removed by washing with 1XPBS buffer with a pH value of 7.0-8.6, to obtain the magnetic beads-exosome-surface mineralization engineering bacteria;

[0060] S3, visual detection of exosomes: the magnetic beads-exosome-surface mineralization engineering bacteria are added to 3,3',5,5'-tetramethylbenzidine (TMB), and the gold platinum nanoscale enzyme modified by the surface mineralization of the engineering bacteria catalyzes the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H+ The catalytic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H

[0061] In this embodiment, the CD63 protein aptamer modified magnetic beads recognize the common target of all exosomes (CD63 protein) through CD63 protein aptamer, generate magnetic bead-exosome complexes combined with all exosomes, then the nucleolin protein aptamer modified surface mineralization engineering bacteria recognize the specific target of tumor exosomes (nucleolin protein) through nucleolin protein aptamer, while normal cell exosomes cannot be recognized by nucleolin protein aptamer, then through magnetic separation, remove the unbound surface mineralization engineering bacteria, finally after color development, the specific tumor exosomes are quantitatively detected.

[0062] Example 2

[0063] An exosome visualization detection method, the steps of which comprise:

[0064] S1, the nucleolin protein aptamer modified magnetic beads after blocking treatment are washed with PBS buffer for multiple times, then the exosome sample is added, and incubated at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the PBS buffer is washed to obtain the magnetic bead-exosome complex;

[0065] S2, the magnetic bead-exosome complex is incubated with the CD63 protein aptamer modified surface mineralization engineering bacteria at room temperature for 10-60 min, then magnetic separation is performed, the supernatant is removed, and the 1XPBS buffer with a pH value of 7.0-8.6 is cleaned to remove the non-target adsorbed engineering bacteria, to obtain the magnetic bead-exosome-surface mineralization engineering bacteria;

[0066] S3, visualization detection of exosomes: the magnetic bead-exosome-surface mineralization engineering bacteria are added to 3,3',5,5'-tetramethylbenzidine (TMB), and the catalytically active nanoparticles on the surface of the engineering bacteria modified by surface mineralization catalyze the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H + The catalytic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H

[0067] In this embodiment, the nucleolin protein aptamer modified magnetic beads recognize the specific target of tumor exosomes (nucleolin protein) through nucleolin protein aptamer, generate magnetic bead-exosome complexes combined with tumor exosomes, while normal cell exosomes cannot be combined and are removed by subsequent magnetic separation. Then, the CD63 protein aptamer modified surface mineralization engineering bacteria recognize the common target of tumor exosomes (CD63 protein) through CD63 protein aptamer, generate magnetic bead-tumor exosome-surface mineralization engineering bacteria, then remove the unbound surface mineralization engineering bacteria by magnetic separation, and finally perform color reaction to quantitatively detect specific tumor exosomes.

[0068] I. Collection and characterization of exosomes

[0069] By culturing leukemia HL-60 cells, cell-secreted exosomes were collected as tumor cell exosomes for experiments, and their morphological structure, particle size and marker proteins were characterized. Transmission electron microscopy (TEM) imaging results show that the size of exosomes is about 150 nm, and they exhibit a typical disc shape (as shown in Figure 2 A). Nanoparticle tracking analysis (NTA) results further confirm that the size of exosomes is about 160 nm, which is consistent with the TEM results (as shown in Figure 2 B). Finally, two marker proteins CD63 and TSG-101 were successfully detected on the exosome sample by Western blotting (WB) (as shown in Figure 2 C). These results show that the exosomes have been successfully obtained.

[0070] II. Preparation and characterization of surface mineralization modified gold platinum nanoparticle engineering bacteria (E. coli-Au-Pt)

[0071] First, the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria were prepared according to the above method. By comparing the synthesis colors of the engineering bacteria at different stages, as shown in Figure 3 A, the color changes from the single E. coli cream color (1) to black (3), indicating that the engineering bacteria are successfully synthesized. Transmission electron microscopy (TEM) shows that the engineering bacteria without gold platinum nanoscale enzyme modification (as shown in Figure 3 B) compared with the engineering bacteria modified with gold platinum nanoscale enzyme (as shown in Figure 3 D), the gold platinum nanoscale enzyme modified engineering bacteria have obvious nanoparticles on the surface, and the average size of E. coli-Au-Pt is about 1209 nm, proving that our gold platinum nanoscale enzyme is successfully encapsulated on the surface of E. coli. The particle size and zeta potential were characterized, and the particle size of NPs was measured by dynamic light scattering DLS method, as shown in Figure 3 E, the DLS analysis results show that the particle size of the engineering bacteria modified with gold platinum nanoscale enzyme is significantly larger than that of the single E. coli and the engineering bacteria modified with gold platinum nanoscale enzyme alone, and at the same timeFigure 3 The potential change of F indicates that the gold platinum nanometer enzyme is successfully combined to the surface of the engineering bacteria, and indicates that the gold platinum nanoparticles modified engineering bacteria are successfully prepared.

[0072] III. Feasibility characterization and optimization

[0073] Different detection systems were used to detect exosomes at the same time to verify the detection effect of the tumor-specific aptamer combined with the engineering bacteria in Example 1. In the verification, four control groups were set, one group was E. coli-Au, the second group of E. coli-Au-Pt did not have an aptamer sequence, the third group of E. coli-Au-Pt did not have exosomes, and the fourth group contained the complete system required for detection. It can be seen that the fourth group has a significant catalytic effect compared to the previous three groups. In addition, different concentrations of aptamers modified on the surface of the engineering bacteria were compared. The results are shown in Figure 4 B, different concentrations of aptamers were added to modify the engineering bacteria, and the same detection steps were performed using the six groups, and finally the color development results and ultraviolet-visible absorption of each group were observed. In addition, the binding time of magnetic beads and exosomes was optimized, as shown in Figure 4 C, by observing the color development results and ultraviolet-visible absorption of each group, it was found that the saturation was basically reached at 30 min. And Figure 4 D, the binding time of the engineering bacteria and the magnetic bead-exosome complex was optimized, and the results showed that the saturation was basically reached at 30 min by observing the color development results and ultraviolet-visible absorption of each group. Therefore, the above results well illustrate that the aptasensor of the present application is feasible and the best reaction conditions are optimized.

[0074] IV. Real sample detection:

[0075] Fourteen groups of healthy serum exosome samples were prepared from the serum of 14 healthy people, and fourteen groups of tumor serum exosome samples were prepared from the serum of 14 tumor patients, and the detection was performed by the method of Example 1 of the present application, and the detection results are shown in Figure 5 As can be seen from the figure, the TMB results of healthy patients are close to colorless, and the color development of tumor patients is deeper. By using a multi-well enzyme marker to detect the absorbance, it can be seen that the absorbance of tumor patients is significantly higher than that of healthy patients, and the experimental results show that the method can detect clinical samples.

Claims

1. An exosome visualized detection biosensor comprising a recognition probe modified magnetic bead for binding with exosomes to generate a magnetic bead-exosome complex, characterized in that, Also comprising: The surface mineralization modified gold platinum nanoscale enzyme engineering bacteria are used for catalyzing oxidation of the color developing solution, and the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria are used for binding with the recognition probe two to generate the surface mineralization engineering bacteria modified by the recognition probe two; The surface mineralization engineering bacteria modified by the recognition probe two are used for binding with the magnetic bead-exosome compound, and after magnetic separation, the magnetic bead-exosome-surface mineralization engineering bacteria compound is generated, the color developing solution is catalyzed by the gold platinum nanoscale enzyme on the compound, and the corresponding exosome concentration is visually detected; At least one of the recognition probe one and the recognition probe two is a specific target recognition probe; The preparation method of the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria is as follows: F1, the escherichia coli is proliferated in the LB liquid culture medium, and when the predetermined concentration is reached, the escherichia coli is collected; first, the escherichia coli bacteria solution is centrifuged at a centrifugal force of 5000 g for 5 min, then the precipitate is collected, and the buffer is used for multiple washing to obtain the concentrated escherichia coli bacteria solution; F2, the escherichia coli bacteria solution is added into the anaerobic M9 low-salt culture medium, resuspended, free oxygen is removed by blowing nitrogen, and then chloroauric acid and sodium borohydride are injected, and the culture is shaken at 37 DEG C and a rotation speed of 200 rpm for 3 h, then the escherichia coli bacteria solution is centrifuged at a centrifugal force of 5000 g for 5 min, the supernatant is removed, and 5% physiological saline is added for washing, and after repeated washing for 2-3 times, pure water is added to obtain the surface modification gold platinum nanoscale enzyme engineering bacteria; F3, the synthesized surface modification gold platinum nanoscale enzyme engineering bacteria are added into pure water, resuspended, and chloroplatinic acid, sodium borohydride and sodium citrate are injected, and stirring treatment is carried out at 1500 rpm / min, until the engineering bacteria become black, indicating that the platinum nanoparticles are successfully modified to the surface of the engineering bacteria, then the engineering bacteria are centrifuged at a centrifugal force of 5000 g for 5 min, the supernatant is removed, and 5% physiological saline is added for washing, and after repeated washing for 2-3 times, pure water is added to obtain the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria.

2. The exosome visualized detection biosensor according to claim 1, characterized in that, The preparation method of the surface mineralization engineering bacteria modified by the recognition probe two is as follows: The concentration of the thiol-modified recognition probe two is 1%-10%, and the concentration of tris(2-carboxyethyl) phosphine is 1%-10%, and the incubation is carried out at room temperature for 10-60 min; Then the incubation product is added into the surface mineralization modified gold platinum nanoscale enzyme engineering bacteria solution, and co-incubation is carried out at room temperature for 10-60 min to obtain the surface mineralization engineering bacteria modified by the recognition probe two.

3. The exosome visualized detection biosensor according to claim 1, characterized in that, The color developing solution is 3,3',5,5'-tetramethylbenzidine.

4. The exosome visualized detection biosensor according to claim 1, characterized in that, The recognition probe one adopts a common target aptamer or antibody, and the recognition probe two adopts a specific target aptamer or antibody.

5. The exosome visualized detection biosensor according to claim 4, characterized in that, The common target aptamer is specifically a CD63 protein aptamer, a CD81 protein aptamer or a CD9 protein aptamer.

6. The exosome visualized detection biosensor according to claim 4, characterized in that, The specific target aptamer is specifically a nucleolin protein aptamer, a PD-L1 protein aptamer, a CD44 protein aptamer, a HER2 protein aptamer, an EpCAM protein aptamer or a MUC1 protein aptamer.

7. A method for detecting exosome visualization for non-diagnostic purposes, characterized in that, The exosome visual detection biosensor according to any one of claims 1-6, comprising the following steps: S1. washing the recognition probe one modified magnetic beads with PBS buffer for multiple times, adding an exosome sample, incubating at room temperature for 10-60 min, magnetically separating, removing the supernatant, washing with PBS buffer, and obtaining a magnetic bead-exosome complex; S2. incubating the magnetic bead-exosome complex with the recognition probe two modified surface mineralization engineering bacteria at room temperature for 10-60 min, magnetically separating, removing the supernatant, washing with PBS buffer to remove non-target adsorbed engineering bacteria, and obtaining a magnetic bead-exosome-surface mineralization engineering bacteria. S3, Visual detection of exosomes: The magnetic bead-exosome-surface mineralization engineering bacteria are added to 3,3',5,5'-tetramethylbenzidine, and the gold platinum nanoscale enzyme modified by surface mineralization of the engineering bacteria catalyzes the reaction of 3,3',5,5'-tetramethylbenzidine in the presence of H + 2O2, so that the color of the solution changes obviously, producing a visible signal with the naked eye. The absorbance is measured by an instrument, and the corresponding exosome concentration is calculated.

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