Exosome visual detection biosensor and detection method

Abstract

The invention discloses an exosome visual detection biosensor and a detection method.The visual detection biosensor comprises a first recognition probe modified magnetic bead used for being combined with an exosome to generate a magnetic bead-exosome compound, and further comprises a gold-platinum nano-enzyme modified engineering bacterium used for being combined with a second recognition probe to generate a gold-platinum nano-enzyme modified magnetic bead-exosome compound, and the gold-platinum nano-enzyme modified engineering bacterium is used for being combined with the second recognition probe to generate a gold-platinum nano-enzyme modified magnetic bead-exosome compound. Generating an engineering bacterium modified by a recognition probe II; the surface mineralization engineering bacteria modified by the recognition probe II are used for being combined with a magnetic bead-exosome compound, after magnetic separation, a magnetic bead-exosome-surface mineralization engineering bacteria compound is generated, and the corresponding exosome concentration is visually detected through a nano-enzyme catalytic oxidation developing solution on the compound. Compared with single gold-platinum nano-enzyme, the gold-platinum nano-enzyme is modified on the surface of escherichia coli through a biomineralization synthesis method, and more gold-platinum nano-enzyme can be accumulated and synthesized on the surface of single escherichia coli due to the fact that the escherichia coli has a large specific surface area, so that the cascade amplification effect is achieved; the visual quantitative detection of the tumor exosome is realized.

Description

Technical Field

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

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

[0003] For example, the Chinese invention patent application with publication number CN118688452A proposed by the applicant is specifically a biosensor and detection method for exosome detection based on engineered bacteria, which includes: magnetic beads modified with recognition elements for binding to exosomes to generate magnetic bead-exosome complexes; engineered bacteria modified with recognition elements for binding to magnetic bead-exosome complexes to generate engineered bacteria combined with magnetic bead-exosomes, removing engineered bacteria adsorbed by non-target sites by washing, and evenly coating the engineered bacteria combined with magnetic bead-exosomes on the corresponding culture medium for cultivation, and calculating the concentration of the corresponding exosomes by observing the number of colonies. However, this method is relatively cumbersome to operate, takes a long time to detect, has many interference items when detecting by colony count, is easily affected by external factors, and cannot analyze the detection data by instruments, so that no intuitive comparison can be obtained between the sample groups.

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

[0005] The object of the present invention is to provide a biosensor and a detection method for visual detection of exosomes to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A biosensor for visual detection of exosomes, comprising a recognition probe-modified magnetic bead, which is used to bind to exosomes to generate a magnetic bead-exosome complex, and also comprising:

[0008] The engineered bacteria with gold-platinum nanozymes modified by surface mineralization are used to catalyze the oxidation of the color developing solution. The engineered bacteria with gold-platinum nanozymes modified by surface mineralization are used to combine with recognition probe 2 to generate surface mineralized engineered bacteria modified with recognition probe 2;

[0009] The surface mineralization engineered bacteria modified with the recognition probe 2 are used to bind to the magnetic bead-exosome complex. After magnetic separation, a magnetic bead-exosome-surface mineralization engineered bacteria complex is generated. The gold platinum nanozyme on the complex catalyzes the oxidation of the color developing solution to visualize the corresponding exosome concentration.

[0010] Among them, at least one of the recognition probe 1 and the recognition probe 2 is a recognition probe with a specific target.

[0011] As a further solution of the present invention, the method for preparing the engineered bacteria of the surface mineralized gold platinum nanozyme is as follows:

[0012] F1. Proliferate E. coli in LB liquid culture medium, and collect the E. coli when the predetermined concentration is reached; first, centrifuge the E. coli culture at 5000g for 5 minutes, then collect the precipitate, and wash it several times with buffer to obtain a concentrated E. coli culture.

[0013] F2. Add the Escherichia coli liquid to the anaerobic M9 low-salt culture medium, resuspend it, remove free oxygen by bubbling nitrogen, inject chloroauric acid and sodium borohydride, and culture it at 37°C and 200 rpm for 3 hours. Then, centrifuge the Escherichia coli liquid at 5000 g for 5 minutes, remove the supernatant, add 5% physiological saline for washing, repeat the washing 2-3 times, and add pure water to obtain the engineered bacteria with surface modified gold nanoparticles.

[0014] F3. Add pure water to the engineered bacteria with surface modified gold-platinum nanozymes synthesized above, resuspend, inject chloroplatinic acid, sodium borohydride and sodium citrate, and stir at 1500rpm / min. When the engineered bacteria turns black, it means that the platinum nanoparticles are successfully modified on the surface of the engineered bacteria. Next, centrifuge the engineered bacteria at 5000g for 5 minutes, remove the supernatant, add 5% physiological saline for washing, repeat the washing 2-3 times, and add pure water to obtain the engineered bacteria with surface mineralized gold-platinum nanoparticles.

[0015] The present invention modifies gold-platinum nanoparticles on engineering bacteria by a biomineralization synthesis method, and the preparation process is simple and environmentally friendly. The obtained gold-platinum nanoparticles can exhibit high catalytic activity due to their clean surface, large specific surface area, and good colloidal stability. The present invention modifies gold-platinum nanozymes on the surface of Escherichia coli by a biomineralization synthesis method. Compared with a single gold-platinum nanozyme, due to the larger surface area of ​​Escherichia coli, the surface of a single Escherichia coli can accumulate and synthesize more gold-platinum nanozymes, thereby playing a role of cascade amplification. Even if the sample to be detected (such as serum) contains a lower concentration of tumor exosomes, its signal can be amplified by less biomineralized synthesized engineering bacteria, thereby achieving quantitative detection.

[0016] As a further solution of the present invention, the preparation method of the surface mineralization engineering bacteria modified with the recognition probe II is as follows: incubating the recognition probe II modified with thiol at a concentration of 1%-10% with tris(2-carboxyethyl)phosphine at a concentration of 1%-10% at room temperature for 10-60 minutes;

[0017] The incubation product is then added to the engineered bacteria solution of surface mineralized gold-platinum nanoparticles, and incubated at room temperature for 10-60 minutes to generate surface mineralized engineered bacteria modified with recognition probe II.

[0018] As a further embodiment of the present invention, the color developing solution is specifically 3,3′,5,5′-tetramethylbenzidine (TMB) or the like.

[0019] As a further solution of the present invention, the recognition probe 1 adopts recognition molecules such as common target aptamers or antibodies; the recognition probe 2 adopts recognition molecules such as specific target aptamers or antibodies.

[0020] As a further embodiment of the present invention, the common target aptamer is specifically a CD63 protein aptamer, a CD81 protein aptamer or a CD9 protein aptamer, etc.

[0021] As a further embodiment of the present invention, 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, etc.

[0022] In the present invention, the exosomes to be detected can be exosomes from different types of tumors, such as early, middle and late stage exosomes from breast cancer cells, lung cancer cells, liver cancer cells, leukemia cells, etc. Exosomes from other diseases can also be exosomes, such as exosomes from cells corresponding to Alzheimer's disease, exosomes from cells corresponding to diabetes, exosomes from cells corresponding to Parkinson's disease, etc.

[0023] A method for visual detection of exosomes, based on the aforementioned biosensor for visual detection of exosomes, comprises the following steps:

[0024] S1. Wash the magnetic beads modified with the recognition probe 1 with PBS buffer for multiple times, then add the exosome sample, incubate at room temperature for 10-60 min, then perform magnetic separation, remove the supernatant, and wash with PBS buffer to obtain a magnetic bead-exosome complex;

[0025] S2, incubating the magnetic bead-exosome complex with the surface mineralized engineered bacteria modified with the recognition probe 2 at room temperature for 10-60 min, then magnetically separating, removing the supernatant, and washing with PBS buffer to remove the engineered bacteria adsorbed on non-target sites, thereby obtaining magnetic bead-exosome-surface mineralized engineered bacteria;

[0026] S3. Visual detection of exosomes: The magnetic beads-exosomes-surface mineralized engineered bacteria were added to 3,3′,5,5′-tetramethylbenzidine (TMB). The catalytically active nanoparticles modified by the surface mineralization of the engineered bacteria were exposed to H + In the presence of 5-nitropropene, 3,3′,5,5′-tetramethylbenzidine (TMB) is catalyzed to cause a significant change in the color of the solution, generating a signal visible to the naked eye. The absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention modifies gold-platinum nanozymes on engineered bacteria through a biomineralization synthesis method, and the preparation process is simple and environmentally friendly. The obtained gold-platinum nanozymes can exhibit high catalytic activity due to their clean surface, large specific surface area, and good colloidal stability;

[0029] 2. The present invention modifies gold-platinum nanozymes on the surface of Escherichia coli through a biomineralization synthesis method. Compared with a single gold-platinum nanozyme, since Escherichia coli has a rod-shaped 3D structure and a larger surface-to-volume ratio, a single Escherichia coli surface can accumulate and synthesize more gold-platinum nanozymes, providing a large number of targets for probe binding, that is, the large surface area of ​​these nanoparticles can capture a large number of probes and fix them on the surface of the nanoparticles, thereby playing a role of cascade amplification. Even if the sample to be detected (such as serum) contains a low concentration of tumor exosomes, its signal can be amplified by a small number of engineered bacteria synthesized by biomineralization, thereby achieving quantitative detection;

[0030] 3. The detection method of the present invention is simple to operate and does not require expensive instruments and complicated operations. Tumor exosomes can be detected using a simple method, which improves the sensitivity, convenience, reaction time and cost of early tumor exosome detection. Compared with the existing technology, the detection results can be visualized and more intuitive, which is conducive to promoting the application of exosome-based liquid biopsy in early tumor screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the process of visually detecting tumor cell exosomes in the present invention.

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

[0033] Figure 3 For the characterization of E.coli-Au-Pt, 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: Potentials of E. coli alone, E. coli-Au, and E. coli-Au-Pt.

[0034] Figure 4 To verify the feasibility and optimize the reaction conditions, Figure 4 A: The effect of the presence or absence of aptamers and exosomes on catalytic efficiency. Figure 4 B: E. coli binding to different concentrations of DNA (μM). Figure 4 C: Optimization of the binding time between exosomes and magnetic beads. Figure 4 D: Binding time of exosomes and E. coli.

[0035] Figure 5 For clinical sample testing, bright field photographs and ultraviolet absorption images of 14 groups of healthy patient serum and 14 groups of tumor patient serum samples tested by the method of the present invention are shown. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] A biosensor for visual detection of exosomes, comprising a magnetic bead modified with a recognition probe and an engineered bacterium with a surface mineralized gold-platinum nanozyme, wherein the magnetic bead modified with the recognition probe is used to bind to the exosome to generate a magnetic bead-exosome complex;

[0038] In the engineered bacteria with surface mineralization-modified gold-platinum nanozymes, the gold-platinum nanozymes are used to catalyze the oxidation of the color-developing solution, and the engineered bacteria with surface mineralization-modified catalytically active nanoparticles are used to combine with the recognition probe 2 to generate the surface mineralization engineered bacteria modified with the recognition probe 2;

[0039] The surface mineralization engineered bacteria modified with the recognition probe 2 are used to bind to the magnetic bead-exosome complex, and after magnetic separation, magnetic bead-exosome-surface mineralization engineered bacteria are generated. The corresponding exosome concentration is visually detected by catalytic oxidation of the color developing solution by the magnetic bead-exosome-surface mineralization engineered bacteria;

[0040] The color developing solution is specifically 3,3′,5,5′-tetramethylbenzidine (TMB), and at least one of the recognition probe 1 and the recognition probe 2 is a recognition probe of a specific target.

[0041] For example: the recognition probe 1 adopts a common target aptamer, specifically a CD63 protein aptamer, a CD81 protein aptamer or a CD9 protein aptamer;

[0042] In order to screen and remove exosomes from normal cells and facilitate accurate detection of the concentration of tumor cell exosomes, the recognition probe 2 adopts 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 another solution, the recognition probe 1 may also use a specific target aptamer, and the corresponding recognition probe 2 may use a common target aptamer.

[0044] In the present invention, the exosomes to be detected can be exosomes from different types of tumors, such as early and middle stage exosomes from breast cancer cells, lung cancer cells, liver cancer cells, leukemia cells, etc. It can also be exosomes from other diseases, such as exosomes from cells corresponding to Alzheimer's disease, exosomes from cells corresponding to diabetes, exosomes from cells corresponding to Parkinson's disease, etc.

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

[0046] In the present invention, the specific preparation method of the engineered bacteria with surface mineralization modified catalytically active nanoparticles is:

[0047] F1. Proliferate E. coli in LB liquid culture medium, and collect the E. coli when the bacterial OD value reaches 0.9; first, centrifuge the E. coli culture at 5000g for 5 minutes, then collect the precipitate, and wash it several times with buffer to obtain a concentrated E. coli culture.

[0048] F2. Add the Escherichia coli liquid to the anaerobic M9 low-salt culture medium, resuspend, remove free oxygen by bubbling nitrogen, inject 50 μL of chloroauric acid and 100 μL of sodium borohydride, and culture at 37°C and 200 rpm for 3 hours. Then, centrifuge the Escherichia coli liquid at 5000 g for 5 minutes, remove the supernatant, add 5% saline for washing, repeat the washing 2-3 times, and add pure water to obtain the engineered bacteria with surface modified gold nanoparticles.

[0049] F3. Add pure water to the engineered bacteria with surface modified gold nanoparticles synthesized above, resuspend, inject 25μL of chloroplatinic acid, 100μL of sodium borohydride and 300μL of sodium citrate, stir at 1500rpm / min, wait until the engineered bacteria turns black, indicating that the platinum nanoparticles are successfully modified on the surface of the engineered bacteria, then centrifuge the engineered bacteria at 5000g centrifugal force for 5min, remove the supernatant, add 5% saline for washing, repeat the washing 2-3 times, add pure water, and you can get the engineered bacteria with surface mineralized gold-platinum nanozymes.

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

[0051] The incubation product is then added to the engineered bacteria solution of the surface mineralized gold-platinum nanozyme, and incubated at room temperature for 10-60 minutes to generate surface mineralized engineered bacteria modified with recognition probe II.

[0052] A method for visual detection of exosomes, based on the aforementioned biosensor for visual detection of exosomes, comprises the following steps:

[0053] S1. The magnetic beads modified with the recognition probe after blocking treatment are washed several times with PBS buffer, and then the exosome sample is added and incubated at room temperature for 10-60 minutes. Then, magnetic separation is performed, the supernatant is removed, and the beads are washed with PBS buffer to obtain a magnetic bead-exosome complex.

[0054] S2, incubating the magnetic bead-exosome complex with the surface mineralized engineered bacteria modified with the recognition probe 2 at room temperature for 10-60 min, then performing magnetic separation, removing the supernatant, and washing with 1XPBS buffer having a pH value of 7.0-8.6 and a concentration of 10-100 nm / mL to remove the engineered bacteria adsorbed on non-target sites, thereby obtaining magnetic bead-exosome-surface mineralized engineered bacteria;

[0055] S3. Visual detection of exosomes: Magnetic beads-exosomes-surface mineralized engineered bacteria were added to 3,3′,5,5′-tetramethylbenzidine (TMB). The gold platinum nanozymes modified by surface mineralization of engineered bacteria were detected in H + In the presence of 5-nitropropene, 3,3′,5,5′-tetramethylbenzidine (TMB) is catalyzed to cause a significant change in the color of the solution, generating a signal visible to the naked eye. The absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0056] Example 1

[0057] A method for visualizing exosomes, see Figure 1 , the steps include:

[0058] S1. The CD63 protein aptamer-modified magnetic beads after blocking treatment were washed with PBS buffer for multiple times, and then the exosome sample was added and incubated at room temperature for 10-60 minutes. Then, magnetic separation was performed, the supernatant was removed, and the beads were washed with PBS buffer to obtain the magnetic bead-exosome complex;

[0059] S2, incubating the magnetic bead-exosome complex with the surface mineralized engineered bacteria modified with the nucleolin protein aptamer at room temperature for 10-60 minutes, then magnetically separating, removing the supernatant, and washing with 1XPBS buffer with a pH value of 7.0-8.6 to remove the engineered bacteria adsorbed on non-target sites, thereby obtaining magnetic bead-exosome-surface mineralized engineered bacteria;

[0060] S3. Visual detection of exosomes: Magnetic beads-exosomes-surface mineralized engineered bacteria were added to 3,3′,5,5′-tetramethylbenzidine (TMB). The gold platinum nanozymes modified by surface mineralization of engineered bacteria were detected in H+ In the presence of 5-nitropropene, 3,3′,5,5′-tetramethylbenzidine (TMB) is catalyzed to cause a significant change in the color of the solution, generating a signal visible to the naked eye. The absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0061] In this embodiment, the magnetic beads modified with CD63 protein aptamers recognize the common target (CD63 protein) of all exosomes through the CD63 protein aptamers to generate magnetic bead-exosome complexes bound to all exosomes. Then, the surface mineralized engineered bacteria modified with nucleolin protein aptamers recognize the specific target (nucleolin protein) of tumor exosomes through the nucleolin protein aptamers, while the exosomes of normal cells cannot be recognized by the nucleolin protein aptamers. Then, the unbound surface mineralized engineered bacteria are removed by magnetic separation, and finally, the specific tumor exosomes are quantitatively detected through a color development reaction.

[0062] Example 2

[0063] A method for visualizing detection of exosomes, comprising the steps of:

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

[0065] S2, incubating the magnetic bead-exosome complex with the surface mineralized engineered bacteria modified with the CD63 protein aptamer at room temperature for 10-60 minutes, then magnetically separating, removing the supernatant, and washing with 1XPBS buffer with a pH value of 7.0-8.6 to remove the engineered bacteria adsorbed on non-target sites, thereby obtaining magnetic bead-exosome-surface mineralized engineered bacteria;

[0066] S3. Visual detection of exosomes: The magnetic beads-exosomes-surface mineralized engineered bacteria were added to 3,3′,5,5′-tetramethylbenzidine (TMB). The catalytically active nanoparticles modified by the surface mineralization of the engineered bacteria were exposed to H + In the presence of 5-nitropropene, 3,3′,5,5′-tetramethylbenzidine (TMB) is catalyzed to cause a significant change in the color of the solution, generating a signal visible to the naked eye. The absorbance is measured by an instrument to calculate the corresponding exosome concentration.

[0067] In this embodiment, the magnetic beads modified with nucleolin protein aptamers recognize the specific target (nucleolin protein) of tumor exosomes through the nucleolin protein aptamers to generate magnetic beads-exosome complexes bound to tumor exosomes, while normal cell exosomes cannot bind and are removed by subsequent magnetic separation. Then, the surface mineralized engineered bacteria modified with CD63 protein aptamers recognize the common target (CD63 protein) of tumor exosomes through CD63 protein aptamers to generate magnetic beads-tumor exosomes-surface mineralized engineered bacteria. Then, the unbound surface mineralized engineered bacteria are removed by magnetic separation, and finally, the specific tumor exosomes are quantitatively detected through a color development reaction.

[0068] 1. Collection and Characterization of Exosomes

[0069] By culturing leukemia line HL-60 cells, the cell-secreted exosomes were collected as tumor cell exosomes for experimental use, and their morphological structure, particle size and marker proteins were characterized. Transmission electron microscopy (TEM) imaging results showed that the size of the exosomes was about 150nm and exhibited a typical disc shape (such as Figure 2 A). Nanoparticle tracking analysis (NTA) results further confirmed that the size of exosomes was approximately 160 nm, which was consistent with the TEM results (e.g. Figure 2 B). Finally, two marker proteins CD63 and TSG-101 were successfully detected on the exosome samples by immunoblotting (WB) (e.g. Figure 2 C) These results indicate that exosomes have been successfully obtained.

[0070] 2. Preparation and characterization of engineered bacteria (E. coli-Au-Pt) for surface mineralization modification of gold-platinum nanoparticles

[0071] First, the engineered bacteria with surface mineralized gold-platinum nanozymes were prepared according to the above method. By comparing the synthetic colors of the engineered bacteria at different stages, such as Figure 3 As shown in A, the color of E. coli changed from milky white (1) to black (3), indicating that the engineered bacteria were successfully synthesized. Transmission electron microscopy (TEM) showed that the engineered bacteria without modified gold platinum nanozymes (such as Figure 3 B) and engineered bacteria modified with gold-platinum nanozymes (such as Figure 3 D), the surface of the engineered bacteria modified with Au-Pt nanozymes has obvious nanoparticles, and the average size of E.coli-Au-Pt is about 1209nm, which proves that our Au-Pt nanozymes are successfully encapsulated on the surface of E. coli. The particle size and potential were characterized, and the particle size of NPs was measured by dynamic light scattering DLS. Figure 3 As shown in E, the DLS analysis results show that the particle size of the engineered bacteria modified with gold platinum nanozymes is significantly larger than that of Escherichia coli alone and its engineered bacteria modified with gold platinum nanozymes alone. Figure 3 The potential change of F indicates that the gold-platinum nanozyme has successfully bound to the surface of the engineered bacteria, indicating that the engineered bacteria modified with gold-platinum nanoparticles have been successfully prepared.

[0072] 3. Feasibility Characterization and Optimization

[0073] Different detection systems were used to simultaneously detect exosomes to verify the detection effect of the engineered bacteria combined with tumor-specific aptamers in Example 1. In the verification, four control groups were set up, one group was E.coli-Au, the second group had no aptamer sequence in E.coli-Au-Pt, the third group had no exosomes in E.coli-Au-Pt, and the fourth group contained the complete system required for detection. It can be seen that group 4 has a significant catalytic effect compared with the previous three groups. In addition, the simultaneous detection of different concentrations of aptamers modified on the surface of engineered bacteria was compared. The results are shown in Figure 2. Figure 4 B. Add different concentrations of aptamers to modify the engineered bacteria, and use these 6 groups to perform the same detection steps, and finally observe the color development results and UV-visible absorption of each group. In addition, the binding time of magnetic beads and exosomes was optimized, such as Figure 4 As shown in C, by observing the color development results and UV-visible absorption of each group, it basically reached saturation at 30 minutes. Figure 4 D optimized the binding time of the engineered bacteria and the magnetic bead-exosome complex, and the results showed that the color development results and UV-visible absorption of each group were basically saturated at 30 minutes. Therefore, the above results well illustrate that the adaptor sensor of the present invention is feasible and optimizes the optimal reaction conditions.

[0074] 4. Real sample testing:

[0075] Corresponding healthy serum exosome samples were prepared from sera of 14 groups of healthy people, and corresponding tumor serum exosome samples were prepared from sera of 14 groups of tumor patients. The samples were tested by the method of Example 1 of the present invention. The test results are as follows: Figure 5 As shown, it can be seen that the TMB results of healthy patients are close to colorless, while those of tumor patients are darker in color. By using a multi-well microplate reader to detect its absorbance, it can be seen that the absorption of tumor patients is significantly higher than that of healthy patients. The experimental results show that this method can be used to detect clinical samples.

Claims

1. A biosensor for visual detection of exosomes, comprising a recognition probe-modified magnetic bead, which is used to bind to exosomes to generate a magnetic bead-exosome complex, characterized in that: Also includes: The engineered bacteria with gold-platinum nanozymes modified by surface mineralization are used to catalyze the oxidation of the color developing solution. The engineered bacteria with gold-platinum nanozymes modified by surface mineralization are used to combine with recognition probe 2 to generate surface mineralized engineered bacteria modified with recognition probe 2; The surface mineralization engineered bacteria modified with the recognition probe 2 are used to bind to the magnetic bead-exosome complex. After magnetic separation, a magnetic bead-exosome-surface mineralization engineered bacteria complex is generated. The gold platinum nanozyme on the complex catalyzes the oxidation of the color developing solution to visualize the corresponding exosome concentration. Among them, at least one of the recognition probe 1 and the recognition probe 2 is a recognition probe with a specific target.

2. The exosome visualization detection biosensor according to claim 1, characterized in that: The method for preparing the engineered bacteria of the surface mineralized gold-platinum nanozyme is as follows: F1. Proliferate E. coli in LB liquid culture medium, and collect the E. coli when the predetermined concentration is reached; first, centrifuge the E. coli culture at 5000g for 5 minutes, then collect the precipitate, and wash it several times with buffer to obtain a concentrated E. coli culture. F2. Add the Escherichia coli liquid to the anaerobic M9 low-salt culture medium, resuspend it, remove free oxygen by bubbling nitrogen, inject chloroauric acid and sodium borohydride, and culture it at 37°C and 200 rpm for 3 hours. Then, centrifuge the Escherichia coli liquid at 5000g for 5 minutes, remove the supernatant, add 5% saline for washing, repeat the washing 2-3 times, and add pure water to obtain the engineered bacteria with surface modified gold-platinum nanozymes. F3. Add pure water to the engineered bacteria with surface modified gold-platinum nanozymes synthesized above, resuspend, inject chloroplatinic acid, sodium borohydride and sodium citrate, and stir at 1500rpm / min. When the engineered bacteria turns black, it means that the platinum nanoparticles are successfully modified on the surface of the engineered bacteria. Next, centrifuge the engineered bacteria at 5000g for 5 minutes, remove the supernatant, add 5% physiological saline for washing, repeat the washing 2-3 times, and add pure water to obtain the engineered bacteria with surface mineralized gold-platinum nanozymes.

3. The exosome visualization detection biosensor according to claim 2, characterized in that: The preparation method of surface mineralization engineering bacteria modified with recognition probe 2 is as follows: Incubate the thiol-modified recognition probe 2 at a concentration of 1%-10% with tris(2-carboxyethyl)phosphine at a concentration of 1%-10% at room temperature for 10-60 minutes; The incubation product is then added to the engineered bacteria solution of the surface mineralized gold-platinum nanozyme, and incubated at room temperature for 10-60 minutes to generate surface mineralized engineered bacteria modified with recognition probe II.

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

5. The exosome visualization detection biosensor according to claim 1, characterized in that: The recognition probe 1 adopts a common target aptamer or antibody; the recognition probe 2 adopts a specific target aptamer or antibody.

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

7. The exosome visualization detection biosensor according to claim 5, 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.

8. A method for visualizing detection of exosomes, characterized in that: A biosensor for visual detection of exosomes according to any one of claims 1 to 7, comprising the steps of: S1. Wash the magnetic beads modified with the recognition probe 1 with PBS buffer for multiple times, then add the exosome sample, incubate at room temperature for 10-60 min, then perform magnetic separation, remove the supernatant, and wash with PBS buffer to obtain a magnetic bead-exosome complex; S2, incubating the magnetic bead-exosome complex with the surface mineralized engineered bacteria modified with the recognition probe 2 at room temperature for 10-60 min, then magnetically separating, removing the supernatant, and washing with PBS buffer to remove the engineered bacteria adsorbed on non-target sites, thereby obtaining magnetic bead-exosome-surface mineralized engineered bacteria; S3. Visual detection of exosomes: Add magnetic beads-exosomes-surface mineralized engineered bacteria to 3,3′,5,5′-tetramethylbenzidine, and the gold-platinum nanozymes modified by the surface mineralization of the engineered bacteria are detected in H + In the presence of 5-nitropropene, 3,3′,5,5′-tetramethylbenzidine is catalyzed to cause a significant change in the color of the solution, generating a signal visible to the naked eye. The absorbance is measured by an instrument to calculate the corresponding exosome concentration.

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