A biosensor and detection method for exosome detection based on engineered bacteria

Abstract

The present invention discloses a biosensor and method for detecting exosomes based on engineered bacteria. The method comprises: magnetic beads modified with recognition elements for binding to exosomes to form a magnetic bead-exosome complex; engineered bacteria modified with recognition elements for binding to the magnetic bead-exosome complex to generate engineered bacteria bound to magnetic beads and exosomes; washing to remove non-target adsorbed engineered bacteria; uniformly coating the engineered bacteria bound to magnetic beads and exosomes on a corresponding culture medium, culturing the culture, and calculating the concentration of the corresponding exosomes by observing the number of colonies. The method is simple to operate, does not require expensive instruments or cumbersome procedures, and can be used for detection using common bacterial culture methods. It can not only detect exosomes from a single source, but also simultaneously detect exosomes from multiple sources by rationally utilizing engineered bacteria containing different resistance genes.

Description

Technical Field

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

[0002] Cancer poses a serious threat to human health and is a major public health issue in contemporary society. Statistics show that early diagnosis of cancer can more than triple a patient's five-year survival rate. However, the proportion of cancers diagnosed in the middle and late stages remains as high as 82%, severely impacting patient survival. This is primarily due to the scarcity of early screening technologies. Therefore, developing precise diagnostic methods that can be used for early cancer detection is crucial. Liquid biopsies offer significant advantages for cancer diagnosis by detecting biomarkers in human fluids. Currently, liquid biopsy markers primarily include circulating tumor cells (CTCs), circulating free tumor DNA (ctDNA), and exosomes. Exosomes are lipid nanovesicles secreted by virtually all cells and widely present in body fluids (blood, urine, saliva, etc.). They carry components of parent cells, such as proteins, nucleic acids, and lipids, on their surfaces and within their interiors. They can serve as diagnostic markers for disease and offer advantages such as high availability and stability. However, the current gold standard for detecting exosome markers is primarily ELISA, flow cytometry, and mass spectrometry, which lack feasibility for early cancer screening due to low sensitivity, expensive equipment, and the need for specialized personnel. Therefore, early cancer screening is more difficult to achieve 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] Bacteria are a major biological group closely linked to human health and the development of disease. Due to their inherent biocompatibility and motility, the engineering of bacteria is gaining increasing attention. Engineered bacteria are artificially modified, designed, and utilized. By modifying wild bacteria, they not only retain their reproductive capacity but also impart new properties, such as target recognition, drug synthesis, and biodegradation. Consequently, engineered bacteria are widely used in diagnostics, therapeutic delivery, and other fields. Summary of the Invention

[0004] The purpose of the present invention is to provide an exosome detection biosensor and detection method based on engineered bacteria to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An exosome detection biosensor based on engineered bacteria, comprising:

[0007] Magnetic beads modified with recognition elements are used to bind to exosomes to generate magnetic bead-exosome complexes;

[0008] The engineered bacteria modified with the identification element are used to bind to the magnetic bead-exosome complex to generate engineered bacteria bound to magnetic beads and exosomes. The engineered bacteria adsorbed to non-target sites are removed by washing. The engineered bacteria bound to magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured. The concentration of the corresponding exosomes is calculated by observing the number of colonies.

[0009] The method for preparing magnetic beads modified with recognition elements is as follows: magnetic beads modified with streptavidin are combined with recognition elements labeled with biotin to generate the recognition elements; the method for preparing engineered bacteria modified with recognition elements is as follows: distearoylphosphatidylethanolamine (DSPE-DBCO) modified with a dibenzocyclooctyne group is first combined with a recognition element modified with an azide group to generate a modification product of the recognition element, and then the modification product of the recognition element is combined with concentrated engineered bacteria to generate engineered bacteria modified with the recognition element.

[0010] As a further embodiment of the present invention, the recognition element includes an aptamer, an antibody, a small molecule and / or a polypeptide.

[0011] As a further embodiment of the present invention, when the recognition element used to modify the magnetic beads adopts the recognition element of the co-expressed target, the recognition element used to modify the engineered bacteria includes the recognition element of at least one specific target.

[0012] As a further embodiment of the present invention, the recognition element used to modify the engineered bacteria also includes a recognition element for a co-expression target.

[0013] As a further embodiment of the present invention, when the recognition element used to modify the magnetic beads adopts the recognition element of the specific target, the recognition element used to modify the engineered bacteria includes the recognition element of the specific target and / or the recognition element of the co-expressed target.

[0014] In the present invention, co-expression targets may specifically include proteins such as CD63 protein, CD81 protein, CD9 protein, or common small molecules such as sialic acid; and specific targets may specifically include proteins such as nucleolin protein, PD-L1 protein, CD44 protein, HER2 protein, EpCAM protein, MUC1 protein, or specific small molecules such as sialic acid.

[0015] In the present invention, the exosomes to be detected can be exosomes from different tumor sources, such as breast cancer cell exosomes, lung cancer cell exosomes, liver cancer cell exosomes, leukemia cell exosomes, etc., and 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.

[0016] A method for detecting exosomes based on engineered bacteria, based on the aforementioned exosome detection biosensor based on engineered bacteria, comprising the following steps:

[0017] Preparation of engineered bacteria modified with recognition elements: Incubate distearoylphosphatidylethanolamine (DSPE-DBCO) modified with a dibenzocyclooctyne group at a concentration of 1%-10% with a recognition element modified with an azide group at a concentration of 2-10 μM at room temperature for 1-6 hours, then add the incubation product to the concentrated engineered bacteria solution and incubate at room temperature for 10-60 minutes to obtain engineered bacteria modified with the recognition element;

[0018] Preparation of magnetic beads modified with recognition elements: Incubate magnetic beads modified with streptavidin at a concentration of 10 mg / mL with a biotin-labeled recognition element at a concentration of 10 μM in a buffer solution such as PBS for 10-60 minutes. Wash the incubation product several times with a buffer solution such as PBS to remove free recognition elements. Block the incubation product with 10% bovine serum albumin at room temperature for 1-2 hours to prevent non-target adsorption. This yields magnetic beads modified with recognition elements.

[0019] Binding of magnetic beads to exosomes: The blocked and modified magnetic beads are washed multiple times with PBS or other buffers, 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 or other buffers to obtain the magnetic bead-exosome complex.

[0020] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex is incubated with the engineered bacteria modified with the recognition element at room temperature for 10-60 minutes. Then, magnetic separation is performed, the supernatant is removed, and the solution is washed with a buffer such as PBS with a pH value of 7.0-8.6 to remove the engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic beads-exosomes.

[0021] Culture observation: The engineered bacteria combined with magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured at 37°C. The number of colonies on the culture medium is observed to calculate the concentration of the corresponding exosomes.

[0022] As a further embodiment of the present invention, the engineered bacteria include engineered bacteria containing resistance genes, and each engineered bacterium containing resistance genes is used to match and bind with a corresponding type of recognition element.

[0023] As a further embodiment of the present invention, the engineered bacteria include engineered bacteria containing ampicillin resistance genes, engineered bacteria containing kanamycin resistance genes and / or engineered bacteria containing genes such as gentamicin resistance, and the corresponding resistance culture media include culture media containing ampicillin, culture media containing kanamycin and / or culture media containing gentamicin, etc.

[0024] As a further embodiment of the present invention, in the step of preparing engineered bacteria modified with recognition elements, the preparation of the concentrated engineered bacterial solution includes the following steps: adding the engineered bacteria to a liquid culture medium, shaking and culturing at 37°C and 200 rpm, until the OD value of the engineered bacterial solution reaches 0.6-0.8; then, centrifuging the engineered bacterial solution at a centrifugal force of 5000g for 5 minutes, removing the supernatant, adding 5-10% physiological saline for washing, repeating the washing 2-3 times, and adding pure water to obtain concentrated engineered bacteria. The concentration of the concentrated engineered bacteria is 10 6 -10 8 CFU / mL.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention generates magnetic bead-exosome complexes by binding magnetic beads modified with recognition elements to exosomes. Engineered bacteria modified with recognition elements bind to the magnetic bead-exosome complexes to generate engineered bacteria bound to magnetic beads and exosomes. By using recognition elements to recognize co-expressed targets, engineered bacteria bound to all exosomes can be generated. By using recognition elements to recognize specific targets, engineered bacteria that only bind to tumor cell exosomes (or exosomes from cells corresponding to other diseases) can be selectively screened. Finally, by observing the number of corresponding colonies, the concentrations of all exosomes and specific tumor cell exosomes (or exosomes from cells corresponding to other diseases) can be calculated.

[0027] 2. In the present invention, each engineered bacterium containing a resistance gene is matched with a specific recognition element of the corresponding type. Engineered bacteria with different specific recognition elements can selectively screen for different tumor cell exosomes (or exosomes corresponding to other diseases). Then, the corresponding resistance culture medium is used to screen and distinguish them. By observing the number of final colonies on the culture dish, the concentration of different specific tumor cell exosomes can be calculated, thereby achieving simultaneous detection of multiple tumor cell exosomes, which is conducive to simplifying the detection steps.

[0028] 3. The present invention is simple to operate and does not require expensive instruments or cumbersome operations. It can be detected using common bacterial culture methods. It can not only detect exosomes from a single source, but also achieve simultaneous detection of exosomes from multiple sources by rationally utilizing engineered bacteria containing different resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the detection method of Example 3 of the present invention, including (A) a schematic diagram of the process of modifying aptamers on the surface of engineered bacteria containing resistance genes, and (B) a schematic diagram of the principle of precise visual detection of tumor-derived exosomes;

[0030] Figure 2 Figure 1 is a graph showing the culture results of engineered bacteria with different resistances;

[0031] Figure 3 This is a visualization diagram of the engineered bacteria modified DNA fluorescent probe;

[0032] Figure 4 This is the fluorescence microscopy result of engineering bacteria to modify DNA fluorescent probe;

[0033] Figure 5 The results of bacterial plate culture with and without modified DNA probes on the surface of engineered bacteria;

[0034] Figure 6 This is a diagram showing the specific detection and culture results of engineered bacteria;

[0035] Figure 7 Figure 3 shows the results of culture of exosomes at different concentrations detected by the method of Example 3 of the present invention;

[0036] Figure 8 Figure 1 is a graph showing the culture results of serum samples from four groups of healthy people tested by the method of Example 3 of the present invention;

[0037] Figure 9 The figure shows the culture results of serum samples from four groups of tumor patients tested by the method of Example 3 of the present invention. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0039] An exosome detection biosensor based on engineered bacteria, comprising: magnetic beads modified with recognition elements for binding to exosomes to form magnetic bead-exosome complexes;

[0040] The engineered bacteria modified with the identification element are used to bind to the magnetic bead-exosome complex to generate engineered bacteria bound to magnetic beads and exosomes. The engineered bacteria adsorbed to non-target sites are removed by washing. The engineered bacteria bound to magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured. The concentration of the corresponding exosomes is calculated by observing the number of colonies.

[0041] The method for preparing magnetic beads modified with recognition elements is as follows: magnetic beads modified with streptavidin are combined with recognition elements labeled with biotin to generate the recognition elements; the method for preparing engineered bacteria modified with recognition elements is as follows: distearoylphosphatidylethanolamine (DSPE-DBCO) modified with a dibenzocyclooctyne group is first combined with a recognition element modified with an azide group to generate a modification product of the recognition element, and then the modification product of the recognition element is combined with concentrated engineered bacteria to generate engineered bacteria modified with the recognition element.

[0042] As some embodiments, the recognition element may specifically include aptamers, antibodies, small molecules and / or polypeptides. Since the exosomes of normal cells have almost no tumor-related proteins, the surfaces of normal cell exosomes and tumor cell exosomes have common surface proteins, which serve as co-expression targets of the recognition element. In order to detect tumor cell exosomes, it is often necessary to screen and remove the exosomes of normal cells. In specific applications, aptamers may specifically include common protein aptamers and specific protein aptamers. Common protein aptamers can bind to the common surface proteins of all exosomes, while specific protein aptamers can only bind to specific surface proteins (i.e., specific targets) of tumor cell exosomes; similarly, antibodies may specifically include common protein antibodies and specific protein antibodies, small molecules may specifically include common protein small molecules and specific protein small molecules, and polypeptides may specifically include common protein polypeptides and specific protein polypeptides.

[0043] In some embodiments, when the recognition element used to modify the magnetic beads is a recognition element for a co-expressed target, the recognition element used to modify the engineered bacteria can include a recognition element for at least one specific target in order to screen out exosomes from normal cells and facilitate accurate detection of the concentration of tumor cell exosomes. As a further solution, to simultaneously detect the concentration of all exosomes, the recognition element used to modify the engineered bacteria can also include a recognition element for a co-expressed target.

[0044] As some embodiments, when the recognition element used to modify the magnetic beads adopts the recognition element of the specific target, the magnetic beads modified with the recognition element of the specific target can only bind to the exosomes of tumor cells, which can screen and remove the exosomes of normal cells. Therefore, the recognition element used to modify the engineered bacteria can adopt the recognition element of the specific target, or the recognition element of the co-expressed target, or the recognition element of the specific target and the recognition element of the co-expressed target can be used at the same time.

[0045] In an embodiment of the present invention, co-expression targets may specifically include proteins such as CD63 protein, CD81 protein, CD9 protein, or common small molecules such as sialic acid; and specific targets may specifically include proteins such as nucleolin protein, PD-L1 protein, CD44 protein, HER2 protein, EpCAM protein, MUC1 protein, or specific small molecules such as sialic acid.

[0046] In the present invention, the exosomes to be detected can be exosomes from different tumor sources, such as breast cancer cell exosomes, lung cancer cell exosomes, liver cancer cell exosomes, leukemia cell exosomes, etc., and 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.

[0047] A method for detecting exosomes based on engineered bacteria, based on the aforementioned exosome detection biosensor based on engineered bacteria, comprising the following steps:

[0048] Preparation of engineered bacteria modified with recognition elements: Incubate distearoylphosphatidylethanolamine (DSPE-DBCO) modified with a dibenzocyclooctyne group at a concentration of 1%-10% with a recognition element modified with an azide group at a concentration of 2-10 μM at room temperature for 1-6 hours, then add the incubation product to the concentrated engineered bacteria solution and incubate at room temperature for 10-60 minutes to obtain engineered bacteria modified with the recognition element;

[0049] Preparation of recognition element-modified magnetic beads: Incubate 10 mg / mL streptavidin-modified magnetic beads with 10 μM biotin-labeled recognition element in PBS buffer for 10-60 minutes. Wash the incubation product several times with PBS buffer to remove free recognition element. Block the incubation product with 10% bovine serum albumin at room temperature for 1-2 hours to prevent non-target adsorption. This yields recognition element-modified magnetic beads.

[0050] Binding of magnetic beads to exosomes: The blocked and modified magnetic beads were washed with PBS buffer several times, 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.

[0051] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex is incubated with the engineered bacteria bound to the recognition element at room temperature for 10-60 minutes. Then, magnetic separation is performed, the supernatant is removed, and the solution is washed with PBS buffer with a pH value of 7.0-8.6 to remove the engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic beads-exosomes.

[0052] Culture observation: The engineered bacteria combined with magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured at 37°C. The number of colonies on the culture medium is observed to calculate the concentration of the corresponding exosomes.

[0053] In some embodiments, to facilitate simultaneous detection of the concentrations of at least two types of tumor cell exosomes and avoid mutual interference between the respective detection processes, the engineered bacteria may include engineered bacteria containing resistance genes, each type of engineered bacteria containing resistance genes being used to match and bind to the specific recognition element of the corresponding type. Specifically, the engineered bacteria may include engineered bacteria containing ampicillin resistance genes, engineered bacteria containing kanamycin resistance genes, and / or engineered bacteria containing genes such as gentamicin resistance, and the corresponding resistance culture media may include culture media containing ampicillin, culture media containing kanamycin, and / or culture media containing gentamicin, etc.

[0054] For example, engineered bacteria containing an ampicillin resistance gene are used to bind to a specific protein aptamer (PD-L1 protein aptamer) to generate engineered bacteria that bind to a specific protein aptamer (PD-L1 protein aptamer) (ampicillin resistance); and engineered bacteria containing a kanamycin resistance gene are used to bind to a specific protein aptamer (nucleolin protein aptamer) to generate engineered bacteria that bind to a specific protein aptamer (nucleolin protein aptamer) (kanamycin resistance); when magnetic beads modified with a common protein aptamer (CD63 protein aptamer) bind to all exosomes, magnetic beads-exosome complexes are generated. complex; the engineered bacteria bound to the two specific protein aptamers are added to the magnetic bead-exosome complex. After incubation and magnetic separation, they are uniformly coated on a culture medium containing ampicillin and a culture medium containing kanamycin, respectively. After culture, the number of colonies observed on the culture medium containing ampicillin can reflect the tumor exosomes corresponding to the PD-L1 protein (such as: lung cancer cell exosomes), while the number of colonies observed on the culture medium containing kanamycin can reflect the tumor exosomes corresponding to the nucleolin protein (such as: gastric cancer, breast cancer or colorectal cancer cell exosomes).

[0055] In the preparation step of the engineered bacteria modified with the recognition element, the preparation of the concentrated engineered bacterial solution includes the following steps: adding the engineered bacteria to the liquid culture medium, shaking and culturing at 37°C and 200 rpm, and waiting for the OD value of the engineered bacterial solution to reach 0.6-0.8; then, centrifuging the engineered bacterial solution at 5000g centrifugal force for 5 minutes, removing the supernatant, adding 5-10% physiological saline for washing, repeating the washing 2-3 times, and adding pure water to obtain the concentrated engineered bacteria. The concentration of the concentrated engineered bacteria is 10 6 -10 8 CFU / mL.

[0056] The following examples of the present invention use protein aptamers (DNA probes) as specific recognition elements. Specifically, engineered bacteria can be engineered bacteria without resistance genes, engineered bacteria containing ampicillin resistance genes, and engineered bacteria containing kanamycin resistance genes. The protein aptamer sequences used are as follows:

[0057] Azide-modified CD63 protein aptamer sequence:

[0058] 5′-N3-TTTTTTTTTTCACCCCACCTCGCTCCCGTGACACTAAT GCTA-3′;

[0059] DNA fluorescent probe sequence:

[0060] 5′-N3-TTTTTTTTTTCACCCCACCTCGCTCCCGTGACACTAATGCTA-FAM-3′;

[0061] Biotin-labeled CD63 protein aptamer sequence:

[0062] 5′-CACCCCACCTCGCTCCCGTGACACTAATGCTAAAAAA-bio-3′;

[0063] Azide-modified nucleolin protein aptamer sequence:

[0064] 5′-N3-TTTTTTTTTTGGTGGTGGTTGTGGTGGTGGTGGT-3′;

[0065] Biotinylated nucleolin protein aptamer sequence:

[0066] 5′-GGTGGTGGTTGTGGTGGTGGTGGTAAAAA-bio-3′;

[0067] Azide-modified PD-L1 protein aptamer sequence:

[0068] 5′-N3-TTTTTTTTTTTACAGGTTCTGGGGGGTGGGTGGGGAA CCTGTT-3′;

[0069] The relevant nucleic acid sequences and their biotin modifications used were synthesized by Bioengineering (Shanghai) Co., Ltd. Streptavidin-modified nanomagnetic beads (65002) were purchased from Thermo Fisher.

[0070] Example 1

[0071] A method for detecting exosomes based on engineered bacteria, comprising the following steps:

[0072] Preparation of engineered bacteria modified with PD-L1 protein aptamers: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and an azide-modified PD-L1 protein aptamer are incubated at room temperature. The incubation product is then added to a concentrated engineered bacteria solution. The PD-L1 protein aptamer-modified engineered bacteria are obtained by co-incubation at room temperature.

[0073] Preparation of CD63 protein aptamer-modified magnetic beads: Incubate streptavidin-modified magnetic beads with biotin-labeled CD63 protein aptamers in PBS buffer. Wash the incubation product multiple times with PBS buffer to remove free CD63 protein aptamers. Block the incubation product with bovine serum albumin at room temperature to prevent non-target adsorption. This yields CD63 protein aptamer-modified magnetic beads.

[0074] Binding of magnetic beads to exosomes: The blocked CD63 protein aptamer-modified magnetic beads were washed multiple times with PBS buffer, then added with the exosome sample and incubated at room temperature. Then, magnetic separation was performed, the supernatant was removed, and the beads were washed with PBS buffer to obtain the magnetic bead-exosome complex.

[0075] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex is incubated with engineered bacteria modified with the PD-L1 protein aptamer at room temperature. The complex is then magnetically separated, the supernatant removed, and washed with PBS buffer to remove engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic beads and exosomes. Because the PD-L1 protein aptamer is a specific aptamer that recognizes PD-L1, the resulting engineered bacteria bound to magnetic beads and exosomes contain only PD-L1-positive exosomes.

[0076] Culture observation: The engineered bacteria combined with magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured at 37°C. The number of colonies on the culture medium is observed to calculate the concentration of PD-L1 protein-positive exosomes.

[0077] Example 2

[0078] A method for detecting exosomes based on engineered bacteria, comprising the following steps:

[0079] Preparation of engineered bacteria modified with PD-L1 protein aptamers: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and an azide-modified PD-L1 protein aptamer are incubated at room temperature. The incubation product is then added to a concentrated solution of engineered bacteria (ampicillin-resistant). Co-incubation at room temperature yields engineered bacteria modified with PD-L1 protein aptamers (ampicillin-resistant).

[0080] Preparation of engineered bacteria modified with nucleolin protein aptamers: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and a nucleolin protein aptamer modified with an azide group are incubated at room temperature, and the incubation product is added to a concentrated solution of engineered bacteria (kanarabinoresistant). Nucleolin protein aptamer-modified engineered bacteria (kanarabinoresistant) are obtained by co-incubation at room temperature.

[0081] Preparation of CD63 protein aptamer-modified magnetic beads: Incubate streptavidin-modified magnetic beads with biotin-labeled CD63 protein aptamers in PBS buffer. Wash the incubation product multiple times with PBS buffer to remove free CD63 protein aptamers. Block the incubation product with bovine serum albumin at room temperature to prevent non-target adsorption. This yields CD63 protein aptamer-modified magnetic beads.

[0082] Binding of magnetic beads to exosomes: The blocked CD63 protein aptamer-modified magnetic beads were washed multiple times with PBS buffer, then added with the exosome sample and incubated at room temperature. Then, magnetic separation was performed, the supernatant was removed, and the beads were washed with PBS buffer to obtain the magnetic bead-exosome complex.

[0083] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex, the engineered bacteria modified with the PD-L1 protein aptamer (ampicillin resistance), and the engineered bacteria modified with the nucleolin aptamer (kanamycin resistance) were incubated at room temperature. Then, magnetic separation was performed, the supernatant was removed, and the cells were washed with PBS buffer to remove the engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic beads and exosomes.

[0084] Culture observation: The engineered bacteria combined with magnetic beads and exosomes were evenly spread on culture medium containing ampicillin and culture medium containing kanamycin, respectively, and cultured at 37°C. The number of colonies on the ampicillin-resistant culture medium and the kanamycin-resistant culture medium was observed, and the concentration of PD-L1 or nucleolin-positive exosomes was calculated.

[0085] Example 3

[0086] A method for detecting exosomes based on engineered bacteria, comprising the following steps:

[0087] Preparation of engineered bacteria modified with CD63 protein aptamers: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and a CD63 protein aptamer modified with an azide group are incubated at room temperature. The incubation product is then added to a concentrated solution of engineered bacteria (ampicillin-resistant). The engineered bacteria modified with the CD63 protein aptamer (ampicillin-resistant) are obtained by co-incubation at room temperature.

[0088] Preparation of engineered bacteria modified with nucleolin protein aptamers: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and a nucleolin protein aptamer modified with an azide group are incubated at room temperature, and the incubation product is added to a concentrated solution of engineered bacteria (kanarabinoresistant). Nucleolin protein aptamer-modified engineered bacteria (kanarabinoresistant) are obtained by co-incubation at room temperature.

[0089] Preparation of CD63 protein aptamer-modified magnetic beads: Incubate streptavidin-modified magnetic beads with biotin-labeled CD63 protein aptamers in PBS buffer. Wash the incubation product multiple times with PBS buffer to remove free CD63 protein aptamers. Block the incubation product with bovine serum albumin at room temperature to prevent non-target adsorption. This yields CD63 protein aptamer-modified magnetic beads.

[0090] Binding of magnetic beads to exosomes: The blocked CD63 protein aptamer-modified magnetic beads were washed multiple times with PBS buffer, then added with the exosome sample and incubated at room temperature. Then, magnetic separation was performed, the supernatant was removed, and the beads were washed with PBS buffer to obtain the magnetic bead-exosome complex.

[0091] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex was divided into two parts. One part of the magnetic bead-exosome complex was incubated with engineered bacteria modified with a CD63 protein aptamer (ampicillin-resistant) at room temperature, and the other part of the magnetic bead-exosome complex was incubated with engineered bacteria modified with a nucleolin protein aptamer (kanamycin-resistant) at room temperature. Then, magnetic separation was performed, the supernatant was removed, and the cells were washed with PBS buffer to remove engineered bacteria adsorbed to non-target sites. Two parts of engineered bacteria bound to magnetic bead-exosomes were obtained, one with ampicillin resistance and the other with kanamycin resistance.

[0092] Culture observation: The engineered bacteria (ampicillin-resistant) bound to magnetic beads and exosomes were evenly spread on a culture medium containing ampicillin and cultured at 37°C. The number of colonies on the culture medium was observed to calculate the concentration of all CD63-positive exosomes in the sample.

[0093] The engineered bacteria (kanamycin-resistant) combined with magnetic beads and exosomes were evenly spread on a culture medium containing kanamycin and cultured at 37°C. The number of colonies on the kanamycin-resistant culture medium was observed, and the concentration of nucleolin-positive exosomes was calculated.

[0094] Example 4

[0095] A method for detecting exosomes based on engineered bacteria, comprising the following steps:

[0096] Preparation of engineered bacteria modified with CD63 protein aptamers: Incubate distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group and a CD63 protein aptamer modified with an azide group at room temperature. Then, add the incubation product to the concentrated engineered bacteria solution, and incubate at room temperature to obtain engineered bacteria modified with CD63 protein aptamers.

[0097] Preparation of nucleolin protein aptamer-modified magnetic beads: Streptavidin-modified magnetic beads are incubated with biotin-labeled nucleolin protein aptamer in PBS buffer. The incubation product is then washed multiple times with PBS buffer to remove free nucleolin protein aptamer. The incubation product is then blocked with bovine serum albumin at room temperature to prevent adsorption of non-target sites, thereby obtaining nucleolin protein aptamer-modified magnetic beads.

[0098] Binding of magnetic beads to exosomes: The blocked nucleolin protein aptamer-modified magnetic beads are washed multiple times with PBS buffer, then the exosome sample is added and incubated at room temperature. Then, magnetic separation is performed, the supernatant is removed, and the beads are washed with PBS buffer to obtain a magnetic bead-exosome complex. Because the nucleolin aptamer is a specific aptamer for exosomes from tumor cells such as gastric cancer, breast cancer, or colorectal cancer, the resulting magnetic bead-exosome complex contains only exosomes from tumor cells such as gastric cancer, breast cancer, or colorectal cancer.

[0099] Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex was incubated with engineered bacteria modified with CD63 protein aptamers at room temperature. Then, the mixture was magnetically separated, the supernatant was removed, and the mixture was washed with PBS buffer to remove engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic bead-exosomes.

[0100] Culture observation: The engineered bacteria combined with magnetic beads and exosomes were evenly spread on the corresponding culture medium and cultured at 37°C. The number of colonies on the culture medium was observed to calculate the concentration of nucleolin-positive exosomes.

[0101] 1. Experiment on culture of engineered bacteria containing different resistance genes:

[0102] Engineered bacteria containing an ampicillin resistance gene and an engineered bacteria containing a kanamycin resistance gene were prepared separately; the engineered bacteria containing the ampicillin resistance gene were added to a culture medium containing ampicillin and a culture medium containing kanamycin, respectively, and were recorded as group 2-1 and group 2-6, respectively; the engineered bacteria containing the kanamycin resistance gene were added to a culture medium containing ampicillin and a culture medium containing kanamycin, respectively, and were recorded as group 2-5 and group 2-2, respectively; the engineered bacteria containing the ampicillin resistance gene and the engineered bacteria containing the kanamycin resistance gene were added together to the culture medium containing ampicillin, and were recorded as group 2-3; the engineered bacteria containing the ampicillin resistance gene and the engineered bacteria containing the kanamycin resistance gene were added together to the culture medium containing kanamycin, and were recorded as group 2-4; after culturing for 12 hours, the culture results were as follows: Figure 2As shown, the engineered bacteria in groups 2-1, 2-2, 2-3, and 2-4 were all growing, while the engineered bacteria in groups 2-5 and 2-6 were not growing (dead). The experimental results show that engineered bacteria containing resistance genes can grow in culture media containing the corresponding resistance, but do not grow in culture media containing other resistances.

[0103] 2. Fluorescence verification of protein aptamer modification on the surface of engineered bacteria:

[0104] Fluorescence detection was performed on group 3-1 of DSPE-DBCO+DNA fluorescent probe, group 3-2 of unmodified engineered bacteria, group 3-3 of engineered bacteria+DNA fluorescent probe, and group 3-4 of engineered bacteria+DSPE-DBCO+DNA fluorescent probe. The detection results are as follows: Figure 3 As shown, group 3-1 has fluorescence, group 3-2 has precipitation but no fluorescence, group 3-3 has precipitation but no fluorescence, and group 3-4 has precipitation and fluorescence. The experimental results show that compared with the engineered bacterial precipitation without protein aptamer binding, obvious green fluorescence can be seen in group 3-4, proving that the protein aptamer has been successfully modified on the surface of the engineered bacteria.

[0105] The engineered bacteria, engineered bacteria + DNA fluorescent probe, and engineered bacteria + DSPE-DBCO + DNA fluorescent probe were analyzed using an inverted fluorescence microscope. The analysis results are shown in Figure 2. Figure 4 As shown, only the engineered bacteria + DSPE-DBCO + DNA fluorescent probe group has obvious green fluorescence under the fluorescent environment. The experimental results show that only when the protein aptamer combines with distearoylphosphatidylethanolamine modified with dibenzocyclooctyne group (DSPE-DBCO) to generate protein aptamer products can the protein aptamer products combine with the concentrated engineered bacteria to generate protein aptamer-bound engineered bacteria.

[0106] 3. Cultivation experiment of the presence or absence of modified DNA probes on the surface of engineered bacteria:

[0107] Engineered bacteria containing ampicillin resistance, engineered bacteria containing kanamycin resistance, engineered bacteria with surface-modified DNA probes and containing ampicillin resistance, and engineered bacteria with surface-modified DNA probes and containing kanamycin resistance were added to the culture medium, respectively, and recorded as group 5-1, group 5-2, group 5-3, and group 5-4, and cultured. The culture results are shown in FIG. Figure 5 As shown, the experimental results show that after the surface of the engineered bacteria is modified with DNA probes, its activity is not affected and it can still grow on the culture dish.

[0108] 4. Specific detection of resistant genetically engineered bacteria modified with protein aptamers:

[0109] The engineered bacteria modified with CD63 protein aptamers and containing resistance to ampicillin and the engineered bacteria modified with nucleolin protein aptamers and containing resistance to kanamycin were co-plated on a culture dish containing kanamycin, and were recorded as group 6-1; the engineered bacteria modified with CD63 protein aptamers and containing resistance to ampicillin were plated on a culture dish containing ampicillin, and were recorded as group 6-2; the engineered bacteria modified with CD63 protein aptamers and containing resistance to ampicillin were plated on a culture dish containing kanamycin, and were recorded as group 6-3; the engineered bacteria modified with nucleolin protein aptamers and containing resistance to ampicillin were plated on a culture dish containing kanamycin, and were recorded as group 6-4. The engineered bacteria containing kanamycin resistance modified by nucleolin protein aptamer were spread on the culture dish containing kanamycin, which was recorded as group 6-4; the engineered bacteria containing ampicillin resistance modified by CD63 aptamer and the engineered bacteria containing kanamycin resistance modified by nucleolin protein aptamer were co-spread on the culture dish containing ampicillin, which was recorded as group 6-5; the engineered bacteria containing kanamycin resistance modified by nucleolin protein aptamer were spread on the culture dish containing ampicillin, which was recorded as group 6-6; after 12 hours of culture, the culture results were as follows: Figure 6 As shown, group 6-1 had colony growth, group 6-2 had colony growth, group 6-3 had no colony growth, group 6-4 had colony growth, group 6-5 had colony growth, and group 6-6 had no colony growth. The experimental results show that the resistant genetically engineered bacteria modified with protein aptamers can grow in culture media containing the corresponding resistance, but do not grow in culture media containing other resistances.

[0110] 5. Sensitivity testing of engineered bacteria

[0111] The following exosomes at different concentrations were detected using the method of Example 3 of the present invention. After coating the plates, the number of colonies at different concentrations was observed:

[0112] 1 group of normal cell exosomes; 2 groups of 1×10 4 particles / μL; 3 groups of 5×10 3 particles / μL; 4 groups of 2×10 3 particles / μL; 5 groups of 1×10 3 particles / μL; 6 groups of 1×10 2 particles / μL.

[0113] The results are as follows Figure 7 As shown, the experimental results showed that the binding of engineered bacteria to exosomes was positively correlated, and the higher the exosome concentration, the greater the number of bound engineered bacteria.

[0114] 6. Real sample testing:

[0115] 1. The corresponding healthy serum exosome samples were prepared from 4 groups of healthy people's serum and tested by the method of Example 3 of the present invention. The test results are as follows: Figure 8As described above, colonies grew on the culture dish containing ampicillin resistance, indicating that the sample contained exosomes, and no colonies grew on the culture dish containing kanamycin resistance, indicating that the serum of healthy people did not contain tumor exosomes of tumor cells such as gastric cancer, breast cancer or colorectal cancer.

[0116] 2. The corresponding tumor serum exosome samples were prepared using the serum of 4 groups of tumor patients and tested by the method of Example 3 of the present invention. The test results are as follows: Figure 9 As described above, colonies grew on both the culture dishes containing ampicillin resistance and kanamycin resistance, indicating that the serum of tumor patients contains tumor exosomes of tumor cells such as gastric cancer, breast cancer and / or colorectal cancer.

Claims

1. A biosensor for detecting exosomes based on engineered bacteria, characterized in that: include: Magnetic beads modified with recognition elements are used to bind to exosomes to generate magnetic bead-exosome complexes; The engineered bacteria modified with the identification element are used to bind to the magnetic bead-exosome complex to generate engineered bacteria bound to magnetic beads and exosomes. The engineered bacteria adsorbed to non-target sites are removed by washing. The engineered bacteria bound to magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured. The concentration of the corresponding exosomes is calculated by observing the number of colonies. The preparation method of the magnetic beads modified with the recognition element is as follows: the magnetic beads modified with streptavidin are combined with the recognition element labeled with biotin; The method for preparing engineered bacteria modified with a recognition element comprises: firstly combining distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group with a recognition element modified with an azide group to generate a modification product of the recognition element; then, combining the modification product of the recognition element with concentrated engineered bacteria to generate engineered bacteria modified with the recognition element; The engineered bacteria include engineered bacteria containing resistance genes, and each engineered bacterium containing resistance genes is used to match and combine with a recognition element of a corresponding type.

2. The exosome detection biosensor based on engineered bacteria according to claim 1, characterized in that: The recognition elements include aptamers, antibodies, small molecules and / or polypeptides.

3. The exosome detection biosensor based on engineered bacteria according to claim 2, characterized in that: When the recognition element used to modify the magnetic beads adopts the recognition element of the co-expressed target, the recognition element used to modify the engineered bacteria includes the recognition element of at least one specific target.

4. The exosome detection biosensor based on engineered bacteria according to claim 3, characterized in that: The recognition elements used to modify engineered bacteria also include recognition elements for co-expressed targets.

5. The exosome detection biosensor based on engineered bacteria according to claim 2, characterized in that: When the recognition element used to modify the magnetic beads is a recognition element of a specific target, the recognition element used to modify the engineered bacteria includes a recognition element of a specific target and / or a recognition element of a co-expressed target.

6. A method for detecting exosomes based on engineered bacteria, characterized in that: An exosome detection biosensor based on engineered bacteria according to any one of claims 1 to 5, comprising the steps of: Preparation of engineered bacteria modified with recognition elements: Distearoylphosphatidylethanolamine modified with a dibenzocyclooctyne group is incubated with a recognition element modified with an azide group at room temperature, and the incubation product is added to a concentrated engineered bacteria solution. The mixture is co-incubated at room temperature to obtain engineered bacteria modified with the recognition element; Preparation of magnetic beads modified with recognition elements: Streptavidin-modified magnetic beads are incubated with biotin-labeled recognition elements in PBS buffer. The incubation product is then washed multiple times with PBS buffer to remove free recognition elements. The incubation product is then blocked with bovine serum albumin at room temperature to prevent adsorption of non-target sites. This yields magnetic beads modified with recognition elements. Binding of magnetic beads to exosomes: The blocked and modified magnetic beads with recognition elements were washed multiple times with PBS buffer, then the exosome sample was added and incubated at room temperature. After that, magnetic separation was performed, the supernatant was removed, and the beads were washed with PBS buffer to obtain the magnetic bead-exosome complex. Magnetic bead-exosome-engineered bacteria binding: The magnetic bead-exosome complex is incubated with the engineered bacteria modified with the recognition element at room temperature. Then, magnetic separation is performed, the supernatant is removed, and the solution is washed with PBS buffer to remove the engineered bacteria adsorbed to non-target sites, thereby obtaining engineered bacteria bound to magnetic bead-exosomes. Culture observation: The engineered bacteria combined with magnetic beads and exosomes are evenly spread on the corresponding culture medium and cultured at 37°C. The number of colonies on the culture medium is observed to calculate the concentration of the corresponding exosomes.

7. The method for detecting exosomes based on engineered bacteria according to claim 6, characterized in that: The engineered bacteria include engineered bacteria containing ampicillin resistance genes, engineered bacteria containing kanamycin resistance genes and / or engineered bacteria containing gentamicin resistance genes, and the corresponding resistance culture media include culture media containing ampicillin, culture media containing kanamycin and / or culture media containing gentamicin.

8. The method for detecting exosomes based on engineered bacteria according to claim 7, characterized in that: In the preparation step of the engineered bacteria modified with the recognition element, the preparation of the concentrated engineered bacterial solution includes the following steps: adding the engineered bacteria to the liquid culture medium, shaking and culturing at 37°C and 200 rpm, and waiting for the OD value of the engineered bacterial solution to reach 0.6-0.8; then, centrifuging the engineered bacterial solution at 5000g centrifugal force for 5 minutes, removing the supernatant, adding 5-10% physiological saline for washing, repeating the washing 2-3 times, and adding pure water to obtain the concentrated engineered bacteria. The concentration of the concentrated engineered bacteria is 10 6 -10 8 CFU / mL.

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