CN117030991A - Extracellular vesicle detection device and detection method - Google Patents
Extracellular vesicle detection device and detection method Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention provides an extracellular vesicle detection device and a detection method. The nitrocellulose has strong adsorption effect on biological molecules, the polypeptide of the targeting extracellular vesicle membrane is adsorbed on the NC membrane, and is firmly combined on the surface of the membrane on the premise of not generating covalent crosslinking, and meanwhile, the function of combining extracellular vesicles is still reserved. The polypeptides are arranged on the NC membrane in the form of a microarray, and can be stored at room temperature after drying while still maintaining the binding activity. Before being used for sample detection, surfactant with proper concentration is directly added to the polypeptide microarray, so that hydrophobic sites on the NC membrane surface can be blocked, non-specific binding of biomolecules is avoided, and the background signal is obviously reduced. After a biological sample containing extracellular vesicles is added to the microarray, the extracellular vesicles can be captured by a polypeptide probe with targeting ability, and detection of the extracellular vesicles can be realized by using a detection probe with a fluorescent group.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an extracellular vesicle detection device and a detection method.
Background
Extracellular vesicles (Extracellular Vesicles, EVs) are a class of membranous vesicles secreted extracellularly by living cells, typically between 20-200nm in diameter. Secretory EVs are physiological functions common to organisms of the eukaryotic, prokaryotic and archaebacterial kingdoms, and EVs generally contain various biomolecules (such as proteins, nucleic acids, lipids, small molecule metabolites, etc.), mediate interactions between cells, and participate in various vital activities of organisms. The stable presence of EVs from the human body itself and the flora in the body fluid of humans, the capture and analysis of these EVs is a great aid in understanding healthy physiological and abnormal disease states. However, EVs have small particle size and complex composition, and are difficult to separate from biological samples, thus making the development of the field difficult.
Microarrays are discrete spots of spatially ordered arrays formed by immobilizing ligands on a solid substrate. Microarray technology can simultaneously identify the binding of multiple ligands to target substances using only trace amounts of samples, and has great potential in the field of extracellular vesicle research. Prior studies have generally immobilized probe molecules with extracellular vesicle binding capacity on a substrate to achieve capture and in situ detection of extracellular vesicles. Antibodies are the most commonly used capture molecules, and by covalent binding to a functionalized surface via chemical bonds, antibodies directed against vesicle surface proteins can be bound to a substrate, followed by detection of the bound vesicles by fluorescent antibody hybridization or spectroscopy. By increasing the variety of capture antibodies and detection probes, multi-index analysis of vesicles can be achieved.
Most extracellular vesicle detection microarrays at present use antibodies as probe molecules to target specific proteins on the surface of vesicles, but the antibodies are high in price and poor in stability, and large differences exist between different manufacturers and different batches of antibodies, so that the comparison and analysis of results are not facilitated.
In addition to antibodies, polypeptides that selectively recognize vesicles are also used in the enrichment of extracellular vesicles and in microarray detection. Compared with the antibody, the polypeptide has the characteristics of low cost and easy storage, is more suitable for practical application, and provides a new tool for the research of extracellular vesicles.
However, in the prior art, when preparing polypeptide microarrays, polypeptides are usually connected to a substrate in a covalent bond form by using a chemical reaction, active groups available for the reaction must exist on the polypeptide sequence, so that modification or sequence modification of the adopted polypeptide probes is required according to requirements, which is not beneficial to the direct application of natural polypeptides. Numerous factors influence the covalent reaction conditions and extensive optimization of reagents and conditions is required to obtain the desired experimental results. In order to solve the above problems, there are also researchers to directly synthesize polypeptides on a substrate, but the reaction requires high demands on technology and instrumentation, and the application of polypeptide microarrays is limited.
Disclosure of Invention
The invention aims to overcome the defects of complex preparation process and severe requirements on reagents and equipment of polypeptide microarrays, and provides an extracellular vesicle detection device and a detection method. The method for combining the polypeptide and the nitrocellulose membrane is adopted, so that the preparation process of the polypeptide microarray is obviously simplified, the polypeptide microarray can be used for detecting extracellular vesicles from various sources, and the preparation cost of extracellular vesicle capture and detection chips is reduced.
The invention is characterized in that: nitrocellulose has strong adsorption effect on biological molecules, is commonly used for fixing proteins, nucleic acids and polypeptides, and the commercialized nitrocellulose membrane is a porous membrane with a three-dimensional structure, and has the advantages of large adsorption area, low price and flexible use. The extracellular vesicle membrane-targeting polypeptide can be adsorbed onto a Nitrocellulose (NC) membrane, and firmly bound to the membrane surface without covalent crosslinking, while still retaining the extracellular vesicle-binding function. The polypeptides are arranged on the NC membrane in the form of a microarray, and can be stored at room temperature after drying while still maintaining the binding activity. Before being used for sample detection, surfactant with proper concentration is directly added to the polypeptide microarray, so that hydrophobic sites on the NC membrane surface can be blocked, non-specific binding of biomolecules is avoided, and the background signal is obviously reduced. After a biological sample containing extracellular vesicles is added to the microarray, the extracellular vesicles can be captured by a polypeptide probe with targeting ability, and detection of the extracellular vesicles can be realized by using a detection probe with a fluorescent group.
In order to achieve the object of the present invention, in a first aspect, the present invention provides an extracellular vesicle testing device comprising a base plate and a nitrocellulose membrane having a dot array immobilized on the base plate.
The detection device is characterized in that polypeptide solution points are added to an NC film of a bottom plate in a contact sample application mode and are arranged into a square matrix to form a microarray chip; the arrays are separated into independent sample cells through fences; each array comprises a plurality of spot units on which a polypeptide capable of specifically recognizing a vesicle is immobilized; each dot cell has a diameter of between 200-220 μm and adjacent dot cells are equally spaced from each other.
The polypeptide can be a polypeptide produced by natural fermentation, such as epsilon-polylysine, polymerized from 25-30 lysines (preferably L-lysine), and has the structural formula:
alternatively, an artificially synthesized extracellular vesicle-targeted polypeptide is employed, the sequence of which is (PEG 8 ) KKKKKRFSFKKSFKLSGFSFKKNKK (SEQ ID NO: 1). Wherein, PEG 8 Is polyethylene glycol chain, and has the following structure:
it should be noted that the present method can be used to construct microarrays from polypeptides of various sequences and sources.
The substrate may be a glass slide or any other solid material that provides support for the nitrocellulose membrane.
Preferably, the slides are 75mm and 25mm in length and width, respectively, and 1mm in thickness.
Preferably, each sample cell has dimensions of 1cm by 1.5cm.
Preferably, the nitrocellulose membrane has a pore size of 0.22 μm.
In a second aspect, the present invention provides a method for preparing the detection device, comprising the steps of:
(1) Fixing the nitrocellulose membrane on the bottom plate by using double faced adhesive tape;
(2) Dissolving polypeptide capable of specifically recognizing vesicle with sample application liquid, and then uniformly applying the sample application liquid on nitrocellulose membrane with sample application instrument to form a matrix to form a microarray chip;
(3) Naturally airing after finishing sample application; then according to the arrangement of the arrays on the chip, pasting fences around each array to form respective independent sample tanks with certain liquid storage capacity (the liquid storage capacity of each sample tank is the same, and the suitable liquid storage capacity is 100-200 mu L);
(4) Adding a proper amount of sealing liquid into each sample pool, and slowly shaking and sealing on a shaking table;
(5) After the sealing is finished, the sealing liquid is discarded, and each sample cell is washed by PBS buffer solution, thus obtaining the sample cell.
Further, in the step (2), the polypeptide is dissolved in the spotting liquid to prepare a solution with a concentration of 0.1-5mg/mL (preferably 0.5-2 mg/mL), and spotting is performed. The residence time of the sample application needle on the surface of the nitrocellulose membrane is 0.01 to 0.1s, preferably 0.01s.
Further, the fence can be selected from a multi-sample chip fence manufactured by Duobo chip biotechnology limited company, or a sample fence made of other biocompatible materials.
Further, a 0.3M phosphate buffer, pH8.5, containing 0.15M NaCl, 0.005v/v% surfactant, and 15v/v% glycerol; wherein the surfactant is at least one selected from Tween-20, triton X-100, SDS, etc.
Wherein, 0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Further, the blocking solution is a low concentration surfactant or other non-protein small molecular weight blocking agent, preferably 0.05-0.1v/v% Tween-20 aqueous solution, more preferably 0.1v/v% Tween-20 aqueous solution.
Further, the pH of the PBS buffer is 7.2-7.4 (preferably 7.4).
And (4) adding a proper amount of sealing liquid into each sample pool, and shaking and sealing on a shaking table at a speed of 10-30rpm for 0.5-2 hours (preferably 20rpm for 1 hour).
In a third aspect, the invention provides the use of the detection device for extracellular vesicle detection (including for non-disease diagnostic purposes).
In a fourth aspect, the present invention provides a method for detecting extracellular vesicles (including for non-disease diagnostic purposes), comprising the steps of:
S1, adding a sample (such as pre-separated and enriched extracellular vesicles, a cell culture medium subjected to simple centrifugation, biological samples such as serum, urine and tissue fluid) into a sample cell of the detection device, and shaking and incubating on a shaking table at a speed of 15-30rpm for 0.5-1 hour (preferably shaking and sealing at 20rpm for 1 hour);
s2, after sucking out the sample, adding a PBS solution containing 5% skimmed milk or a PBS solution containing 1% Bovine Serum Albumin (BSA) into the sample pool, and washing on a shaker for 2-3 times each for 5-10 minutes (preferably 3 times each for 5 minutes);
s3, adding a detection probe solution containing fluorescent markers (such as a polypeptide, an antibody, a lipid dye, an aptamer and the like for targeting extracellular vesicles) into the sample pool, and shaking the sample pool at a speed of 20rpm in a dark place for incubation for a period of time;
s4, after incubation, sucking out the probe solution, adding PBS buffer solution into the sample cell, and washing 3-5 times in a dark place on a shaking table for 5-10 minutes each time (preferably 3 times in a dark place each time for minutes);
s5, placing the cleaned chip into a scanner (slide scanner), and detecting the fluorescence intensity of each point unit in the point array.
Preferably, the detection probe containing fluorescent label used in step S3 is 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 ；
Wherein FITC is fluorescein isothiocyanate and Acp is aminocaproic acid.
Preferably, in step S1, the sample is centrifuged to remove large particle impurities and intact cells, and then added to the sample cell.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
compared with the existing preparation method of the polypeptide microarray, the method does not need to couple active groups on the polypeptide additionally, so that the method can be used for artificially synthesizing the polypeptide and fixing the natural polypeptide, and the whole process does not need harsh reaction and toxic chemical reagents, and does not need experimenters to design and optimize the reaction conditions in a large amount. The materials and reagents used are cheap and easy to obtain, and the prepared microarray can still keep detection activity at room temperature for several months, and has high practicability and economy. By adjusting the sequence and array arrangement of the polypeptide probes, the polypeptide microarray can be used for detecting extracellular vesicles from various sources and can be used for various biological samples.
Drawings
FIG. 1 is a process for preparing a microarray chip according to the present invention.
FIG. 2 is a schematic diagram of the capture and detection of extracellular vesicles by a microarray of the invention.
FIG. 3 is a physical view of a nitrocellulose-based polypeptide microarray of the present invention.
FIG. 4 shows an example of the design of the probe arrangement (A) and the results (B) directly observed by a fluorescence scanner after spotting.
FIG. 5 shows the detection and quantification of pre-enriched mammalian cell (293T) derived vesicles in accordance with a preferred embodiment of the present invention.
FIG. 6 shows the detection and quantification of pre-enriched bacterial (E.coli) derived vesicles in a preferred embodiment of the invention.
FIG. 7 shows the quantitative results of detecting extracellular vesicles directly from a bacterial (E.coli) medium in a preferred embodiment of the present invention.
FIG. 8 shows the effect of different spotting fluids on the results in a preferred embodiment of the invention.
FIG. 9 shows the effect of different spotting concentrations of polypeptide probes on the detection results in a preferred embodiment of the invention.
FIG. 10 shows the effect of various components of the blocking fluid on the results in a preferred embodiment of the present invention.
FIG. 11 shows the effect of varying concentrations of Tween-20 on the results in a preferred embodiment of the present invention.
FIG. 12 shows the result of resealing the chip prior to adding fluorescent probes in a preferred embodiment of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
NC membranes used in the following examples are available from beijing sonebao technologies, inc, and preferably have a pore size of 0.22 μm.
Example 1 polypeptide microarray for extracellular vesicle detection and method of preparing the same
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Dissolving the polypeptide with sample application liquid, diluting to a concentration of 2mg/mL, and adding into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution (blocking solution), adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Capture and detection of extracellular vesicles in a sample
(1) Adding a proper amount of sample (such as pre-separated and enriched extracellular vesicles, cell culture medium subjected to simple centrifugation, biological samples such as serum and urine) into a pre-treated sample cell, placing a slide on a rocker, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe (e.g., a polypeptide targeting extracellular vesicles, an antibody, a lipid dye, an aptamer, etc.) containing a fluorescent label to a sample cell, placing a slide on a rocker, and incubating at 20rpm in the absence of light for a suitable time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
6. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The preparation process of the microarray chip is shown in FIG. 1.
The principle of microarray capture and detection of extracellular vesicles is shown in FIG. 2.
A physical diagram of the nitrocellulose-based polypeptide microarray of the present invention is shown in FIG. 3.
Example of probe array design (A) and results (B) directly observed with a fluorescence scanner after spotting are shown in FIG. 4.
Example 2 detection of mammalian cell (293T) derived vesicles
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Dissolving the polypeptide with sample application liquid, diluting to a concentration of 2mg/mL, and adding into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour-sample enclosureColumns, each sample cell having dimensions of 1cm by 1.5cm, suitable for holding a sample in an amount of 100-200. Mu.L;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution, adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of human 293T cell-derived extracellular vesicles
(1) Collecting a culture medium of the 293T cells after culture, firstly centrifuging at a centrifugal speed of 300g for 5 minutes to remove the cells, then centrifuging at a temperature of 4 ℃ for 10 minutes to remove large cell fragments at 3000g, and finally centrifuging at a temperature of 4 ℃ for 30 minutes at 10000g to further remove the fragments to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 70 minutes under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 70 min at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. capture and detection of mammalian extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid. The results of detection and quantification of pre-enriched mammalian cell (293T) derived vesicles are shown in FIG. 5.
Example 3 detection of E.coli derived vesicles
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Dissolving the polypeptide with sample application liquid, diluting to a concentration of 2mg/mL, and adding into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adopting Four sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution, adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of E.coli-derived extracellular vesicles
(1) Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 2 hours under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 2 hr at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. Capture and detection of bacterial derived extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The detection and quantification results of the E.coli-derived vesicles after pre-enrichment are shown in FIG. 6.
Example 4 detection of extracellular vesicles from E.coli Medium
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Dissolving the polypeptide with sample application liquid, diluting to a concentration of 2mg/mL, and adding into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution, adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pretreatment of E.coli Medium
Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
6. capture and detection of bacterial derived extracellular vesicles
(1) Adding the pretreated escherichia coli culture medium into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The quantitative results of detecting extracellular vesicles directly from E.coli medium are shown in FIG. 7.
Example 5 optimization of spotting fluids
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKK SFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM 16 person sample application instrumentThe diameter of each point is between 200 and 220 mu m, the layout of the sample points on a slide (microarray chip) is designed according to the requirement, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and the NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Dissolving the polypeptide by using a sample application liquid or PBS buffer solution, diluting to the concentration of 2mg/mL, and adding the solution into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution, adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of E.coli-derived extracellular vesicles
(1) Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 2 hours under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 2 hr at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. capture and detection of bacterial derived extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
As shown in FIG. 8, other conditions were the same, and the effect of the different spotting liquids on the results was (A) the spotting liquid used in the present method, and (B) the spotting liquid using PBS buffer. It can be seen that the spotting solution of the invention is significantly better than direct dissolution of the polypeptide in PBS buffer.
Example 6 optimization of polypeptide Probe concentration
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Respectively diluting the polypeptides to concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/mL by using a sample application liquid, and then adding the diluted polypeptides into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of sealing buffer solution into a sample pool, wherein the composition of the sealing buffer solution is a surfactant water solution without protein, placing a slide on a rocker, and slowly shaking and sealing at a speed of 20rpm for 1 hour;
(3) Sucking and discarding Tween-20 solution, adding a proper amount of PBS buffer solution into a sample cell, and cleaning for 5 minutes on a shaker;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of E.coli-derived extracellular vesicles
(1) Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 2 hours under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 2 hr at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. capture and detection of bacterial derived extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The effect of different spotting concentrations of the polypeptide probes on the detection results is shown in FIG. 9. The signal intensity is gradually enhanced along with the increase of the concentration, and after the concentration reaches 0.8mg/mL, the increase of the concentration has no obvious influence on the signal.
Example 7 optimization of the Components of the blocking fluid
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Respectively diluting the polypeptides to the concentration of 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/mL by using a sample application liquid, and then adding the diluted polypeptides into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of blocking buffer solutions with different components into a sample pool, wherein the blocking buffer solutions comprise 5% of skimmed milk, 5% of skimmed milk containing 0.05% of Tween-20 and PBS buffer solutions containing different concentrations of Tween-20 which are commonly used in NC membrane blocking, placing a slide on a rocker, and slowly shaking and blocking at a speed of 20rpm for 1 hour;
(3) Sucking and discarding the sealing buffer solution, adding a proper amount of PBS buffer solution into the sample cell, and cleaning for 5 minutes on a shaking table;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of E.coli-derived extracellular vesicles
(1) Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 2 hours under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 2 hr at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. capture and detection of bacterial derived extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After sucking out the sample, adding 5% skimmed milk dissolved in PBS into the sample cell, and washing on a shaker for 3 times, each for 5 minutes;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The effect of the different composition of the blocking solution on the results is shown in FIG. 10. The three blocking solutions were (A) 5% skim milk, (B) 5% skim milk plus 0.05% Tween-20 and (C) 0.05% Tween-20, respectively. Skim milk is the most commonly used blocking agent in the case of performing a western blot experiment using nitrocellulose membrane, but in a polypeptide microarray experiment, the presence of a large molecular weight protein prevents the extracellular vesicles from binding to a polypeptide probe, and a blocking solution containing only a small molecular surfactant is beneficial to obtaining a result signal.
The effect of varying concentrations of Tween-20 on the results is shown in FIG. 11. The three concentrations were (A) 0.025%, (B) 0.05% and (C) 0.1%, respectively. When the concentration of the surfactant Tween-20 is too low, the blocking cannot be completely realized, so that the background is too high; if the concentration is too high, the polypeptide probe may be washed away, so that the concentration of Tween-20 in the blocking solution is preferably 0.05-0.1%.
Example 8 Effect of re-blocking with skimmed milk before addition of fluorescence-labeled probes on detection results
1. Polypeptide probes for preparing microarrays
(1) The polypeptide epsilon-polylysine produced by natural fermentation of actinomycetes is polymerized by 25-30 lysines (L-lysines).
(2) An artificially synthesized extracellular vesicle targeting polypeptide has the sequence: (PEG) 8 )-KKKKKRFSFKKSFKLSGFSFKKNKK。
(3) An extracellular vesicle targeting polypeptide modified by a fluorescent group has the sequence: 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 。
2. Preparation of buffer solution
0.3M phosphate buffer: 0.057M NaH 2 PO 4 ，0.243M Na 2 HPO 4 The pH was adjusted to 8.5 with HCl.
Sample application buffer (sample application liquid): 0.3M phosphate buffer, pH8.5, was added with NaCl at a final concentration of 0.15M, tween-20 at 0.005% (v/v) and glycerol at 15% (v/v).
Blocking buffer (blocking solution): 0.1% (v/v) Tween-20 in water.
PBS buffer: pH7.4.
3. Preparation of polypeptide microarray
(1) Sample application usingPersonalArrayer TM The contact sample application mode of the 16 personal sample application instruments, the diameter of each point is between 200 and 220 mu m, the layout of sample points on a slide (microarray chip) is designed according to the requirements, the slide is a standard slide, the length and the width are 75mm and 25mm respectively, the thickness is 1mm, and an NC film is fixed on the corresponding position of the slide by double-sided adhesive tape;
(2) Respectively diluting the polypeptides to the concentration of 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/mL by using a sample application liquid, and then adding the diluted polypeptides into a sample hole of a sample application instrument;
(3) Setting a sample application program of a sample application instrument, and applying the polypeptide solution to the NC film on the glass slide in a contact sample application mode; the residence time of the sample application needle on the surface of the nitrocellulose membrane is about 0.01 s;
(4) After the sample application is finished, the glass slide is placed in a clean place for natural airing for standby.
4. Pretreatment of polypeptide microarrays
(1) According to the arrangement of the sample application areas, pasting fences around the sample application areas to form a sample pool with a certain liquid storage capacity, if adoptingFour sample rails, each sample cell having dimensions of 1cm by 1.5cm, suitable for accommodating samples in an amount of 100-200 μl;
(2) Adding a proper amount of blocking buffer solutions with different components into a sample pool, wherein the blocking buffer solutions comprise 5% of skimmed milk, 5% of skimmed milk containing 0.05% of Tween-20 and PBS buffer solutions containing different concentrations of Tween-20 which are commonly used in NC membrane blocking, placing a slide on a rocker, and slowly shaking and blocking at a speed of 20rpm for 1 hour;
(3) Sucking and discarding the sealing buffer solution, adding a proper amount of PBS buffer solution into the sample cell, and cleaning for 5 minutes on a shaking table;
(4) Sucking and discarding PBS buffer solution, adding a proper amount of PBS buffer solution, and cleaning for 5 minutes on a shaker;
(5) The PBS buffer in the sample cell was blotted off for further use.
5. Pre-enrichment of E.coli-derived extracellular vesicles
(1) Collecting a culture medium for culturing escherichia coli, firstly centrifuging at a centrifugal speed of 3000g for 10 minutes to remove thalli, and further removing fragments by centrifuging at 10000g for 30 minutes at 4 ℃ to obtain a pretreated culture medium;
(2) Centrifuging the pretreated cell culture medium at 4 ℃ for 2 hours under 160000g conditions, discarding the supernatant, and re-suspending the pellet with PBS buffer;
(3) Re-centrifuging the resuspended pellet at 4deg.C for 2 hr at 160000g, discarding the supernatant, re-suspending the pellet with a small amount of PBS buffer to obtain enriched mammalian extracellular vesicles;
6. capture and detection of bacterial derived extracellular vesicles
(1) Adding the extracellular vesicles separated and enriched in advance into a pretreated sample cell, placing a slide on a rocker shaking table, and slowly shaking and incubating at a speed of 20rpm for 1 hour;
(2) After aspirating the samples, PBS or 5% skim milk dissolved in PBS was added to the sample cell and washed 3 times, 5 minutes each on a shaker;
(3) Adding a detection probe containing fluorescent markers into a sample cell, placing a slide on a rocker, and shaking at 20rpm in a dark place for incubation for a proper time;
(4) After aspiration of the probe, PBS buffer was added to the cuvette and washed 3 times, 5 minutes each, in the absence of light on a shaker.
7. Detection of signals
The microarray was placed in a LuxScan 10K/D microarray chip scanner manufactured by Dubo chip Biotechnology Co., ltd, and the fluorescence intensities of each point in the microarray were scanned and recorded.
The fluorescence intensity values of each point are processed, the background value is deducted, the average value and standard deviation are calculated by using the signal value of each repeated point, and the target extracellular vesicles are known to exist in the sample by comparing with the negative point only with blank sample application liquid.
The result of resealing the chip before adding the fluorescent probe is shown in FIG. 12. (A) For the result of washing with PBS alone, (B) for the result of 3 times of resealing with 5% skimmed milk. It can be seen that adding skim milk to reseal can reduce the background.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Reference is made to:
[1]Gill S,Catchpole R,Forterre P.Extracellular membrane vesicles in the three domains of life and beyond[J].FEMS microbiology reviews,2019,43(3):273-303.
[2]Kalluri R,LeBleu V S.The biology,function,and biomedical applications of exosomes[J].Science,2020,367(6478):eaau6977.
[3]Hendrix A,De Wever O.Systemically circulating bacterial extracellular vesicles:Origin,fate,and function[J].Trends in Microbiology,2022,30(3):213-6.
[4]Matsumoto A,Takahashi Y,Chang H-Y,et al.Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance[J].Journal of extracellular vesicles,2020,9(1):1696517.
[5]Liangsupree T,Multia E,Riekkola M-L.Modern isolation and separation techniques for extracellular vesicles[J].Journal of Chromatography A,2021,1636:461773.
[6]Chen Y,Meng X,Zhu Y,et al.Rapid detection of four mycotoxins in corn using a microfluidics and microarray-based immunoassay system[J].Talanta,2018,186:299-305.
[7]M,/>R,Pedersen S,et al.Extracellular Vesicle(EV)Array:microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping[J].Journal of extracellular vesicles,2013,2(1):20920.
[8]Martel R,Shen M L,DeCorwin-Martin P,et al.Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis[J].ACS sensors,2022.
[9]Wang Y,Zhang Q,Yuan W,et al.Hyperspectral imaging-based exosome microarray for rapid molecular profiling of extracellular vesicles[J].Lab on a Chip,2021,21(1):196-204.
[10]Gagni P,Cretich M,Benussi L,et al.Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray platform[J].Analytica Chimica Acta,2016,902:160-7.
[11]Flynn AD,Yin H.Lipid-Targeting Peptide Probes for Extracellular Vesicles[J].Journal of cellular physiology,2016,231(11):2327-32.
[12]Wei S,Jiao D,Xing W.A rapid method for isolation of bacterial extracellular vesicles from culture media using epsilon-poly-L–lysine that enables immunological function research[J].Frontiers in Immunology,2022,13.
[13]Gori A,Romanato A,Bergamaschi G,et al.Membrane-binding peptides for extracellular vesicles on-chip analysis[J].Journal of Extracellular Vesicles,2020,9(1):1751428.
Claims (10)
1. an extracellular vesicle detection device, comprising a base plate and a nitrocellulose membrane fixed on the base plate and containing a dot array;
the detection device is characterized in that polypeptide solution points are added to an NC film of a bottom plate in a contact sample application mode and are arranged into a square matrix to form a microarray chip; the arrays are separated into independent sample cells through fences; each array comprises a plurality of spot units on which a polypeptide capable of specifically recognizing a vesicle is immobilized; each dot cell has a diameter of between 200-220 μm and adjacent dot cells are equally spaced from each other.
2. The device according to claim 1, wherein the polypeptide is a naturally occurring fermentation-produced epsilon-polylysine polymerized from 25-30 lysines, preferably from 25-30L-lysines; or,
the polypeptide is synthetic (PEG 8 ) -KKKKKRFSFKKSFKLSGFSFKKNKK; wherein, PEG 8 Is polyethylene glycol chain.
3. The detection apparatus according to claim 1 or 2, wherein the bottom plate is a slide glass;
preferably, the length and width of the slide are 75mm and 25mm, respectively, and the thickness is 1mm;
preferably, each sample cell has dimensions of 1cm by 1.5cm.
4. A method of manufacturing a detection device according to any one of claims 1 to 3, comprising the steps of:
(1) Fixing the nitrocellulose membrane on the bottom plate by using double faced adhesive tape;
(2) Dissolving polypeptide capable of specifically recognizing vesicle with sample application liquid, and then applying the sample application liquid onto nitrocellulose membrane with sample application instrument to form a matrix to form a microarray chip;
(3) Naturally airing after finishing sample application; then pasting fences around each array according to the arrangement of the arrays on the chip to form independent sample cells with a certain liquid storage amount;
(4) Adding a proper amount of sealing liquid into each sample pool, and slowly shaking and sealing on a shaking table;
(5) After the sealing is finished, the sealing liquid is discarded, and each sample cell is washed by PBS buffer solution, thus obtaining the sample cell.
5. The method according to claim 4, wherein the step (2) is performed by dissolving the polypeptide in a spotting solution to prepare a solution having a concentration of 0.1-5 mg/mL; and/or
The sample application liquid is as follows: 0.3M phosphate buffer, pH8.5, containing 0.15M NaCl, 0.005v/v% surfactant and 15v/v% glycerol; wherein the surfactant is at least one selected from Tween-20, triton X-100 and SDS; and/or
The blocking solution is a low-concentration surfactant or other non-protein small molecular weight blocking agent, preferably 0.05-0.1v/v% Tween-20 water solution; and/or
The pH value of the PBS buffer solution is 7.2-7.4.
6. The method according to claim 4 or 5, wherein in step (4), a proper amount of a blocking liquid is added to each sample cell, and the mixture is blocked by shaking on a shaker at a speed of 10 to 30rpm for 0.5 to 2 hours.
7. Use of the detection device according to any one of claims 1-3 for extracellular vesicle detection, said use being for non-disease diagnostic purposes.
8. A method for detecting extracellular vesicles, comprising the steps of:
s1, adding a sample into a sample cell of the detection device according to any one of claims 1-3, and incubating for 0.5-1 hour by shaking at a speed of 15-30rpm on a shaking table;
s2, after sucking out the sample, adding a PBS solution containing 5% skimmed milk or a PBS solution containing 1% bovine serum albumin into the sample pool, and cleaning for 2-3 times on a shaker for 5-10 minutes each time;
s3, adding a detection probe solution containing fluorescent markers into the sample cell, and shaking and incubating for a period of time at 20rpm in a dark place on a shaking table;
s4, after incubation is finished, sucking out the probe solution, adding PBS buffer solution into the sample pool, and cleaning for 3-5 times on a shaking table in a dark place; 5-10 minutes each time;
s5, placing the cleaned chip into a scanner, and detecting the fluorescence intensity of each point unit in the point array;
The method is for non-disease diagnostic purposes.
9. The method of claim 8, wherein the detection probe of step S3 is a polypeptide, an antibody, a lipid dye, an aptamer that specifically targets extracellular vesicles;
preferably, the detection probe containing fluorescent label is 5-FITC- (Acp) -KKKKKRFSFKKSFKLSGFSFKKNKK (PEG) 8 )-NH 2 ；
Wherein FITC is fluorescein isothiocyanate and Acp is aminocaproic acid.
10. The method of claim 8 or 9, wherein the sample is from cell culture medium, serum, urine, interstitial fluid;
preferably, the sample is centrifuged to remove large particle impurities and intact cells before being added to the sample cell.
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