CN114184662A - MOF electrochemical sensor for exosome analysis and preparation and application thereof - Google Patents
MOF electrochemical sensor for exosome analysis and preparation and application thereof Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention belongs to the technical field of immunoassay and diagnosis, and particularly relates to an electrochemical exosome analysis method based on a metal organic framework. The detection method provided by the invention is characterized in that the used materials comprise a gold electrode assembled by a metal organic framework material, a mouse anti-human epithelial cell adhesion molecule antibody marked with glucose oxidase, a glucose solution, a Triton-X solution, an anti-fouling polypeptide solution, a ferrocene marking signal DNA chain, an acidic buffer solution and a cleaning buffer solution. The detection method can use a gold electrode made of a metal organic framework material to capture exosomes and identify proteins and internal microRNA molecules on the surface of the exosomes. The method has mild reaction conditions, does not need an ultracentrifugation step, and is suitable for rapid analysis.
Description
Technical Field
The invention relates to the technical field of immunoassay and diagnosis, in particular to an MOF electrochemical sensor for exosome analysis and preparation and application thereof.
Background
The exosome is a lipid bilayer structure vesicle with uniform size and diameter of 30-100 nanometers, which is actively secreted outside a cell after a multivesicular body in the cell is fused with a cytoplasmic membrane. Exosomes are released by shedding of different types of cells (including tumor cells) and are detectable in most body fluids such as peripheral blood, urine, saliva, ascites, milk, and the like. Recent research finds that exosomes derived from tumors play important roles in causing tumor metastasis, shaping conditions suitable for tumor metastasis, helping tumors escape immune surveillance and the like, and the exosomes contain lipids, proteins, microRNAs and the like related to the tumors and can be used as one of main detection objects for liquid biopsy.
At present, the international analysis method for exosomes mainly comprises reverse transcription polymerase chain reaction, second-generation sequencing, immunoblotting, proteomics analysis and the like. Although polymerase chain hybridization can analyze nucleic acid molecules with low abundance in exosomes with high sensitivity, the technology has complex operation steps and high requirements on the technical level of detection personnel. The second generation sequencing technology is long in time consumption, needs expensive professional instruments and is difficult to meet routine detection. Although the western blotting method has good specificity, because of its low analysis efficiency, a large amount of exosome samples need to be proposed, which increases the difficulty of analysis.
What is more troublesome is that the main means for obtaining exosomes still depends on an ultra-high-speed centrifuge at present, the whole process usually takes several to more than ten hours, and the analysis efficiency of exosomes is greatly reduced. Thus, exosome-based detection analysis studies still face a number of challenges.
The electrochemical detection technology has the main principle that specific or non-specific biological recognition events generated on the electrodes are converted into electric signals to realize the detection of the target object, and has the advantages of sensitivity, rapidness, low cost, light detection device, low energy consumption, easiness in miniaturization and integration and the like. In recent years, the existing exosome electroanalysis method mainly relies on an electrode chip modified by recognition molecules such as aptamers, polypeptides or antibodies to recognize exosome vesicles in a sample.
However, the complexity of biological samples and the heterogeneity of interfacial assembly severely interfere with the recognition function of these biomolecules, resulting in a decrease in the capture efficiency of exosomes in complex samples. In addition, the current analysis method can only analyze the protein or nucleic acid information in the exosome each time, and cannot simultaneously analyze two substances, so that the analysis efficiency is low, and the application of the electroanalysis technology in exosome detection is greatly limited.
Metal-organic framework (MOF) is an organic-inorganic hybrid material with intramolecular pores formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds. The diversity of organic ligands and metal ions or clusters in MOFs determines the structural diversity of metal organic framework materials, and has been widely used in many fields such as gas storage and separation, drug delivery, and biosensing. As the metal organic framework material surface generally has uncoordinated metal ions, and the surface of the biological vesicle structure has a large number of phospholipid molecules and electronegative protein molecules, the phospholipid molecules and the electronegative protein molecules can strongly interact through electrostatic adsorption force and metal coordination, so that the metal organic framework material is utilized to adsorb exosome vesicles in a biological sample.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an MOF electrochemical sensor for exosome analysis and preparation and application thereof.
One of the purposes of the invention is to provide a preparation method of an MOF electrochemical sensor for exosome analysis, which comprises the steps of incubating DNA molecules marked by sulfydryl and methylene blue with a clean gold electrode (the diameter is 3 mm) to obtain a DNA modified gold electrode, and then using mercaptohexanol to close the vacant position of the electrode. And putting the DNA modified electrode into a precursor solution of the MOF, incubating for a period of time, and then thoroughly washing the surface of the electrode by using a washing buffer solution to obtain a MOF assembled gold electrode and recording a methylene blue electric signal.
The concentration of the sulfydryl labeled DNA molecule solution is 1-3 mu mol/L, and the modification time of the DNA and the electrode is 2-4 hours; the concentration of the mercaptohexanol is 0.5-1mmol/L, and the modification time of the mercaptohexanol and the electrode is 0.5-2 hours; a zinc source in the precursor solution of the MOF is acetic acid, zinc chloride or zinc nitrate, and an organic ligand is 2-methylimidazole; the molar ratio of zinc ions to 2-methylimidazole in the precursor solution of the MOF is 1: 4-1: 100, the incubation time of the precursor solution of the MOF and the electrode is 0.5-2 hours; the wash buffer component was 10mmol/L HEPES, pH 7.4.
The invention also aims to provide an MOF electrochemical sensor for exosome analysis, which is prepared by adopting the preparation method of the MOF electrochemical sensor for exosome analysis.
The invention also aims to provide application of the MOF electrochemical sensor for exosome analysis in capturing exosome vesicles and detecting proteins and RNA contained in exosomes.
The fourth purpose of the invention is to provide an exosome analysis method, which comprises the following steps:
(I) capture of exosomes in samples using MOF-assembled gold electrodes: biological products, such as serum, plasma, urine, etc., are centrifuged at low speed in order to remove residual cells or cell fibers, and then a 0.22 μm filter is used to further remove large particles from the sample. And directly dripping the treated sample on the surface of the electrode, and thoroughly cleaning the surface of the electrode by using a cleaning buffer solution after incubation to obtain the MOF assembled gold electrode adsorbed by the exosome.
The volume of the required sample is 10-20 mu L, the rotation speed used in the low-speed centrifugation treatment is 1000-2000 r/min, and the centrifugation time is 5-15 min; the amount of the exosome sample is 5-20 mu L, and the incubation time is 10-25 minutes.
(II) antigen-antibody adsorption reaction: and (3) dropwise adding an anti-fouling polypeptide solution to the surface of the electrode in the step (I), adding a mouse anti-human epithelial cell adhesion molecule antibody marked by glucose oxidase after incubation, and thoroughly cleaning the electrode after full reaction.
The concentration of the anti-fouling polypeptide is 10-100 mu mol/L, and the incubation time is 15-60 minutes; the antibody concentration is 200-500 mu g/mL, and the incubation time is 30-60 minutes.
(III) electrochemical detection of exosome surface proteins: and (3) adding a glucose solution into the step (II), completely cleaning the surface of the electrode after the catalytic reaction is finished, reading a methylene blue electric signal, and judging the exosome or the protein concentration information on the surface of the exosome according to the change of the electric signal.
The glucose solution is prepared by pure water with the pH value of 7.0-7.4, the concentration of the contained glucose is 10-50mmol/L, and the catalytic reaction time is 10-60 minutes.
(IV) electrochemical detection of internal microRNA molecules of the exosome: and (4) after the capture of exosomes in the step (I) is finished, treating the surface of the electrode for 5 minutes by using an acidic buffer solution, and completely degrading all MOF structures on the surface of the electrode. And adding a mixed solution of Triton-X and ferrocene labeled DNA signal chains, wherein the Triton-X is used for cracking the exosome vesicle and releasing the microRNA molecules contained in the exosome, and the signal chains, the microRNA chains and the DNA chains on the surface of the electrode can form a triplet structure. At the moment, the ferrocene molecules are very close to the electrodes to generate obvious ferrocene signals, so that whether the exosomes contain target microRNA molecules or not is judged.
The component of the acidic buffer solution is 20mmol/L acetic acid, and the pH value is 5.0; the concentration of the Triton-X is 0.05-0.2%, the concentration of the signal chain is 20-50nmol/L, and the incubation time of the mixed solution and the electrode is 80-100 minutes; the reaction temperature is 25-50 ℃.
The principle of the exosome electrochemical analysis method of the invention is as follows: the MOF on the surface of the electrode can restrict the collision of nucleic acid molecules on the surface of the electrode and the surface of the electrode to inhibit methylene blue electric signals, and can capture exosomes in a solution to the surface of the electrode through strong interaction between various acting forces, such as electrostatic force, van der Waals force, metal coordination acting force and the like, and exosomes in the sample. After the adsorption is finished, adding the anti-fouling polypeptide to shield the vacancy on the surface of the electrode, and preventing the non-specific adsorption of substances such as subsequent antibodies and the like. The characteristic protein on the surface of the exosome is identified by adding the specific antibody marked by the glucose oxidase, so that the accurate identification of the tumor exosome is completed. After glucose is added, glucose oxidase converts glucose into gluconic acid, the acidic substance can rapidly decompose the MOF structure on the surface of the electrode, and at the moment, nucleic acid molecules can freely collide with the surface of the electrode, so that methylene blue signal is increased. Thus, the intensity of the electrical signal is positively correlated with the exosome concentration or protein abundance at its surface.
Since exosomes can be adsorbed directly to the electrode surface, the addition of a membrane-disrupting agent such as Triton-X causes the rupture of the exosome vesicles, resulting in the release of their contents. After a signal chain marked with a ferrocene signal is introduced, the signal chain, a target microRNA molecule and a DNA molecule on the surface of the electrode form a triplet structure through hybridization reaction. At the moment, the ferrocene molecules are very close to the electrodes to generate obvious ferrocene signals, so that whether the exosomes contain target microRNA molecules or not is judged.
Compared with the prior art, the invention has at least the following beneficial effects:
1. the electrode modified by the metal organic framework material can directly collect exosomes from a complex sample, the reaction time is controlled within 30 minutes, an expensive large-scale centrifuge and a complicated and time-consuming separation process are not needed, and the time for analyzing the exosomes is greatly shortened.
2. The detection method can simultaneously analyze the protein information on the surface of the exosome and the RNA information in the exosome, and remarkably improves the analysis efficiency.
3. The detection method utilizes a metal organic framework to rapidly collect exosomes, has high-efficiency enzyme catalytic reaction and an electrochemical sensitive analysis platform, greatly reduces the use amount of samples, improves the analysis effect of the exosomes, and can detect about 250 exosome vesicles in 10 mu L samples.
Drawings
FIG. 1 is a process diagram of the MOF electrochemical sensor assembly and exosome capture for exosome analysis obtained in the present invention.
FIG. 2 is a schematic diagram of MOF electrochemical sensor for exosome analysis used for detecting tumor exosomes.
FIG. 3 is a scanning electron microscope image of MOF electrochemical sensors for capturing exosomes secreted by MCF-7 cells and analyzing the exosomes after the completion of the catalytic reaction.
FIG. 4 is a diagram showing the result of using the MOF electrochemical sensor for exosome analysis obtained in the present invention to detect MCF-7 extracellular exosomes.
FIG. 5 is a result chart of the MOF electrochemical sensor for exosome analysis obtained in the invention for detecting miR-21 contained in MCF-7 cell exosomes.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
(1) Assembly of MOF/AuE
As shown in FIG. 1, DNA molecules labeled with methylene blue at a concentration of 1 to 3. mu. mol/L were immobilized to the surface of a gold electrode (3 mm in diameter) via gold-thiol bonds,
the DNA chain sequence is: 5 '-mercapto-TTTTCTCGATCCCTGATAAGCTA-methylene blue-3'.
The buffer solution used for fixing the DNA strand was 10mmol/L HEPES (pH 7.4), NaCl 1mol/L, and the fixing time was 2 to 4 hours.
And then, soaking the electrode for modifying the DNA molecules in mercaptohexanol sealing solution with the concentration of 0.5-1mmol/L for 0.5-2 hours, sealing the vacant positions on the surface of the electrode, and recording square wave voltammetric signals of methylene blue.
And finally, dripping precursor liquid of the MOF on the surface of the prepared electrode, and completely cleaning the electrode after the MOF is formed. The zinc source in the MOF precursor solution is acetic acid, zinc chloride or zinc nitrate, and the organic ligand is 2-methylimidazole; the molar ratio of zinc ions to 2-methylimidazole in the precursor solution of the MOF is 1: 4-1: 100, the incubation time of the precursor solution of the MOF and the electrode is 0.5-2 hours; the wash buffer component was 10mmol/L HEPES (pH 7.4).
(2) Capturing exosomes in a sample using MOF/AuE
The biological product was centrifuged at low speed for 5-15 minutes at 1000-.
And (3) directly dripping the treated sample on the surface of the electrode, and completely washing the surface of the electrode by using a washing buffer solution after incubation to obtain the MOF/AuE adsorbed by the exosome. As indicated by the arrows in fig. 3, many round vesicle structures were adsorbed on MOFs with polygonal structures, indicating successful capture of exosomes.
The biological sample can be liquid such as serum, plasma, urine, cell culture solution, etc., and the volume of the required sample is 10-20 μ L; the amount of the exosome sample is 5-20 mu L, and the incubation time is 10-25 minutes.
(3) Antigen-antibody adsorption reaction
Dripping 10-20 mu L of an anti-fouling polypeptide (amino acid sequence is EKEKEKEPPPPHHHHHH) solution with the concentration of 10-100 mu mol/L on the surface of the electrode in the step (2), and incubating for 15-60 minutes to fully occupy the vacant sites of the MOF on the surface of the electrode; then 10-20 μ L of glucose oxidase labeled mouse anti-human epithelial cell adhesion molecule antibody solution (GOx-Ab) with the concentration of 200-.
The reaction buffer was 10mmol/L HEPES (pH 7.4), NaCl 137mmol/L, and the reaction temperature was 25-37 ℃.
(4) Electrochemical detection of exosome surface proteins
And (4) dripping 10-20 mu L of glucose solution with the concentration of 10-50mmol/L on the surface of the electrode treated in the step (3), preparing the glucose solution by pure water with the pH value of 7.0-7.4, carrying out catalytic reaction for 10-60 minutes, completely cleaning the surface of the electrode after the catalytic reaction is finished, reading a methylene blue electric signal, and judging the concentration information of exosomes through the change of the electric signal.
As shown in FIG. 3, the MOF structure on the electrode surface appeared to have many void structures after glucose addition, indicating that the MOF had corroded.
As shown in FIG. 4, the detection limit of this assay is an exosome vesicle of about 250 MCF-7 cells in 10. mu.L of serum sample.
(5) Electrochemical detection of exosome internal microRNA molecules
And (3) after the capture of the exosomes in the step (2) is finished, treating the surface of the electrode for 5 minutes by using an acidic buffer solution, completely degrading all MOF structures on the surface of the electrode, and releasing the exosomes on the surface of the electrode. Adding mixed solution of Triton-X with the concentration of 0.05-0.2% and a signal chain with the concentration of 20-50nmol/L, and incubating the mixed solution and the electrode for 80-100 minutes at the reaction temperature of 25-50 ℃. The acidic buffer component was 20mmol/L acetic acid (pH 5.0).
The Triton-X is used for cracking the exosome vesicle and releasing microRNA molecules contained in the exosome, and the signal chain, the microRNA chain and the DNA chain on the surface of the electrode can form a triplet structure. At the moment, the ferrocene molecules are very close to the electrodes to generate obvious ferrocene signals, so that whether the exosomes contain target microRNA molecules or not is judged.
As shown in FIG. 5, the detection method can detect about 21600 microRNA molecules in 10 μ L of sample.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A preparation method of an MOF electrochemical sensor for exosome analysis is characterized by comprising the following steps:
carrying out primary incubation on a DNA molecular solution marked by sulfydryl and methylene blue and a clean gold electrode, and then adopting a sulfydryl hexanol solution to seal the vacant position of the gold electrode to obtain a DNA modified gold electrode;
and putting the DNA modified electrode into the MOF precursor solution for secondary incubation, and then cleaning the surface of the electrode to obtain the MOF electrochemical sensor for exosome analysis.
2. The method for preparing the MOF electrochemical sensor for exosome analysis according to claim 1, wherein the concentration of a sulfydryl and methylene blue labeled DNA molecule solution is 1-3 μmol/L, and the time of one incubation is 2-4 hours;
the concentration of the mercaptohexanol solution is 0.5-1mmol/L, and the time for blocking the gold electrode with mercaptohexanol is 0.5-2 hours.
3. The preparation method of the MOF electrochemical sensor for exosome analysis according to claim 1, characterized in that acetic acid, zinc chloride or zinc nitrate are adopted as a zinc source in MOF precursor liquid, 2-methylimidazole is adopted as an organic ligand, and the molar ratio of zinc ions to 2-methylimidazole is 1: 4-100.
4. The method for preparing the MOF electrochemical sensor for exosome analysis according to claim 1, wherein the secondary incubation time is 0.5-2 hours, and the electrode surface is washed by 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with the concentration of 10mmol/L, pH-7.4.
5. An MOF electrochemical sensor for exosome analysis, characterized in that it is produced by the production method of the MOF electrochemical sensor for exosome analysis according to any one of claims 1 to 4.
6. Use of an MOF electrochemical sensor for exosome analysis according to claim 5 as a sensor for capturing exosome vesicles and detecting proteins and RNAs contained in exosomes.
7. A method of trapping exosome vesicles, comprising the steps of: centrifuging the biological product, and then filtering to obtain a pretreatment sample; dripping a pretreatment sample on the surface of the MOF electrochemical sensor for exosome analysis according to claim 5, incubating, and cleaning the surface of the MOF electrochemical sensor for exosome analysis to obtain an MOF gold electrode adsorbed by exosome.
8. The method of capturing exosome vesicles according to claim 7, wherein the bioproduct volume is 10-20 μ L; the centrifugal processing rotating speed is 1000-2000 r/min, and the centrifugal processing time is 5-15 min; the dripping volume of the pretreated sample is 5-20 mu L; the incubation time was 10-25 minutes.
9. A method of analyzing protein and RNA information of exosomes, comprising the steps of:
s1, dropping an anti-fouling polypeptide solution on the surface of the MOF gold electrode adsorbed by the exosome obtained in the method for capturing the exosome vesicle of claim 7 or 8, adding a glucose oxidase-labeled mouse anti-human epithelial cell adhesion molecule antibody solution after incubation, and thoroughly cleaning the electrode after full reaction to obtain a first electrode;
adding a glucose solution into the first electrode, thoroughly cleaning the surface of the electrode after the catalytic reaction is finished, reading a methylene blue electric signal, and judging the protein concentration information of the exosome or the exosome surface through the change of the electric signal;
s2, treating the MOF gold electrode surface adsorbed by the exosomes obtained in the method for capturing exosome vesicles according to claim 7 or 8 by using an acidic buffer solution for 5 minutes, adding a mixed solution of Triton-X and a ferrocene labeling signal DNA chain for incubation, and judging whether the exosomes contain target microRNA molecules or not according to the obtained ferrocene signals.
10. The method for analyzing protein and RNA information of exosomes according to claim 9, wherein in S1, the anti-fouling polypeptide concentration solution is 10-100 μmol/L, and the incubation time is 15-60 minutes; the concentration of the glucose oxidase labeled mouse anti-human epithelial cell adhesion molecule antibody solution is 200-; the concentration of the glucose solution is 10-50mmol/L, and the catalytic reaction time is 10-60 minutes;
in S2, the acidic buffer solution is acetic acid buffer solution with pH of 5.0 and concentration of 20mmol/L, the mass fraction of Triton-X in the mixed solution is 0.05-0.2%, the concentration of ferrocene labeling signal DNA chain in the mixed solution is 20-50nmol/L, the incubation time is 80-100 minutes, and the incubation temperature is 25-50 ℃.
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