Biomarker detection system based on gas generation

Abstract

A biomarker detection system based on gas generation comprises a closed container, a capillary tube and a scale mark, a magnetic bead-nucleic acid aptamer compound is used for specific recognition and capture of target protein, a platinum nanoparticle-nucleic acid aptamer compound catalyzes hydrogen peroxide to generate oxygen, a gas pressure difference is formed to push a liquid column to move, and the target protein is detected through the scale mark. The concentration of the target protein is measured by reading the length of a liquid column in a capillary tube with naked eyes, and quantitative detection is realized. The detection system improves the detection sensitivity and speed, reduces the operation complexity and cost, has high expansibility, can detect various biomarkers, and is suitable for diagnosis of various diseases such as tumors and the like. The system has the advantages of high sensitivity, rapidness, low cost and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection, and in particular to a biomarker detection system based on gas generation. Background Art

[0002] Existing biological protein target detection methods mainly include protein blotting, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, colloidal gold immunochromatography and other technologies.

[0003] (1) Western blotting (WB) is the most commonly used protein analysis technique and is widely used in the analysis of protein components in various samples. The conventional WB process includes electrophoresis, transfer, incubation, color development and other operation steps. This method uses antibodies to recognize specific proteins in the sample and achieves signal output (such as luminescence and color development) through a second antibody. It is a universal method for biological protein determination. However, this method is cumbersome and time-consuming (>12h), and has low sensitivity and cannot be used for accurate quantitative detection. It is not suitable for large-scale clinical sample determination.

[0004] (2) Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for quantitative protein detection. It has a variety of reaction modes: sandwich method, competitive method, indirect method, etc., to suit different types of target molecules. Taking the classic sandwich method as an example, a "solid phase-capture antibody-target protein-enzyme-labeled antibody" sandwich complex is formed with the target protein in the sample. After washing and removing other unreacted substances, a substrate is added to the system, and the target protein is determined by luminescence or color development. Since ELISA is a solid-phase reaction, its reaction kinetics and detection sensitivity have certain limitations, and the operation is relatively cumbersome and time-consuming (>3h). It has not yet been widely used for exosome protein detection.

[0005] (3) Chemiluminescent immunoassay (CLIA) is the current mainstream high-throughput biological target detection technology. Its basic principle is similar to that of ELISA technology. CLIA technology also includes multiple reaction modes such as sandwich method, competitive method, and indirect method. Taking the classic sandwich method as an example, by forming a "magnetic bead-capture antibody-target protein-enzyme-labeled antibody" sandwich complex with the target protein in the sample, after washing and removing other unreacted substances, an enzymatic luminescent substrate is added to the system, and the target protein can be determined by detecting the intensity of the luminescent signal. CLIA technology has the advantages of high degree of automation and high throughput, but it requires large-scale detection instruments. This method is usually used in large laboratories or large hospital laboratories.

[0006] (4) Colloidal gold immunochromatography technology uses colloidal gold as a tracer marker and nitrocellulose membrane as a carrier, so that the antigen-antibody reaction and washing are carried out on the same permeation membrane. This process uses the capillary action of the microporous membrane to allow the liquid at one end of the membrane to slowly penetrate and move to the other end, thereby driving the movement, binding and washing of the antigen and antibody on the membrane, and finally observing the color change of the detection line to achieve the target protein determination. This method is fast and convenient, but because it is a solid-phase reaction, it has low sensitivity and cannot achieve quantitative detection.

[0007] The existing related detection technologies mainly have the following disadvantages:

[0008] (1) High detection cost and non-portability: Existing biomarker detection technologies usually require large instruments such as spectrophotometers, fluorescence analyzers, microplate readers, and chemiluminescence analyzers. The detection system is expensive, and large instruments cannot be applied to various instant detection application scenarios, which has great application limitations.

[0009] (2) Complex operation and long detection time: Detection technologies such as WB and ELISA have complex operation procedures and usually take more than 3 hours to detect. Developing a detection method with fast reaction and simple operation has a wide range of application scenarios.

[0010] (3) Detection sensitivity and inability to quantify: In many biomarker detection application scenarios, there are usually high requirements for detection speed, sensitivity, and quantitative determination. The existing colloidal gold immunochromatography technology used for rapid detection has low sensitivity due to the limitations of solid-phase reactions and can only be used for qualitative detection. It cannot simultaneously meet the requirements of the above application scenarios for detection speed, sensitivity, and quantification.

[0011] In summary, the existing technology has problems such as complex operation, long detection time, low sensitivity, high cost, and high equipment requirements, and cannot meet application needs well.

[0012] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0013] The main purpose of the present invention is to overcome the defects existing in the above-mentioned background technology and provide a biomarker detection system based on gas generation.

[0014] To achieve the above object, the present invention adopts the following technical solutions:

[0015] A biomarker detection system based on gas generation, comprising:

[0016] Magnetic bead-aptamer complex, used to specifically identify and capture target proteins in samples;

[0017] Platinum nanoparticle-aptamer complex, used to catalyze the decomposition of hydrogen peroxide to produce oxygen, connected to the sandwich complex formed by the magnetic bead-aptamer complex;

[0018] A closed container, the interior of which is used to contain the sandwich compound and the hydrogen peroxide solution, and forms a closed space with the external atmosphere through the container cover;

[0019] a capillary tube connected to the closed container and used to measure the change in length of the liquid column caused by the generation of oxygen;

[0020] A scale mark, matched with the capillary tube, for naked eye reading;

[0021] When the airtight container is sealed, the platinum nanoparticles on the sandwich composite catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the gas pressure in the container to increase;

[0022] The pressure difference between the inside and outside of the container presses the liquid into the capillary, and the length of the liquid column in the capillary is positively correlated with the concentration of the target protein to be detected in the sample;

[0023] Therefore, by reading the length of the liquid column in the capillary with the naked eye, rapid quantitative detection of the target protein concentration can be achieved.

[0024] Furthermore, the magnetic bead-nucleic acid aptamer complex specifically comprises:

[0025] Magnetic beads whose surface is modified with streptavidin;

[0026] The CD63 nucleic acid aptamer is modified with biotin at its end and connected to the magnetic beads through the binding of biotin and streptavidin.

[0027] Furthermore, the platinum nanoparticle-nucleic acid aptamer complex specifically comprises:

[0028] Platinum nanoparticles;

[0029] The MUC1 nucleic acid aptamer is modified with a thiol group at its end and is connected to the surface of the platinum nanoparticles through the formation of a chemical covalent bond between the thiol group and the platinum atom.

[0030] Furthermore, the formation process of the sandwich complex specifically includes: mixing the sample to be tested with the magnetic beads-CD63 nucleic acid aptamer, reacting at an appropriate temperature to form a magnetic bead-CD63 nucleic acid aptamer-exosome complex; removing unreacted substances by magnetic separation and washing; adding platinum nanoparticles-MUC1 nucleic acid aptamer to form a magnetic bead-CD63 nucleic acid aptamer-exosome-MUC1 nucleic acid aptamer-platinum nanoparticle sandwich complex.

[0031] Furthermore, the specific configuration of the sealed container and the capillary tube includes: using a glass bottle as the sealed container; a rubber plug cooperates with the glass bottle to form a sealed space; and a glass capillary tube is vertically inserted into the rubber plug and communicated with the interior of the glass bottle.

[0032] Furthermore, the bottom of the capillary tube is higher than the liquid level in the container, so as to discharge part of the air in the container and keep the air pressure inside and outside the container balanced.

[0033] Furthermore, the inner diameter of the capillary is 0.5 mm, the length is 120 mm, and the lower end of the capillary slightly protrudes from the rubber plug by 1 mm.

[0034] Furthermore, after reacting for a certain period of time at an appropriate temperature, the platinum nanoparticles catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the air pressure in the glass bottle to increase, thereby pressing the colored liquid inside into the capillary.

[0035] The present invention has the following beneficial effects:

[0036] The present invention provides a biomarker detection system based on gas generation, which can realize rapid, highly sensitive, low-cost and instrument-free quantitative detection of biomarkers. The system forms a sandwich complex by utilizing the specific recognition ability of nucleic acid aptamers, and uses platinum nanoparticles to catalyze the decomposition of hydrogen peroxide to produce oxygen to drive the movement of the liquid column, thereby realizing accurate measurement of the concentration of the target protein. Compared with traditional techniques such as protein blotting, enzyme-linked immunosorbent assay, chemiluminescent immunoassay and colloidal gold immunochromatography, the present invention significantly improves the sensitivity and speed of detection, while reducing the complexity and cost of operation. In addition, the detection system of the present invention has extremely high scalability, and can not only detect different nucleic acid sequences by changing the nucleic acid aptamer sequence, but also replace it with antibodies or antigens to identify the corresponding antigens / antibodies. This flexibility makes the present invention not only suitable for the detection of tumor cell-derived exosomes, but also can be extended to the detection of various biomarkers, and has a wide range of application prospects.

[0037] Compared with the prior art, the main advantages of the present invention are:

[0038] (1) Quantitative detection: Traditional colloidal gold immunochromatography can only detect target molecules qualitatively. The present invention uses the correspondence between the length of the pushed liquid column and the concentration of the biomarker to achieve quantitative detection of the target molecules, which can be applied to a wider range of application scenarios.

[0039] (2) Highly sensitive detection: The reaction process of the present invention is carried out in solution, which has better reaction kinetics than immunochromatography, making the reaction process faster and more thorough. Compared with traditional detection methods, the present invention achieves highly sensitive determination of MUC1-positive exosomes, with LoD reaching 0.68fM.

[0040] (3) Rapid detection and simple operation: The present invention can achieve rapid detection of tumor exosomes within 40 minutes, which is easy to operate and greatly improves the practicality of the present invention.

[0041] (4) No instruments required, low cost: The present invention does not require any instruments or equipment, involves a small number of raw materials with low prices (such as glass bottles, capillaries, and hydrogen peroxide solutions), and the reagent synthesis and device assembly methods are simple, and the detection cost is greatly reduced compared to traditional detection technologies.

[0042] (5) Strong application expandability: By replacing the aptamer sequence with the desired sequence, or replacing it with an antigen / antibody, the determination of multiple biomarkers can be achieved. The present invention can not only use PtNP to decompose hydrogen peroxide to produce oxygen, but also use other substances that can catalyze the decomposition of hydrogen peroxide (such as catalase, nanozyme) to replace PtNP for detection, which has strong application expandability.

[0043] The detection system of the present invention utilizes the specific recognition of the target protein by the nucleic acid aptamer to form a "magnetic bead-nucleic acid aptamer 1-target protein-nucleic acid aptamer 2-platinum nanoparticle" sandwich complex, and the oxygen generated by the decomposition of hydrogen peroxide (H2O2) catalyzed by the labeled PtNP generates a pressure difference in a closed container to drive the liquid column to move, and finally realizes the quantitative determination of the target protein through the moving distance of the liquid column in the capillary. The detection system of the present invention has important application value in the detection of biological protein markers, and at the same time meets the detection requirements of rapid quantitative, low-cost, high-sensitivity, and no instrument required, and is suitable for the detection of various biological protein targets.

[0044] Other beneficial effects of the embodiments of the present invention will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of a biomarker detection system based on gas generation according to an embodiment of the present invention.

[0046] Figure 2 This is a schematic diagram of the synthesis principle of the "magnetic bead-CD63 nucleic acid aptamer" complex according to an embodiment of the present invention.

[0047] Figure 3 This is a schematic diagram of the synthesis principle of the "platinum nanoparticle-MUC1 nucleic acid aptamer" complex according to an embodiment of the present invention.

[0048] Figure 4 Schematic diagram of the closed container and capillary structure of the detection system according to an embodiment of the present invention.

[0049] Figure 5 This is a graph showing the agarose gel electrophoresis results of the "magnetic bead-CD63 nucleic acid aptamer" complex of an embodiment of the present invention.

[0050] Figure 6 This is a graph showing the agarose gel electrophoresis results of the “platinum nanoparticle-MUC1 nucleic acid aptamer” complex of an embodiment of the present invention.

[0051] Figure 7 : is a calibration curve of the MUC1 protein positive exosome concentration and the liquid column movement distance of the embodiment of the present invention.

[0052] Figure 8 Graph showing the detection results of exosomes derived from breast cancer (MCF-7) or non-breast cancer (MCF-10A) cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0053] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention.

[0054] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for fixing as well as for coupling or communication.

[0055] It should be understood that the orientation or position relationship indicated by terms such as "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0056] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0057] See also Figures 1 to 4The embodiment of the present invention provides a biomarker detection system based on gas generation, including: a magnetic bead 2-nucleic acid aptamer 1 complex, which is used to specifically identify and capture a target protein 4 in a sample; a platinum nanoparticle 6-nucleic acid aptamer 7 complex, which is used to catalyze the decomposition of hydrogen peroxide to produce oxygen, and is connected to a sandwich complex formed by the magnetic bead-nucleic acid aptamer complex; a sealed container 11 (such as a glass bottle), the interior of which is used to accommodate the sandwich complex and the hydrogen peroxide solution, and forms a closed space with the external atmosphere through the container cover; a capillary 9, which is connected to the sealed container 11 is connected to measure the change in liquid column length caused by the generation of oxygen; the scale mark 12 cooperates with the capillary 9 and is used for naked eye reading 13; when the closed container 11 is sealed, the platinum nanoparticles on the sandwich composite catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the air pressure in the container to increase; the pressure difference between the inside and outside of the container presses the liquid into the capillary 9, and the length of the liquid column in the capillary 9 is positively correlated with the concentration of the target protein to be measured in the sample; thus, by reading the length of the liquid column in the capillary 9 with the naked eye, rapid quantitative detection of the target protein concentration can be achieved.

[0058] During the detection process of the present invention, magnetic separation technology can be used to quickly separate the target molecules in the sample, and an immune sandwich complex is formed in the reaction system. The molecules with catalase function marked on the complex catalyze the decomposition of hydrogen peroxide to produce oxygen, and the pressure difference is generated to push the liquid column to move to achieve reading. The detection reagent components directly affect the identification, signal conversion and amplification of the biomarker to be tested. The detection reagent converts the biomarker concentration signal into an air pressure intensity signal in the closed container 11. The air pressure difference between the closed container 11 and the atmosphere can be used to push the liquid in the container out of the capillary 9, and the above-mentioned air pressure intensity signal is further converted into a liquid column length value in the capillary 9, thereby realizing the concentration measurement of the target to be tested.

[0059] like Figures 1 to 3 As shown, in a preferred embodiment, the magnetic bead-aptamer complex specifically includes: magnetic bead 2, whose surface is modified with streptavidin; CD63 aptamer 1, whose terminal is modified with biotin, and connected to the magnetic bead through the combination of biotin and streptavidin. The platinum nanoparticle-aptamer complex specifically includes: platinum nanoparticle 6; MUC1 aptamer 7, whose terminal is modified with thiol, and connected to the surface of platinum nanoparticle through the formation of chemical covalent bond between thiol and platinum atom. The formation process of the sandwich complex specifically includes: mixing the sample to be tested (exosome 3 containing CD63 protein 4 and MUC1 protein 5 on the surface) with the magnetic bead-CD63 aptamer, reacting at an appropriate temperature to form a magnetic bead-CD63 aptamer-exosome complex; removing unreacted substances by magnetic separation and washing; adding platinum nanoparticle-MUC1 aptamer to form a magnetic bead-CD63 aptamer-exosome-MUC1 aptamer-platinum nanoparticle sandwich complex.

[0060] like Figure 4 As shown, in a preferred embodiment, the specific configuration of the sealed container 11 and the capillary 9 includes: a glass bottle is used as the sealed container 11; the rubber plug 10 cooperates with the glass bottle to form a closed space; the glass capillary 9 is vertically inserted into the rubber plug 10 and communicates with the inside of the glass bottle. The bottom of the capillary 9 is higher than the liquid level in the container to discharge part of the air in the container so that the air pressure inside and outside the container remains balanced. Preferably, the inner diameter of the capillary 9 is 0.5 mm and the length is 120 mm. The lower end of the capillary 9 slightly protrudes 1 mm outside the rubber plug 10. After reacting for a certain period of time at an appropriate temperature, the platinum nanoparticles catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the air pressure in the glass bottle to increase, thereby pressing the colored liquid inside into the capillary 9.

[0061] In the biomarker detection system based on gas generation of the present invention, a nucleic acid aptamer 1 (Aptamer) that can recognize the target protein is connected to the surface of a magnetic bead (MB), and another nucleic acid aptamer 2 that can recognize the target protein is connected to the surface of a platinum nanoparticle (PtNP). When the target protein exists in the sample, the above two nucleic acid aptamers will specifically recognize the target protein to form a "magnetic bead-nucleic acid aptamer 1-target protein-nucleic acid aptamer 2-platinum nanoparticle" sandwich complex. Unbound substances in the system are removed by magnetic separation and cleaning, and the above sandwich complex is transferred to a closed container 11 connected to a capillary 9, and a hydrogen peroxide solution is added. At this time, the PtNP on the above sandwich complex will catalyze the decomposition of H2O2 in the solution to produce water and oxygen, and the generated oxygen accumulates in the container, resulting in an increase in the air pressure in the container. Since the container is connected to the external atmosphere through the capillary 9, the pressure difference between the inside and outside of the container presses the liquid into the capillary 9. Within a certain period of time, the length of the liquid column in the capillary 9 is positively correlated with the concentration of the target protein to be detected in the sample. The length of the liquid column in the capillary 9 is read, and then the concentration of the target protein in the sample can be calculated through the corresponding relationship of "liquid column length-target protein concentration". The present invention can be used for rapid quantitative detection of biomarkers.

[0062] The present invention can not only use nucleic acid aptamers to identify and measure protein targets, but also can realize the determination of specific nucleic acid sequences by designing reasonable nucleic acid sequences and utilizing nucleic acid base complementary pairing. Or the nucleic acid sequence is replaced with an antibody / antigen to realize the recognition and determination of the corresponding antigen / antibody. The present invention can not only use PtNP to realize the decomposition of hydrogen peroxide to produce oxygen, but also use other substances (such as catalase) that can catalyze the decomposition of hydrogen peroxide to replace PtNP for detection. The present invention has many advantages such as no need for instruments, naked eye reading, high sensitivity, quantitative determination, rapid detection, low cost, etc., and can be expanded to the detection of multiple targets, which is of great significance in the diagnosis of various diseases such as tumors.

[0063] The specific embodiments of the present invention are further described below.

[0064] The overall detection principle of this detection system is as follows Figure 1 As shown: the magnetic beads 2 modified with CD63 nucleic acid aptamer 1 recognize CD63 protein 4 (exosome characteristic protein) on the surface of exosome 3 in the sample to be tested through CD63 nucleic acid aptamer 1 to achieve the capture of exosomes. Other unreacted substances in the system are removed by magnetic separation operation, and then platinum nanoparticles 6 modified with MUC1 nucleic acid aptamer 7 are added. At this time, MUC1 nucleic acid aptamer 7 binds to MUC1 protein 5 (tumor-related protein) on the surface of captured tumor exosome 3 to form a "magnetic bead-CD63 nucleic acid aptamer-exosome-MUC1 nucleic acid aptamer-platinum nanoparticle" sandwich complex. Unbound substances in the system are removed by magnetic separation and washing, and the above sandwich complex is transferred to the detection container, and H2O2 is added, and then the rubber stopper 10 with capillary 9 is covered to seal the container. In the process of covering the rubber plug 10, it should be noted that the bottom of the capillary 9 must be higher than the liquid level in the container. During the sealing process of the rubber plug 10, part of the air in the container will be discharged from the container through the capillary 9, and finally the air pressure inside and outside the container is balanced (if the bottom of the capillary 9 is lower than the liquid level in the solution in this step, the increase in the internal air pressure of the container caused by the sealing process of the rubber plug 10 will cause the solution to be pressed into the capillary 9, causing the measurement result deviation). After the rubber plug 10 is sealed, the capillary 9 is quickly and gently inserted a short distance below the liquid level. At this time, the pressure difference between the inside and outside of the container is zero. At this time, the PtNP on the sandwich composite will catalyze the decomposition of H2O2 in the solution to produce water and oxygen, and the generated oxygen accumulates in the container, causing the air pressure in the container to increase. Since the container is connected to the external atmosphere through the capillary 9, the pressure difference between the inside and outside of the container presses the liquid into the capillary 9. Within a certain period of time, the length of the liquid column in the capillary 9 is positively correlated with the concentration of the target protein to be measured in the sample. The length of the liquid column in the capillary 9 is read, and then the concentration of the target protein in the sample can be calculated through the corresponding relationship of "liquid column length-target protein concentration". The present invention can be used for rapid quantitative detection of biomarkers.

[0065] Figure 1 A biomarker detection system based on gas generation is shown (1. CD63 nucleic acid aptamer; 2. magnetic beads; 3. exosomes; 4. CD63 protein; 5. MUC1 protein; 6. platinum nanoparticles; 7. MUC1 nucleic acid aptamer; 8. magnetic separation operation; 9. capillary; 10. rubber stopper (container cover); 11. sealed container; 12. scale; 13. reading process).

[0066] (1) Preparation of detection reagents

[0067] The detection reagent mainly includes two complex components: "magnetic beads-CD63 nucleic acid aptamer" and "platinum nanoparticles-MUC1 nucleic acid aptamer". Among them, the CD63 nucleic acid aptamer sequence end is modified with biotin, and through the combination of biotin and streptavidin (SA), it is connected to the magnetic beads with SA modified on the surface to form a "magnetic beads-CD63 nucleic acid aptamer" complex. The unbound substances are removed by magnetic separation to obtain the corresponding reagent components. The synthesis principle is as follows Figure 2 The MUC1 nucleic acid aptamer sequence with a thiol group at the end forms a chemical covalent bond with the platinum atom through the thiol group and is connected to the surface of the platinum nanoparticle to form a "platinum nanoparticle-MUC1 nucleic acid aptamer" complex component. The unbound substances are removed by centrifugation to obtain the corresponding reagent components. The synthesis principle is as follows Figure 3 shown.

[0068] Figure 2 The synthesis principle of the "magnetic bead-CD63 nucleic acid aptamer" complex is shown (1. Magnetic beads modified with streptavidin (SA); 2. CD63 nucleic acid aptamer sequence modified with biotin at the end; "magnetic bead-CD63 nucleic acid aptamer" complex ( Figure 2 on the right)).

[0069] Figure 3 The synthesis principle of the "platinum nanoparticle-MUC1 nucleic acid aptamer" complex is shown (1. platinum nanoparticles; 2. MUC1 nucleic acid aptamer sequence with terminal thiol modification; "platinum nanoparticle-MUC1 nucleic acid aptamer" complex ( Figure 3 on the right)).

[0070] (2) Assembly of the detection device

[0071] The detection device mainly includes a sealed container (glass bottle and rubber plug) and a liquid column flow channel (capillary). Use a glass bottle as a sealed container, punch a hole in the matching rubber plug and vertically insert a glass capillary, which can slide slightly up and down in the rubber plug. The sealing between the capillary and the rubber plug, and between the rubber plug and the glass bottle is good and leak-proof, and the assembly of the detection device is completed. The structure of the detection device is as follows: Figure 4 shown.

[0072] (3) Using the present invention to detect exosome samples

[0073] After the sample to be tested is mixed with the "magnetic beads-CD63 nucleic acid aptamer" component, it is added to the EP tube and reacted at an appropriate temperature for a certain period of time to form a "magnetic beads-CD63 nucleic acid aptamer-exosome" complex. The remaining unreacted substances are cleaned by magnetic separation to complete the capture of the exosome target. Then, the "platinum nanoparticles-MUC1 nucleic acid aptamer" component is added to the above-mentioned complex system and mixed, and reacted at an appropriate temperature for a certain period of time to form a "magnetic beads-CD63 nucleic acid aptamer-exosome-MUC1 nucleic acid aptamer-platinum nanoparticle" sandwich complex. The remaining unreacted substances are cleaned by magnetic separation to complete the identification of the exosome target. The above sandwich complex is transferred to the glass bottle of the detection device, and a hydrogen peroxide solution containing a color dye is added, and a rubber stopper with a capillary is covered. After the rubber stopper is sealed, the capillary is gently slid down a short distance below the liquid surface to completely seal the reaction device. After reacting at an appropriate temperature for a certain period of time, platinum nanoparticles catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the air pressure in the glass bottle to increase, thereby pressing the colored liquid inside into the capillary. The length of the liquid column in the capillary is read, and the concentration of MUC1-positive exosomes in the sample to be tested can be calculated through the calibration curve.

[0074] Examples

[0075] The detection system was used to measure exosomes from breast cancer cells MCF-7 (MUC1 protein expression positive) and non-breast cancer cells MCF-10A (MUC1 protein expression negative), and the exosomes from the above two cell sources can be effectively distinguished. The specific implementation is as follows:

[0076] 1. Preparation of Detection Reagents

[0077] Synthesis of "magnetic beads-CD63 nucleic acid aptamer" complex: Dissolve the CD63 nucleic acid aptamer sequence (5'-CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTT-biotin-3') in ultrapure water to obtain a 100μM storage solution. Take 5μL×100μM CD63 nucleic acid aptamer storage solution and add it to 45μL ultrapure water to obtain a 10μM CD63 nucleic acid aptamer working solution. Adjust the concentration of magnetic beads with a particle size of 2.8μm and surface modified with streptavidin to 1mg / mL with Tris-HCl solution, take 1mL of the magnetic beads and mix with the above 50μL CD63 nucleic acid aptamer working solution, react at 37℃ for 30min, then use Tris-HCl solution for magnetic separation and washing 4 times, and adjust the volume to 1mL to obtain the "magnetic beads-CD63 nucleic acid aptamer" complex solution, which is placed at 4℃ for use. The successful synthesis of “magnetic beads-CD63 nucleic acid aptamer” was confirmed by agarose gel electrophoresis. Figure 5 shown.

[0078] Figure 5The agarose gel electrophoresis results of the "magnetic beads-CD63 nucleic acid aptamer" complex are shown (band 1: nucleic acid marker; band 2: CD63 nucleic acid aptamer without biotin modification; band 3: a mixture of CD63 nucleic acid aptamer without biotin modification and magnetic beads; band 4: CD63 nucleic acid aptamer modified with biotin; band 5: "magnetic beads-CD63 nucleic acid aptamer" complex)

[0079] Synthesis of "platinum nanoparticle-MUC1 nucleic acid aptamer" complex: Dissolve the MUC1 nucleic acid aptamer sequence (5'-GGTCCCATAGGTTTCCTAGTTGACGAATTTTTT-SH-3') in ultrapure water to obtain a 100μM stock solution. Take 5μL×100μM MUC1 nucleic acid aptamer stock solution and add it to 50μL×10mM TCEP aqueous solution and activate it at room temperature for 2 hours. Mix 1mL of PtNP aqueous solution with a particle size of about 60nm and a concentration of 0.1mg / mL with the above-activated MUC1 nucleic acid aptamer solution, and place it on a shaker at room temperature for 12 hours. Then add 90μL of 0.3M NaCl aqueous solution to the above solution every 30 minutes, and add it 10 times in total to complete the salting process. After salting, continue to place the above solution on a shaker at room temperature for 18 hours. After the reaction, the sample was centrifuged and washed three times (11000g×10min) using Tris-HCl solution, and the PtNP component at the bottom was collected. Finally, the sample was fixed to 1mL with Tris-HCl solution to obtain a "platinum nanoparticle-MUC1 nucleic acid aptamer" complex solution, which was placed at 4°C for later use. The successful synthesis of "platinum nanoparticle-MUC1 nucleic acid aptamer" was confirmed by agarose gel electrophoresis. Figure 6 shown.

[0080] Figure 6 The agarose gel electrophoresis results of the "platinum nanoparticle-MUC1 nucleic acid aptamer" complex are shown (lane 1: nucleic acid marker; lane 2: MUC1 nucleic acid aptamer without thiol modification; lane 3: mixture of MUC1 nucleic acid aptamer without thiol modification and PtNP; lane 4: MUC1 nucleic acid aptamer modified with thiol; lane 5: "platinum nanoparticle-MUC1 nucleic acid aptamer" complex)

[0081] 2. Assembly of the Detection Device

[0082] A glass bottle with a volume of about 3 mL was used as the detection container. A steel needle was used to pre-puncture a hole in the matching rubber stopper. Then a glass capillary with an inner diameter of 0.5 mm and a length of 120 mm was inserted into the rubber stopper through the puncture hole. The lower end of the capillary slightly protruded from the rubber stopper by 1 mm, and the assembly of the detection device was completed.

[0083] 3. Detection of exosome samples

[0084] Acquisition of cell-derived exosome samples: The exosome particles in the supernatant of MCF-7 and MCF-10A cells were extracted by differential centrifugation, dissolved in Tris-HCl solution, and the concentration of exosome samples was measured using a nanoparticle tracking analysis system. The MCF-7 exosomes with positive MUC1 expression were quantitatively measured using a nanoflow cytometer, and the results were recorded for the establishment of a standard curve. The concentration of MUC1 protein-positive exosomes = total exosome concentration × ratio of MUC1 protein-positive exosomes.

[0085] Detection steps of exosome samples: Take 150μL of the above-extracted exosome sample and mix it evenly with 50μL of the "magnetic bead-CD63 nucleic acid aptamer" complex, react at 37°C for 10 minutes, and then remove the unreacted substances by magnetic separation and washing. Add 50μL of the "platinum nanoparticle-MUC1 nucleic acid aptamer" complex to the above components and mix evenly, react at 37°C for 20 minutes, and then remove the unreacted substances by magnetic separation and washing. Transfer the above components to the detection container with 100μL of phosphate buffer solution (PBS), and then add 2.7mL of 15% hydrogen peroxide solution containing color dye. Quickly close the container with a rubber stopper and slide the capillary down to 5mm below the liquid surface. React at 25°C for 5 minutes, immediately read the length of the liquid column in the capillary, and substitute it into the standard curve to calculate the concentration value of MUC1 positive exosomes in the sample to be tested.

[0086] Steps for establishing the calibration curve: MUC1-positive exosomes derived from MCF-7 cells were prepared with Tris-HCl solution at concentrations of 0, 2.5, 5, 7.5 and 10 fM, respectively. According to the above-mentioned detection steps of exosome samples, each gradient sample was measured three times, and the calibration curve was obtained by fitting the 4-parameter equation. The 4-parameter equation is: y = A2 + (A1-A2) / (1 + (x / x0)^p). Where y is the distance the liquid column moves, x is the concentration of MUC1-positive exosomes, and the other fitting parameters are: A1 = 3.41, A2 = 262889.57, x0 = 198.65, p = 2.61, correlation coefficient R 2 =9911. The calibration curve after fitting is as follows Figure 7 From the above data, it can be seen that the minimum detection limit (LoD) of the present invention for MUC1 positive exosomes is about 0.68 fM.

[0087] According to the above detection steps, the exosomes derived from MCF-7 and MCF-10A with a total concentration of 40 fM were measured three times, and the results showed that the readings of the exosomes derived from the two cell sources were significantly different: the readings of the exosomes derived from MCF-7 were significantly higher than those of the exosomes derived from MCF-10A, proving that the present invention can effectively distinguish exosomes derived from breast cancer (MCF-7) or non-breast cancer (MCF-10A) through the MUC1 tumor-specific protein. Figure 8 Therefore, the present invention has excellent sensitivity for exosome determination and is expected to be used for rapid diagnosis of diseases such as tumors.

[0088] Advantages analysis:

[0089] The present invention realizes the rapid determination of tumor cell-derived exosomes with high sensitivity, simple operation, no instrumentation, and naked eye quantification at low cost. In addition, the present invention has extremely high scalability: the nucleic acid aptamer sequence is replaced with the desired nucleic acid sequence, and the determination of the specific nucleic acid sequence can be achieved by using nucleic acid base complementary pairing; or the nucleic acid aptamer sequence is replaced with an antibody / antigen to achieve the recognition and determination of the corresponding antigen / antibody. The present invention can not only use PtNP to decompose hydrogen peroxide to produce oxygen, but also use other substances that can catalyze the decomposition of hydrogen peroxide (such as catalase, nanozyme) instead of PtNP for detection. It is expected to be used for the detection of a variety of biomarkers and has a wide range of application value in the field of biomedicine.

[0090] Compared with the prior art, the present invention has the following technical advantages:

[0091] (1) Quantitative detection: Traditional colloidal gold immunochromatography can only detect target molecules qualitatively. The present invention uses the correspondence between the length of the pushed liquid column and the concentration of the biomarker to achieve quantitative detection of the target molecules, which can be applied to a wider range of application scenarios.

[0092] (2) Highly sensitive detection: The reaction process of the present invention is carried out in solution, which has better reaction kinetics than immunochromatography, making the reaction process faster and more thorough. Compared with traditional detection methods, the present invention achieves highly sensitive determination of MUC1-positive exosomes, with LoD reaching 0.68fM.

[0093] (3) Rapid detection and simple operation: The present invention can achieve rapid detection of tumor exosomes within 40 minutes, which is easy to operate and greatly improves the practicality of the present invention.

[0094] (4) No instruments required, low cost: The present invention does not require any instruments or equipment, involves a small number of raw materials with low prices (such as glass bottles, capillaries, and hydrogen peroxide solutions), and the reagent synthesis and device assembly methods are simple, and the detection cost is greatly reduced compared to traditional detection technologies.

[0095] (5) Strong application expandability: By replacing the aptamer sequence with the desired sequence, or replacing it with an antigen / antibody, the determination of multiple biomarkers can be achieved. The present invention can not only use PtNP to decompose hydrogen peroxide to produce oxygen, but also use other substances that can catalyze the decomposition of hydrogen peroxide (such as catalase, nanozyme) to replace PtNP for detection, which has strong application expandability.

[0096] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A biomarker detection system based on gas generation, characterized in that: include: Magnetic bead-aptamer complex, used to specifically identify and capture target proteins in samples; Platinum nanoparticle-aptamer complex, used to catalyze the decomposition of hydrogen peroxide to produce oxygen, connected to the sandwich complex formed by the magnetic bead-aptamer complex; A closed container, the interior of which is used to contain the sandwich compound and the hydrogen peroxide solution, and forms a closed space with the external atmosphere through the container cover; a capillary tube connected to the closed container and used to measure the change in length of the liquid column caused by the generation of oxygen; A scale mark, matched with the capillary tube, for naked eye reading; When the airtight container is sealed, the platinum nanoparticles on the sandwich composite catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the gas pressure in the container to increase; The pressure difference between the inside and outside of the container presses the liquid into the capillary, and the length of the liquid column in the capillary is positively correlated with the concentration of the target protein to be detected in the sample; Therefore, by reading the length of the liquid column in the capillary with the naked eye, rapid quantitative detection of the target protein concentration can be achieved.

2. The gas generation-based biomarker detection system according to claim 1, characterized in that: The magnetic bead-nucleic acid aptamer complex specifically comprises: Magnetic beads whose surface is modified with streptavidin; The CD63 nucleic acid aptamer is modified with biotin at its end and connected to the magnetic beads through the binding of biotin and streptavidin.

3. The gas generation-based biomarker detection system according to claim 1, characterized in that: The platinum nanoparticle-nucleic acid aptamer complex specifically comprises: Platinum nanoparticles; The MUC1 nucleic acid aptamer is modified with a thiol group at its end and is connected to the surface of the platinum nanoparticles through the formation of a chemical covalent bond between the thiol group and the platinum atom.

4. The gas generation-based biomarker detection system according to claim 1, characterized in that: The formation process of the sandwich complex specifically includes: mixing the sample to be tested with the magnetic bead-CD63 nucleic acid aptamer, reacting at an appropriate temperature to form a magnetic bead-CD63 nucleic acid aptamer-exosome complex; removing unreacted substances by magnetic separation and washing; adding platinum nanoparticles-MUC1 nucleic acid aptamer to form a magnetic bead-CD63 nucleic acid aptamer-exosome-MUC1 nucleic acid aptamer-platinum nanoparticle sandwich complex.

5. The biomarker detection system based on gas generation according to any one of claims 1 to 4, characterized in that: The specific configuration of the sealed container and the capillary tube includes: using a glass bottle as the sealed container; a rubber plug cooperates with the glass bottle to form a sealed space; and a glass capillary tube is vertically inserted into the rubber plug and communicated with the interior of the glass bottle.

6. The biomarker detection system based on gas generation according to any one of claims 1 to 5, characterized in that: The bottom of the capillary tube is higher than the liquid level in the container, so as to discharge part of the air in the container and keep the air pressure inside and outside the container balanced.

7. The biomarker detection system based on gas generation according to any one of claims 1 to 6, characterized in that: The inner diameter of the capillary is 0.5 mm, and the length is 120 mm. The lower end of the capillary slightly protrudes from the rubber plug by 1 mm.

8. The biomarker detection system based on gas generation according to any one of claims 1 to 7, characterized in that: After reacting for a certain period of time at an appropriate temperature, the platinum nanoparticles catalyze the decomposition of hydrogen peroxide to produce oxygen, causing the gas pressure in the glass bottle to increase, thereby pressing the colored liquid inside into the capillary.

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