CN111208297A - Method for detecting exosome GPC1 protein by using microfluidic chip and application of exosome GPC1 protein in early diagnosis of pancreatic cancer - Google Patents
Method for detecting exosome GPC1 protein by using microfluidic chip and application of exosome GPC1 protein in early diagnosis of pancreatic cancer Download PDFInfo
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- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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Abstract
The invention provides a kit for detecting exosome GPC1 protein by a microfluidic chip, which comprises the microfluidic chip, wherein the microfluidic chip comprises a base layer and a top layer positioned on the base layer, and the upper layer and the lower layer are communicated; the basal layer and the top layer are both provided with a micro-flow channel and a hole, the micro-flow channel is communicated with the hole, and the bottom layer and the top layer are communicated through a mixing flow channel; the basal layer and the top layer are respectively provided with a sample adding hole, a reaction hole, a detection hole, a quality control hole and a waste liquid discharge hole; the reaction hole of the basal layer is coated with a monoclonal antibody of galactose coacervate-1 or an IgG antibody of GPC 1; spraying polyethylene glycol (PEG) 6000 polymer on the sampling holes and the reaction holes of the substrate layer; the reaction hole of the base layer is immobilized with capture nano magnetic beads coupled with quantum dot labeled exosome anti-GPC 1 specific antibody. By combining with the quantum dot fluorescence immunoassay system detection technology of the matched equipment, the method has the advantages of high sensitivity, small required sample amount, low cost of the equipment used for detection, high flux, high specificity and high accuracy.
Description
Technical Field
The invention belongs to the field of medical biological detection technology and in-vitro diagnostic reagents, and particularly relates to a method for detecting exosome GPC1 protein by using a microfluidic chip and application of the method in early diagnosis of pancreatic cancer.
Background
Pancreatic cancer is called "cancer king" and has a mortality rate of about 90%, which is one of the most common malignant tumors in the world. Pancreatic cancer has no specific symptoms, has occult onset and rapid progression, is difficult to diagnose early and easy to miss diagnosis and misdiagnose, and usually tumor is transferred to other organs when being diagnosed, so that the success rate of operation and the survival rate of 5 years after operation are both low and are only 3 percent. The research on pancreatic cancer in both China and abroad is significantly delayed from that of other gastrointestinal cancers. According to the data published by the national cancer center of China, the incidence and mortality of pancreatic cancer in China have been increasing continuously in nearly 10 years; the results of ' Chinese cancer statistical data in 2018 ' show that the number of new cases of pancreatic cancer in 2018 ' in China is 6.74/10 ten thousand, wherein 7.45/10 ten thousand of male patients and 3.47/10 ten thousand of pancreatic cancer death patients comprise 6.64/10 ten thousand of male patients and 5.18/10 ten thousand of female patients. Pancreatic cancer lacks specific tumor markers, and although the serum CA19-9 level of most pancreatic cancer patients is obviously increased, the pancreatic cancer patients can only be used for monitoring the disease condition and reflecting prognosis, and because the specificity is poor, especially the differential diagnosis of the pancreatic cancer patients and biliary diseases is difficult, and the pancreatic cancer patients can not be diagnosed early by using CA19-9 alone because the pancreatic cancer patients are normal in early stage. Early diagnosis and large-scale population screening of pancreatic cancer are difficult to achieve if only traditional imaging means are relied on. Therefore, the research on pancreatic cancer is currently the focus and the hot spot of research of scholars at home and abroad, and a sensitive, specific and noninvasive method for early diagnosis of pancreatic cancer is urgently sought, and a detection technology capable of early diagnosis of pancreatic cancer and prognosis monitoring is urgently needed.
Exosomes are involved in regulating various diseases such as tumors and become a new research hotspot. Exosomes (exosomes) are present in various human body fluids, including blood, urine, saliva, milk, etc., and contain cell-specific proteins, lipids and nucleic acids that can be used as markers for disease diagnosis. Exosomes are important vesicles in human bodies, have the diameter of about 30-200nm and the density of 1.13-1.21g/ml, have a cup-shaped form and a double-layer membrane structure, and have more and more attention on biological functions. It has been found that tumor-derived exosomes promote tumor growth, invasion and metastasis and suppress immune responses by transferring their genetic information to recipient cells. In view of its function in tumor progression, exosomes are considered to be the most promising biomarkers for early clinical detection of human malignancies. Tumor-derived exosomes are a heterogeneous population and produce unique tumor microenvironments. Tumors are formed by complexes of tumor cells with different genes and phenotypes, such as stem cells, stromal cells, fibroblasts, immune cells and the like, exosomes secreted by all the cells form a special microenvironment of the tumor cells, and form cell-to-cell communication, promote tumor angiogenesis and tumor metastasis, directly act on the tumor cells and other pathways, and influence the tumor progression.
Glypican-1 (Glypican-1, GPC1 for short) is a specific marker of pancreatic tumor exosomes, and GPC1 is found to be expressed in a large amount in pancreatic cancer cell exosomes, and the content of the exosome GPC1 is in direct proportion to the size of tumors. The detection of the exosome GPC1 protein can be used for clinical cancer diagnosis and treatment monitoring, and has important significance. 2015 from the U.S. M.D. Anderson cancer center investigator, analysis of large numbers of samples of pancreatic cancer patient sera revealed a significant increase in the proportion of GPC 1-positive exosomes in the pancreatic cancer patient sera compared to normal. Further research shows that the GPC1 positive exosome is significantly more abundant in serum of patients with early pancreatic cancer than normal population, and can diagnose early and late pancreatic cancer with 100% accuracy and sensitivity, and the finding suggests that the GPC1 positive exosome can be used as a new detection method for early diagnosis of pancreatic cancer.
The CN 105974122A invention discloses a method for detecting exosome GPC1 protein, which detects pancreatic cancer exosome GPC1 by combining two immunomagnetic beads of exosome specific antibodies, namely an anti-CD 9 antibody, an anti-CD 63 antibody, an anti-CD 81 antibody and an anti-Flotillin-1 antibody, and an electrochemical sensor technology, and extracts exosome by adopting magnetic bead immunocapture. However, the method is low in efficiency, is not suitable for obtaining exosomes from a large number of samples, interference proteins exist in the obtained exosomes due to non-specific adsorption of a matrix, magnetic beads and antibodies are expensive, the storage conditions are harsh, the biological activity of the exosomes is easily influenced by the pH value and the salt concentration, downstream experiments are not facilitated, and the method is difficult to popularize.
The invention patent CN 103344464A discloses a microfluidic lectin chip for glycosyl separation and a preparation method thereof. The invention mainly uses regenerated bacterial cellulose as chip filler, the lectin fixation step is simple and convenient, the spectrum monitoring is convenient, and the glycosyl is used for separating glycoprotein of microorganism. This invention is not applicable to the extraction of exosomes and the detection of tumor markers on the surface of exosomes.
The invention patent of CN 103018437A discloses a quantum dot micro-fluidic chip used for glycosyl expression analysis on the surface of a single-cell horizontal membrane. The invention is also not applicable to the extraction of exosomes and the detection of tumor markers on the surface of exosomes.
Currently, differential centrifugation is considered the gold standard for exosome separation, but has some drawbacks. The method is time-consuming, labor-consuming, highly dependent on manpower, low in recovery rate, prone to damage to the vesicle of the exosome due to repeated centrifugation operation, thereby reducing the quality of the exosome, prone to aggregation and blocking to cause pollution, and unfavorable for downstream analysis. The current methods of exosome extraction and enrichment used are limited because each cell secretes at least 10 different types of nanovesicles, which are difficult or impossible to distinguish based on similar size, density and surface markers. Therefore, at present, scholars at home and abroad actively explore an experimental method capable of quickly and efficiently separating exosomes for early diagnosis of pancreatic cancer.
Galectin is closely related to malignant tumor, and participates in the processes of tumor occurrence, development, invasion, metastasis, immune escape, drug resistance and the like, and becomes a hot spot for the research in the treatment of tumors and a possible new target for prevention and treatment.
Galectin (Galectin) is a glycoprotein, also known as tumor-associated protein. Glycoproteins are molecules consisting of proteins covalently linked to oligosaccharide chains by glycosylation. Glycosylation is a common post-translational modification of proteins and is classified into 4 types: n-glycosylation, O-glycosylation, glycosylphosphatidylinositol (GP 1) anchored cells and C-glycosylation. Since changes in glycosylation levels are closely related to many diseases such as cancer, specifically expressed sugar chains are expected to be novel biomarkers for diagnosing diseases such as cancer, but studies on the changes are currently reported in a few countries. Galectin can interact with glycosylated extracellular matrix proteins and membrane proteins to regulate cell adhesion; galectin can also be combined with cell surface glycosylated CD98 to up-regulate the expression of integrin so as to promote the combination of cells and matrixes;
galectin can also interact with T antigen on the surface of tumor cells, mediate the adhesion of the tumor cells and endothelial cells, and participate in glycosyl-mediated homotypic and heterotypic adhesion of tumor metastasis cells. It is this specific glycosylation that suggests that it can be used to enrich for tumor exosomes.
Although the particle-shaped exosome has a smaller particle size than a cell, a large number of glycoproteins, glycolipids and sugar chains exist on the surface of the particle-shaped exosome, the outermost layer of the particle-shaped exosome is covered with the sugar chains like the cell, the sugar chains can be used as appropriate targets in exosome marker development, a tumor marker applied to clinical application can recognize a plurality of different sugar chain structures, and if the change of the sugar chains on a disease-specific exosome can be captured, the exosome can be applied to biomarker development.
The invention patent of CN 109324186A discloses an Exo-CD82+ (exosome CD82 protein expression level) combined with an Exo-GPC1+ (exosome GPC1 protein expression level) and a CA19-9 detection kit, which is used for pancreatic cancer diagnosis and/or curative effect monitoring. The exosome GPC1 protein and exosome CD82 protein are marked by an indirect immunofluorescence method, and the Exo-GPC1+ (exosome GPC1 protein expression level) and Exo-CD82+ (exosome CD82 protein expression level) are measured by a flow cytometry method. The method is complex to operate, time-consuming, labor-consuming and high in detection cost, and is limited in clinical application. At present, Western Western protein blotting, ELISA, mass spectrometry and flow cytometry methods are also adopted as exosome GPC1 protein detection methods, and the nucleic acid analysis technology mainly comprises second-generation sequencing, gene chips, real-time fluorescence quantitative PCR and the like. These traditional protein and nucleic acid analysis techniques are all deficient in clinical application, and are difficult to popularize and apply clinically. At present, a high-throughput detection platform with strong specificity, high sensitivity, accuracy and rapidness is still lacking, so that the research of a detection technology for rapidly determining pancreatic tumor markers with high sensitivity, high specificity, low price and simple preparation is a hot spot and a key point of the current domestic and foreign research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for detecting exosome GPC1 protein by using a microfluidic chip, and a quantum dot fluorescence immunoassay system and a detection method which are matched with the kit.
The kit comprises a microfluidic chip, wherein the microfluidic chip comprises a substrate layer and a top layer positioned on the substrate layer, and the upper layer and the lower layer are communicated; the basal layer and the top layer are both provided with a micro-flow channel and a hole, the micro-flow channel is communicated with the hole, and the bottom layer and the top layer are communicated through a mixing flow channel; the basal layer and the top layer are respectively provided with a sample adding hole, a reaction hole, a detection hole, a quality control hole and a waste liquid discharge hole; the reaction hole of the substrate layer is coated with a monoclonal antibody of galactose coacervate-1 or an IgG antibody of GPC 1; spraying polyethylene glycol (PEG) 6000 polymer on the sample adding holes and the reaction holes of the substrate layer, wherein each hole is sprayed with 10 mu l of the polyethylene glycol (PEG) 6000 polymer; the reaction hole of the substrate layer is immobilized with capture nanometer magnetic beads, and the magnetic beads are coupled with quantum dot labeled exosome anti-GPC 1 specific antibody.
The monoclonal antibody of the galactose condensate-1 is coated on the basal layer of the microfluidic chip, and the exosome in the serum is effectively captured through the immunoaffinity of the galactose condensate-1 antigen expressed on the surface of the serum exosome and the monoclonal antibody of the galactose condensate-1 coated on the basal layer. Meanwhile, the sample adding holes and the reaction holes of the substrate layer are sprayed with polyethylene glycol (PEG) 6000 polymer, 10 mu l of polyethylene glycol (PEG 6000) polymer is sprayed on each hole, and the amphiphilic copolymer containing polyethylene glycol is adsorbed and trapped on the surface of the medical high polymer material, so that the biocompatibility of the high polymer material contacted with serum can be improved. Polyethylene glycol (PEG) can be combined with hydrophobic protein and lipid molecules for coprecipitation, and the precipitation principle is applied to extract exosome. Since the exosome is a nano-membrane vesicle, the lipid bilayer of the exosome has certain hydrophobic characteristics, and the exosome can be precipitated by combining with hydrophobic protein and lipid molecules.
The substrate layer is also added with ethylene glycol monomer polymerization which is a water-soluble non-ionic polymer with strong hydrophilicity and can dehydrate biological macromolecules to precipitate and can form compound precipitation with the biological macromolecules through hydrogen bond interaction.
Preferably, the substrate layer has 2 reaction wells, the first reaction well is coated with a monoclonal antibody to galactose condensate-1, and the second reaction well is coated with an IgG antibody to GPC 1.
Preferably, the diameter of each hole is 1.5-5.5 mm, and the depth of each hole is 80-1000 microns; the length of the micro-channel is 6-18 mm, the width of the micro-channel is 80-800 micrometers, and the depth of the micro-channel is 100-2200 micrometers.
Preferably, the driving force of the microfluidic chip is a syringe pump.
Preferably, the material of the microfluidic chip comprises a high molecular polymer of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, Polycarbonate (PC) or cyclic olefin copolymer resin (COC).
Preferably, the micro-fluidic chip is made of a silicon nano-structure matrix of dimethyl siloxane alkanol (PDMS) which can simulate the biological characteristics and functions of extracellular matrix and effectively adhere to tumor exosomes.
The invention also provides a quantum dot fluorescence immunoassay system matched with the kit for detecting the exosome GPC1 protein by using the microfluidic chip as claimed in claim 1, which comprises a chip driving platform and a photoelectric detection system, wherein the photoelectric detection system comprises an LED light source, a photoelectric converter, a photoelectric sensor, a signal processing system, a computer analysis system and main detection parts of an injection pump control system.
Preferably, the excitation wavelength of the quantum dot fluorescence is within the range of 400-550, and the emission wavelength is within the range of 550-650.
The third objective of the invention is to provide a method for detecting exosome GPC1 protein by using the microfluidic chip fluorescence immunoassay rapid detection kit as claimed in claim 1, which comprises the following steps:
(1) adding a substance solution to be detected into a top layer sample adding hole of the microfluidic chip, driving the solution to be detected to reach a reaction hole through injection, and capturing and enriching exosomes in the substance to be detected by a monoclonal antibody of galactose condensate-1 in a basal layer reaction hole;
(2) under the drive of fluid, continuously conveying all the solution in the reaction hole to the next reaction hole, adding immunomagnetic beads of quantum dot fluorescence labeling antibodies into the reaction hole at the top layer, and carrying out immunoreaction with anti-GPC 1 IgG antibodies coated by the reaction hole at the bottom layer to form an immune compound of exosome GPC1 antigen-quantum dot fluorescence labeling anti-GPC 1 antibody-magnetic bead microspheres;
(3) under the action of an external magnetic field, the magnetic compound is adsorbed at the bottom of the reaction detection tank, the solution without the magnetic microspheres is conveyed to a waste liquid pool under the centrifugal drive, and the magnetic fluorescent compound is left in the detection hole.
(4) And (3) detecting by using a fluorescence detector, irradiating the magnetic fluorescent microsphere compound in the reaction detection tank with light with a certain wavelength, and detecting the luminous intensity of the fluorescent microsphere, wherein the fluorescent intensity is correlated with the concentration of the substance to be detected.
Preferably, the luminescence wavelength range of the fluorescence detection microsphere is 400-550, and the emission wavelength range is 550-650.
Preferably, the quantum dots are water-soluble activated quantum dots with carboxyl groups, and the carboxyl groups on the surfaces of the CdSe/ZnS quantum dots are activated by adopting N-hydroxy thiosuccinimide.
Preferably, the immunomagnetic beads are nanometer magnetic beads with epoxy groups, and the diameters of the nanometer magnetic beads are 2.0-6.0 μm. The epoxidized magnetic beads can be covalently bonded with amino groups or thiol groups on the surfaces of proteins and other biological ligands, and are particularly suitable for immobilizing bioactive macromolecules by covalently coupling biological amino ligands such as proteins, such as antibodies, streptavidin and the like.
The invention has the beneficial effects that: the kit for detecting the exosome GPC1 protein by the microfluidic chip is combined with a quantum dot fluorescence immunoassay system detection technology of matched equipment thereof, and combines the advantages of multiple aspects, the method has high sensitivity and small required sample amount, simultaneously has low cost of the used equipment, can detect the exosome specific to the pancreatic tumor expressed by the GPC1 protein with high flux and high specificity, and has important clinical significance for early diagnosis and treatment detection of pancreatic cancer.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip according to the present invention;
FIG. 2 is a schematic view of a quantum dot fluorescence immunoassay system of the kit of the present invention;
FIG. 3 is a schematic diagram of the mechanism of detecting the exosome GPC1 protein according to the present invention;
FIG. 4 serum exosome characteristics and quantum-tagged exosome cancer cells (A is a serum exosome; B is a quantum-tagged exosome cancer cell);
FIG. 5 is a graph of the fluorescence signals of the method of the invention for detecting the expression of the exosome GPC1 protein and carbohydrate antigen CA-199;
FIG. 6 is a schematic diagram of the results of comparison between 45 cases of pancreatic cancer exosome GPC1 protein detected by the microfluidic chip and the supporting equipment of the present invention and the results of ELISA detection.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1 preparation of exosome GPC1 protein detection kit reagents
1. Preparation of microfluidic chip
The microfluidic chip comprises a substrate layer and a top layer positioned on the substrate layer, and the upper layer and the lower layer are communicated; the basal layer and the top layer are both provided with a micro-flow channel and a hole, the micro-flow channel is communicated with the hole, and the bottom layer and the top layer are communicated through a mixing flow channel; the top layer is provided with a sample adding hole (1), reaction holes (2, 3), a detection hole (4), a negative quality control hole (5), a positive quality control hole (6), a standard substance hole (7), a detection hole (8) and a waste liquid discharge hole (9); the basal layer is provided with a buffer liquid inlet hole (10), a sample adding hole (11), reaction holes (12, 13), a detection hole (14), a negative quality control hole (15), a positive quality control hole (16), a standard substance hole (17) and a detection hole (18); the substrate layer hole (12) is coated with the galectin-1 antibody, and the substrate layer hole (13) is coated with the IgG antibody of the anti-GPC 1; the sample adding holes (11) and the reaction holes (12, 13) of the substrate layer are sprayed with polyethylene glycol PEG6000 polymer, and each hole is sprayed with 10 mu l of polyethylene glycol PEG6000 polymer.
The diameter of each microfluidic chip hole in the embodiment is 1.5 mm, and the depth is 80 microns; the microchannel has a length of 6 mm, a width of 80 microns and a depth of 100 microns. In this embodiment, the material of the microfluidic chip is a high molecular polymer, namely, Polydimethylsiloxane (PDMS).
2. Preparation of human galectin 1 antibody
The human galectin 1 antibody product of the present invention is available from ProSpec of Israel. White lyophilized powder supplied by the company for filtration sterilization; the lyophilized powder was lyophilized from a 10mM sodium phosphate buffer (pH-7.5) containing 1mg/ml human galectin 1 antibody, sterile 18M omega-cm water was added to make up the working stock to 100. mu.g/ml, and further diluted during the course of the particular experiment according to the experimental requirements. The experimental galectin-1 antibody concentration was 0.5-2.5 mg/ml, the buffer was also 0.01m TBS (pH7.4), 1% bovine serum albumin, 0.03% proclin300 and 50% glycerol.
Mu.l of the obtained human galectin 1 antibody was added to the well (12), and dried and solidified at 37 ℃.
3. Preparation of anti-GPC 1 monoclonal antibody
Mu.l of the obtained anti-GPC 1 monoclonal antibody was added to well (13), and dried and solidified at 37 ℃.
4. And spraying medical high polymer material of polyethylene glycol (PEG) 6000 on the sampling holes and the reaction holes of the substrate layer.
5. Preparation of quantum dot labeled anti-GPC 1 antibody and nano magnetic bead compound
Adding a proper amount of water-soluble activated quantum dots, 10 mu g of EDC, 15 mu g of NHS solution and 10-30 mu g of anti-GPC 1 monoclonal antibody solution into a phosphate buffer solution, uniformly mixing, reacting at room temperature for 4 hours, and adding 1mg of glycine for blocking. Separating and purifying by using a chromatographic column or a chromatographic column to obtain the quantum dot labeled GPC1 antibody. To the phosphate buffer was added 1mg of magnetic particles (2 μm in size), 10 μ g of EDC and 15 μ g of NHS solution and quantum dot-GPC 1 monoclonal antibody to form an immunocomplex, which was blocked by the addition of 2mg of glycine. And preparing 10-30 mu g of IgG monoclonal antibody (different from the quantum dot marked antibody) solution of anti-GPC 1, uniformly mixing, reacting at room temperature for 2h, and adding 2mg of glycine for blocking.
The concentration of the activated quantum dots is 1-10 mu M; the concentration of the GPC1 antibody is 10-100 mu g/ml; the concentration of the BSA solution is 20-200 mg/mL, and the mass ratio of the activated quantum dots to the GPC1 antibody is (1:2) - (1: 10).
When the activated quantum dots are coupled with a GPC1 antibody, the pH value of the solution is 9-11, the concentration of a1 ethyl-3-carbodiimide hydrochloride solution is 9.38% (w/v), the concentration of an N-hydroxysuccinimide solution is 9.38% (w/v), the mass ratio of the activated quantum dots to the added ethyl-3-carbodiimide hydrochloride is 5-10, and the mass ratio of the activated quantum dots to the added N-hydroxysuccinimide is 5-10.
Example 2
The embodiment provides a quantum dot fluorescence immunoassay system matched with the kit for detecting the exosome GPC1 protein by using the microfluidic chip, and the system comprises a chip driving platform and a photoelectric detection system; the photoelectric detection system comprises an LED light source, a photoelectric converter, a photoelectric sensor, a signal processing system, a computer analysis system and main detection components of an injection pump control system.
The quantum dot fluorescence of the analysis system requires that the excitation wavelength range is 400-550, and the emission wavelength range is 550-650.
Example 3
The embodiment provides a method for detecting exosome GPC1 protein by using a microfluidic chip fluorescence immunoassay rapid detection kit, which comprises the following steps:
(1) adding 50 mu l of centrifuged serum into the top layer sample adding hole (1) which is communicated with the bottom layer (11) hole; driving a serum solution through a buffer solution in the hole (10) to the reaction hole (2), wherein the hole (2) is communicated with the hole (12), the serum solution is subjected to immunoreaction with the galectin-1 antibody in the hole (12), the antigen on the surface of the exosome is combined with the galectin-1 antibody through glycosylation, and the exosome in the serum is captured and enriched in the hole (12); the method comprises the following specific steps:
the serum sample and the galectin-1 antibody are incubated in the hole (12) for 1 hour, then the water-soluble quantum dot is added for incubation for 1 hour, and the characteristics of exosomes and quantum-labeled pancreatic cancer cell exosomes are seen under an electron microscope and are shown in detail in fig. 4A and 4B.
(2) Driven by buffer solution, the solution reaches a reaction well (3) through a microchannel, the well (3) is communicated with a well (13), an IgG antibody resisting GPC1 is coated in the well (13), magnetic beads coupled with quantum dot fluorescent-labeled anti-GPC 1 antibody are added into the well (3), and immunoreaction is carried out by a competition method to form an immunomagnetic complex (an immunomagnetic complex of an exosome GPC1 antigen-quantum dot fluorescent-labeled anti-GPC 1 antibody-magnetic beads) formed by GPC1 antigen and quantum dot fluorescent-labeled anti-GPC 1 antibody on the surface of an exosome, as shown in figure 3;
(3) under the action of an external magnetic field, the magnetic compound is adsorbed at the bottom of the detection hole (4), the solution without the magnetic microspheres is conveyed to a waste liquid pool under the drive of a buffer solution, and the magnetic fluorescent compound is left in the detection hole (4);
(4) the detection hole (4) is connected with an LED, a fluorescence detector is used for detecting, an immune magnetic compound of an exosome GPC1 antigen-quantum dot fluorescence labeling anti-GPC 1 antibody in a serum sample enters the detection area of the hole (4) through a micro-channel under the drive of a buffer solution, the fluorescence anti-GPC 1 antibody of the immune compound is captured, and the intensity of a fluorescence signal emitted by the compound reflects the number of captured GPC1, namely the number of exosomes;
(5) in the detection area, the excitation spectrum (350-450) and the emission spectrum (550-650) of the quantum dots control the intensity of fluorescence through an LED fluorescence light-emitting system, the fluorescence light-emitting system amplifies signals, and a photoelectric converter converts fluorescence into digital signals to realize quantitative detection of the analyte.
In the embodiment, the flow rate of the buffer solution is 5-15 mul/min, and the buffer solution is phosphate buffered saline solution.
Example 4
45 serum exosome GPC1 proteins of patients pathologically diagnosed as pancreatic cancer are detected by using the microfluidic chip kit, the detection result of 44 serum exosome GPC1 is increased, only 1 serum exosome GPC1 is in the range of critical values, the detection rate reaches 97.78%, the sensitivity and the specificity are 97% and 100% respectively, and detailed data are shown in FIG. 5. While 45 patients pathologically diagnosed as pancreatic cancer were tested by ELIAS for serum GPC1 protein, only 26 serum exosome GPC1 had higher results, and 19 had normal values, with a detection rate of 57.78% and sensitivity and specificity of 56% and 63%, respectively, as shown in FIG. 6. Therefore, the detection kit has more accurate detection result.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A kit for detecting exosome GPC1 protein by a microfluidic chip is characterized by comprising the microfluidic chip, wherein the microfluidic chip comprises a basal layer and a top layer positioned on the basal layer, and the upper layer and the lower layer are communicated; the basal layer and the top layer are both provided with a micro-flow channel and a hole, the micro-flow channel is communicated with the hole, and the bottom layer and the top layer are communicated through a mixing flow channel; the basal layer and the top layer are respectively provided with a sample adding hole, a reaction hole, a detection hole, a quality control hole and a waste liquid discharge hole; the reaction hole of the substrate layer is coated with a monoclonal antibody of galactose coacervate-1 or an IgG antibody of GPC 1; spraying polyethylene glycol (PEG) 6000 polymer on the sampling holes (11) and the reaction holes (12 and 13) of the substrate layer, wherein each hole is sprayed with 10 mu l of polyethylene glycol (PEG) 6000 polymer; the reaction hole of the substrate layer is immobilized with capture nanometer magnetic beads, and the magnetic beads are coupled with quantum dot labeled exosome anti-GPC 1 specific antibody.
2. The kit for detecting the exosome GPC1 protein by the microfluidic chip according to claim 1, wherein the substrate layer has 2 reaction wells, a first reaction well is coated with the monoclonal antibody of galactose condensate-1, and a second reaction well is coated with the IgG antibody against GPC 1.
3. The kit for detecting exosome GPC1 protein according to claim 1, wherein the diameter of each of the holes is 1.5-5.5 mm, and the depth of each of the holes is 80-1000 microns; the length of the micro-channel is 6-18 mm, the width of the micro-channel is 80-800 micrometers, and the depth of the micro-channel is 100-2200 micrometers.
4. The kit for detecting an exosome GPC1 protein according to claim 1, wherein a driving force of the microfluidic chip is a syringe pump.
5. The kit for detecting exosome GPC1 protein according to claim 1, wherein the material of the microfluidic chip comprises a high molecular polymer of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, Polycarbonate (PC) or cyclic olefin copolymer resin (COC).
6. The kit for detecting an exosome GPC1 protein according to claim 1, wherein the material of the microfluidic chip is dimethiconol (PDMS).
7. A quantum dot fluorescence immunoassay system used in combination with the kit for detecting exosome GPC1 protein according to claim 1, comprising a chip driving platform and a photoelectric detection system; the photoelectric detection system comprises an LED light source, a photoelectric converter, a photoelectric sensor, a signal processing system, a computer analysis system and main detection components of an injection pump control system.
8. The quantum dot fluorescence immunoassay system of claim 6, wherein the quantum dot fluorescence requires an excitation wavelength in the range of 400 to 550 and an emission wavelength in the range of 550 to 650.
9. A method for detecting exosome GPC1 protein by using the microfluidic chip fluorescence immunoassay rapid detection kit of claim 1, which is characterized by comprising the following steps:
(1) adding a substance solution to be detected into a top layer sample adding hole of the microfluidic chip, driving the solution to be detected to reach a reaction hole through injection, and capturing and enriching exosomes in the substance to be detected by a monoclonal antibody of galactose condensate-1 in a basal layer reaction hole;
(2) under the drive of fluid, continuously conveying all the solution in the reaction hole to the next reaction hole, adding immunomagnetic beads of quantum dot fluorescence labeling antibodies into the reaction hole at the top layer, and carrying out immunoreaction with anti-GPC 1 IgG antibodies coated by the reaction hole at the bottom layer to form an immune compound of exosome GPC1 antigen-quantum dot fluorescence labeling anti-GPC 1 antibody-magnetic bead microspheres;
(3) under the action of an external magnetic field, the magnetic compound is adsorbed at the bottom of the reaction detection tank, the solution without the magnetic microspheres is conveyed to a waste liquid pool under centrifugal drive, and the magnetic fluorescent compound is left in the detection hole;
(4) and (3) detecting by using a fluorescence detector, irradiating the magnetic fluorescent microsphere compound in the reaction detection tank with light with a certain wavelength, and detecting the luminous intensity of the fluorescent microsphere, wherein the fluorescent intensity is correlated with the concentration of the substance to be detected.
10. The method for detecting exosome GPC1 protein according to claim 1, wherein the emission wavelength range of the fluorescent detection microspheres is 400 to 550, and the emission wavelength range is 550 to 650; the quantum dots used in the immunomagnetic beads of the quantum dot fluorescence labeling antibody are water-soluble activated quantum dots with carboxyl; the immunomagnetic beads are nanometer magnetic beads with epoxy groups, and the diameters of the nanometer magnetic beads are 2.0-6.0 mu m.
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