A detection system for early tumor screening and its application in preparing tumor antigen detection products

Abstract

The present invention provides a detection system for early tumor screening and its application in the preparation of tumor antigen detection products, belonging to the technical field of early tumor screening. The present invention provides the application of a micro-pot array chip combined with micron-sized magnetic beads in the preparation of an ELISA detection product for early tumor screening. The present invention uses micron-sized hydrophilic micro-pots as reaction carriers for ELISA detection, and uses micron-sized magnetic beads to encapsulate capture antibodies used to capture tumor antigens. By limiting the fluorescent groups produced by a single enzyme-catalyzed substrate to an extremely small volume, a high local concentration of fluorescent product molecules is ensured to generate a sufficiently strong optical signal to be detected, thereby realizing the detection of extremely low concentrations of tumor antigens. At the same time, the present invention can reduce the amount of sample collection, such as the amount of blood collection, and the amount of serum required for detection can be achieved by drawing blood from the fingertips, greatly reducing the degree of trauma to the patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of early tumor screening, and in particular relates to a detection system for early tumor screening and its application in preparing tumor antigen detection products. Background Art

[0002] Malignant tumors are one of the major diseases that threaten human health. Tumor screening refers to a series of targeted medical examinations conducted under healthy conditions, which helps detect tumors that are already present in the body at an early or treatable stage. The methods currently used for early tumor screening generally include tumor marker examinations, imaging examinations, endoscopic examinations, pathological examinations, etc. Among them, tumor marker examinations generally include serum carcinoembryonic antigen, alpha-fetoprotein, prostate-specific antigen, chorionic gonadotropin, etc. However, at present, the above-mentioned methods for tumor marker examinations still have certain limitations, such as large blood consumption and low detection sensitivity. Summary of the invention

[0003] The purpose of the present invention is to provide a detection system for early screening of tumors and its application in the preparation of tumor antigen detection products. The detection system of the present invention has the advantages of small blood consumption and high detection sensitivity.

[0004] The present invention provides an application of a micro-pot array chip combined with micron-sized magnetic beads in the preparation of an ELISA detection product for early tumor screening. The micro-pot array chip uses a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the caliber of each micro-pot is 1.2 to 1.8 times the diameter of the magnetic beads; the inner wall of the micro-pot is made of a hydrophilic material; the size of the micro-pot is in the micron level; the micro-pot serves as a reaction carrier for ELISA detection; the micron-sized magnetic beads are coated with capture antibodies; and the capture antibodies are used to capture tumor antigens.

[0005] The present invention also provides a detection system for early screening of tumors, comprising an antigen capture system, an antibody-coupled enzymatic reaction system and a fluorescence detection system; the antigen capture system comprises micron-sized magnetic beads coated with capture antibodies; the capture antibodies are used to capture tumor antigens; the antibody-coupled enzymatic reaction system comprises a micro-pot array chip and ELISA detection reagents; the micro-pot array chip uses a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the caliber of each micro-pot is 1.2 to 1.8 times the diameter of the magnetic beads; the inner wall of the micro-pot is made of a hydrophilic material; the size of the micro-pot is in the micron level; the ELISA detection reagents comprise: a biotin-labeled detection antibody, β-galactosidase coupled with streptavidin, and a fluorescent substrate for β-galactosidase.

[0006] Preferably, each micron-sized magnetic bead is coated with 1×10 4 ~1×10 6The diameter of the micron-sized magnetic beads is 2.7 μm.

[0007] Preferably, the shape of the micro-pot array chip is square; the specification of the micro-pot array chip is 10 mm×10 mm; the micro-pots on the micro-pot array chip are arranged in a matrix; the number of micro-pots on each micro-pot array chip is 1×10 6 indivual.

[0008] Preferably, the opening of each micro-pot is a circular opening; the diameter of the opening of each micro-pot is 4.5 μm; the distance between two adjacent micro-pots is 4.5 μm; and the depth of the micro-plate is 1 μm.

[0009] Preferably, the ratio of the number of micron-sized magnetic beads coated with capture antibodies to the volume of the serum sample to be tested is 2×10 6 ~2×10 8 Piece: 20μl.

[0010] The present invention also provides the use of the detection system described in the above scheme in the preparation of tumor antigen detection products.

[0011] Preferably, the tumor comprises lung cancer.

[0012] Preferably, the tumor antigens include one or more of CYFRA21-1, CEA, NSE, SCC and CA125.

[0013] The present invention also provides a method for detecting tumor antigens for non-diagnostic purposes based on the detection system described in the above scheme, comprising the following steps:

[0014] Mixing the micron-sized magnetic beads coated with the capture antibody and the sample to be tested, performing a first incubation, and obtaining a first incubation product;

[0015] Separating the reacted magnetic beads from the first incubation product, washing them, mixing them with the biotin-labeled detection antibody, and performing a second incubation to obtain a second incubation product;

[0016] The reacted magnetic beads are separated from the second incubation product, washed, mixed with β-galactosidase coupled with streptavidin, and incubated for the third time to obtain a third incubation product;

[0017] The reacted magnetic beads are separated from the third incubation product, and the beads are washed and resuspended to obtain a resuspended solution; the resuspended solution is added to the micro-pot array chip, and after absorbing the excess solution, a fluorescent substrate of β-galactosidase is added to the micro-pot array chip to perform a fluorescent reaction, and then the micro-pot array chip is oil-sealed;

[0018] The fluorescence detection system is used to perform fluorescence detection on the product after the fluorescence reaction, and the number of fluorescence signals is recorded;

[0019] The concentration of the tumor antigen in the sample to be tested is calculated according to the number of the fluorescent signals.

[0020] The present invention provides an application of a micro-pot array chip combined with micron-sized magnetic beads in the preparation of an ELISA detection product for early tumor screening. The micro-pot array chip uses a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the caliber of each micro-pot is 1.2 to 1.8 times the diameter of the micron-sized magnetic beads; the inner wall of the micro-pot is made of a hydrophilic material; the size of the micro-pot is in the micron level; the micro-pot serves as a reaction carrier for ELISA detection; the micron-sized magnetic beads are coated with capture antibodies; and the capture antibodies are used to capture tumor antigens.

[0021] The present invention uses the micron-sized hydrophilic micro-pots set on the micro-pot array chip as the reaction carrier for ELISA detection, and uses micron-sized magnetic beads to encapsulate the capture antibody used to capture tumor antigens. By limiting the fluorescent group generated by a single enzyme-catalyzed substrate to a very small volume, it ensures a high local concentration of the fluorescent product molecules to generate a sufficiently strong optical signal to be detected, thereby realizing the detection of extremely low concentrations of tumor antigens. At the same time, the present invention uses micron-sized hydrophilic micro-pots as the reaction carrier for ELISA detection, which can reduce the amount of sample collection, such as the amount of blood collection. The amount of serum required for detection can be achieved by drawing blood from the fingertips, greatly reducing the degree of trauma to the patient. Streptavidin and enzyme conjugates are used to react with the biotin-labeled detection antibody (secondary antibody) bound to the antigen, thereby overcoming steric hindrance. In addition, with the increase of streptavidin-enzyme conjugates, the signal intensity can be increased, and therefore the measurement sensitivity can also be increased. In summary, the present invention combines the micro-pot array chip with ELISA detection for the first time and uses it for early screening of tumor antigens. Compared with the traditional ELISA detection of tumor antigens, the present invention can realize fingertip blood collection and can be used for the detection of ultra-low concentration antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The figure is a flow chart of the preparation of micro-pot array chip;

[0024] Figure 2 This is an electron microscope photo of the micro-pot array chip;

[0025] Figure 3The key components and operation flow chart of the detection of the present invention; wherein, a represents that the micron-sized magnetic beads are coated with capture antibodies, wherein the microspheres that have captured antigen molecules can be further coupled with biotin-labeled detection antibodies, and then coupled with streptavidin-β-galactosidase, mixed, and a third incubation is performed to obtain a third incubation product; b represents that the magnetic beads (microspheres) are quickly and efficiently separated into microtubs on a microtub chip, c represents an electron microscope photo of the magnetic beads dispersed in the microtubs, and d represents that the magnetic beads that have captured antigen molecules can convert substrates into fluorescent products by linking enzymes to achieve luminescence;

[0026] Figure 4 This is a comparison of the time required to separate magnetic beads (microspheres) using the micro-basin and micro-well methods;

[0027] Figure 5 This is the calibration experiment result diagram. DETAILED DESCRIPTION

[0028] The present invention provides an application of a micro-pot array chip combined with micron-sized magnetic beads in the preparation of an ELISA detection product for early tumor screening. The micro-pot array chip uses a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the caliber of each micro-pot is 1.2 to 1.8 times the diameter of the magnetic beads; the inner wall of the micro-pot is made of a hydrophilic material; the size of the micro-pot is in the micron level; the micro-pot serves as a reaction carrier for ELISA detection; the micron-sized magnetic beads are coated with capture antibodies; and the capture antibodies are used to capture tumor antigens.

[0029] In the present invention, each micron-sized magnetic bead is preferably coated with 1×10 4 ~1×10 6 The diameter of the micron-sized magnetic beads is preferably 2.7 μm.

[0030] In the present invention, the shape of the micro-pot array chip is preferably square; the specification of the micro-pot array chip is preferably 10 mm×10 mm; the micro-pots on the micro-pot array chip are preferably arranged in a matrix; the number of micro-pots on each micro-pot array chip is preferably 1×10 6 The micro-basin array chip is preferably a glass micro-basin array chip; the diameter of the glass micro-basin array chip is.

[0031] In the present invention, the opening of each micro-pot is a circular opening; the diameter of the opening of each micro-pot is 4.5 μm; the distance between two adjacent micro-pots is 4.5 μm; and the depth of the micro-plate is 1 μm.

[0032] In the present invention, the preparation method of the micro-basin array chip is preferably carried out with reference to the prior art, for details, see [Tian F, Li M, Pu D, et al. A Versatile and Environmentally Friendly Microfabrication Process for Producing Micro-Basin Array for Single Cell Analysis [C] / / 2021 IEEE 34th International Conference on Micro ElectroMechanical Systems (MEMS). IEEE, 2021. DOI: 10.1109 / MEMS51782.2021.9375448.]

[0033] In the specific implementation process of the present invention, the preparation method of the micro-basin array chip is:

[0034] The photoresist is spin-coated on a hydrophobic glass substrate; standard ultraviolet lithography and development are performed to produce specific micropore sizes and desired micropore sidewall shapes on the photoresist; a silicon dioxide film is deposited by physical sputtering; and acetone is used for stripping to remove the photoresist, leaving behind a micro-basin composed of silicon dioxide in contact with the hydrophobic glass substrate and silicon dioxide in contact with the micropore sidewalls of the photoresist.

[0035] In the present invention, the height of the micro-basin is determined by the thickness of the photoresist, which can be controlled by selecting a specific type of photoresist and the rotation speed of the spin-coating glue. The preparation method of the micro-basin array chip is to form a unique side wall after the photoresist is developed, which is not a traditional vertical side wall, but a side wall with a curvature. After the silicon dioxide film is sputtered, there is a discontinuity of the film in the undercut area, thereby generating an opening for acetone to penetrate and completely dissolve the photoresist to achieve successful stripping. The micrometer-scale hydrophilic micro-basin array chip is produced in this way. The biggest feature of this method is that it does not require toxic hydrofluoric acid to etch silicon dioxide, and only requires acetone for the stripping process to achieve a three-dimensional micro-basin structure. Since silicon dioxide is hydrophilic and the substrate is a hydrophobicized glass, the difference between hydrophilic and hydrophobic is utilized, and when it contacts the aqueous solution, millions of independent small droplets will be formed in a few seconds to complete the physical separation of the target solution.

[0036] The micro-basin array chip of the present invention can be mass-produced in a low-cost manner, which can reduce the cost of detection equipment and is conducive to clinical promotion.

[0037] The present invention also provides a detection system for early screening of tumors, comprising an antigen capture system, an antibody-coupled enzymatic reaction system and a fluorescence detection system; the antigen capture system comprises micron-sized magnetic beads coated with capture antibodies; the capture antibodies are used to capture tumor antigens; the antibody-coupled enzymatic reaction system comprises a micro-pot array chip and ELISA detection reagents; the micro-pot array chip uses a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the caliber of each micro-pot is 1.2 to 1.8 times the diameter of the magnetic beads; the inner wall of the micro-pot is made of a hydrophilic material; the size of the micro-pot is in the micron order; the ELISA detection reagents comprise: a detection antibody labeled with biotin, β-galactosidase (SβG) coupled with streptoavidin, and a fluorescent substrate for β-galactosidase.

[0038] In the present invention, each micron-sized magnetic bead is preferably coated with 1×10 4 ~1×10 6 capture antibodies, more preferably 2.5×10 4 The diameter of the micron-sized magnetic beads is preferably 2.7 μm. In the present invention, the micron-sized magnetic beads are preferably purchased from Variant Company, and the surface of the micron-sized magnetic beads is modified with acetate (carboxyl end).

[0039] In the present invention, the fluorescent substrate of β-galactosidase is preferably resorufin-β-D-galactopyranoside (RGP).

[0040] In the present invention, the working concentration of the biotin-labeled detection antibody is preferably 1 nM.

[0041] In the present invention, the working concentration of the streptavidin-coupled β-galactosidase is preferably 1 to 50 pM.

[0042] In the present invention, the fluorescence detection system is preferably a fluorescence microscope camera system; the fluorescence microscope camera system preferably includes a mercury lamp, an optical fiber, a microscope and a CCD camera. The present invention has no special restrictions on the fluorescence microscope camera system, and a conventional fluorescence microscope camera system in the art can be used.

[0043] In one embodiment of the present invention, the tumor is lung cancer; the tumor antigens are CYFRA21-1, CEA, NSE, SCC and CA125; in the present invention, the capture antibodies are purchased from Elabscience, wherein the CYFRA21-1 antibody is available in E-KAB-0282, the CEA antibody is available in E-KAB-0016, the NSE antibody is available in E-KAB-0059, the SCC antibody is available in E-KAB-0286, and the CA125 antibody is available in E-KAB-0139.

[0044] In the present invention, the shape of the micro-pot array chip is preferably square; the specification of the micro-pot array chip is preferably 10 mm×10 mm; the micro-pots on the micro-pot array chip are preferably arranged in a matrix; the number of micro-pots on each micro-pot array chip is preferably 1×10 6 indivual.

[0045] In the present invention, the opening of each micro-pot is a circular opening; the diameter of the opening of each micro-pot is 4.5 μm; the distance between two adjacent micro-pots is 4.5 μm; and the depth of the micro-plate is 1 μm.

[0046] The present invention also provides the use of the detection system described in the above scheme in the preparation of tumor antigen detection products.

[0047] In the present invention, the tumor preferably includes lung cancer. In the present invention, the tumor antigen preferably includes one or more of CYFRA21-1 (cytokeratin 19 fragment), CEA (carcinoembryonic antigen), NSE (neuron-specific enolase), SCC (squamous cell carcinoma antigen) and CA125 (carbohydrate antigen 125).

[0048] The detection system of the present invention can be used for tumor antigen detection to achieve early detection and early treatment, thereby improving the cure rate of cancer. The sensitivity of the present invention is greatly improved, and the concentration of disease factors below 1pg / ml can be detected. For certain high-risk groups, early tumor screening can help them get treatment and diagnosis before the disease is discovered, shorten the treatment time, reduce the side effects of treatment, and reduce the cost of treatment. Through early detection and treatment, the expensive treatment costs required for advanced cancer can be avoided.

[0049] The present invention also provides a method for detecting tumor antigens for non-diagnostic purposes based on the detection system described in the above scheme, comprising the following steps:

[0050] Mixing the micron-sized magnetic beads coated with the capture antibody and the sample to be tested, performing a first incubation, and obtaining a first incubation product;

[0051] Separating the reacted magnetic beads from the first incubation product, washing them, mixing them with the biotin-labeled detection antibody, and performing a second incubation to obtain a second incubation product;

[0052] The reacted magnetic beads are separated from the second incubation product, washed, mixed with β-galactosidase coupled with streptavidin, and incubated for the third time to obtain a third incubation product;

[0053] The reacted magnetic beads are separated from the third incubation product, and the beads are washed and resuspended to obtain a resuspended solution; the resuspended solution is added to the micro-pot array chip, and after absorbing the excess solution, a fluorescent substrate of β-galactosidase is added to the micro-pot array chip to perform a fluorescent reaction, and then the micro-pot array chip is oil-sealed;

[0054] The fluorescence detection system is used to perform fluorescence detection on the product after the fluorescence reaction, and the number of fluorescence signals is recorded; and the concentration of the tumor antigen in the sample to be tested is calculated based on the number of fluorescence signals.

[0055] The detection principle of the present invention is: the micron-sized magnetic beads coated with the capture antibody are mixed with the sample to be tested. If there is a disease factor (tumor antigen) in the sample to be tested, the antigen on the surface of the magnetic beads can specifically bind to the disease factor (biomarker). Then the magnetic beads are mixed with the detection antibody labeled with biotin, and only the magnetic beads combined with the disease factor can be combined with the detection antibody labeled with biotin; then the magnetic beads are mixed with the β-galactosidase molecules coupled with streptavidin, biotin and avidin have very good adsorption force, and only the magnetic beads combined with the detection antibody labeled with biotin can be combined with the β-galactosidase coupled with streptavidin; then the magnetic beads are mixed with the fluorescent substrate of β-galactosidase, and only the β-galactosidase combined with streptavidin can react with the fluorescent substrate of β-galactosidase to continuously convert the subsequent substrate into a fluorescent product, and the detection result is obtained by performing fluorescence detection.

[0056] The present invention first mixes the micron-sized magnetic beads coated with the capture antibody with the sample to be tested, and performs a first incubation to obtain a first incubation product. In the present invention, the first incubation time is preferably 2 hours.

[0057] In the present invention, the sample to be tested is preferably a serum sample obtained by centrifugation after fingertip blood collection; the amount of the sample to be tested is preferably 20 to 50 μl; the ratio of the number of micron-sized magnetic beads coated with capture antibodies to the volume of the serum sample to be tested is preferably 2×10 6 ~2×10 8 Piece: 20μl.

[0058] In the present invention, the method for preparing the micron-sized magnetic beads coated with the capture antibody preferably comprises the following steps: immersing the magnetic beads in a capture antibody solution, coating, and obtaining the micron-sized magnetic beads coated with the capture antibody. The present invention controls the number of modifications on the surface of the micron-sized magnetic beads by adjusting the capture antibody concentration.

[0059] After obtaining the first incubation product, the present invention separates the reacted magnetic beads from the first incubation product, washes them, mixes them with the biotin-labeled detection antibody, and performs a second incubation to obtain a second incubation product. In the present invention, the second incubation time is preferably 10 to 60 minutes, more preferably 45 minutes.

[0060] In the present invention, the working concentration of the biotin-labeled detection antibody is preferably 1 nM; the ratio of the concentration of the biotin-labeled detection antibody to the number of micron-sized magnetic beads coated with the capture antibody is preferably 1 nM: 6×10 7 ~6×10 9 indivual.

[0061] After obtaining the second incubation product, the present invention separates the reacted magnetic beads from the second incubation product, washes them, mixes them with β-galactosidase coupled with streptavidin, and performs a third incubation to obtain a third incubation product. In the present invention, the third incubation time is preferably 30 minutes.

[0062] In the present invention, the working concentration of the β-galactosidase coupled with streptavidin is preferably 1 to 50 pM. In the present invention, the ratio of the concentration of the β-galactosidase coupled with streptavidin to the number of micron-sized magnetic beads coated with capture antibodies is preferably 1 pM: 6×10 4 ~6×10 6 indivual.

[0063] In the present invention, the temperatures of the first incubation, the second incubation and the third incubation are preferably 23°C, respectively.

[0064] In the present invention, the cleaning reagent is preferably a PBS solution containing 0.1% Tween-20 by volume; in each step, the cleaning is preferably performed 3 times.

[0065] After obtaining the third incubation product, the present invention separates the reacted magnetic beads from the third incubation product, washes and resuspends them to obtain a resuspension; adds the resuspension to the micro-pot array chip, absorbs the excess solution, adds a fluorescent substrate of β-galactosidase to the micro-pot array chip, performs a fluorescent reaction, and then oil-seals the micro-pot array chip; performs fluorescence detection on the product after the fluorescence reaction using a fluorescence detection system, and records the number of fluorescence signals; and calculates the concentration of tumor antigens in the sample to be tested according to the number of fluorescence signals.

[0066] In the present invention, the solvent used for resuspension is preferably PBS solution.

[0067] In the present invention, the amount of the fluorescent substrate for β-galactosidase used is preferably based on the amount of the micro-pot that is immersed.

[0068] In the present invention, the excitation wavelength of the fluorescence detection is preferably 514 nm or 558 nm; the emission wavelength of the fluorescence detection is preferably 577 nm.

[0069] In the present invention, after the resuspension is added to the micro-pot array chip, the magnetic beads can be sorted in a few seconds using micron-sized micro-pots, thereby greatly improving the capture efficiency and shortening the processing time.

[0070] The method of the present invention is relatively simple to operate, easy to standardize and automate, and manual operation is more convenient.

[0071] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0072] Example 1

[0073] 1. Preparation of micro-basin array chip

[0074] The preparation method of the micro-basin array chip is carried out with reference to the existing technology, for details, see [Tian F, Li M, Pu D, et al. A Versatile and Environmentally Friendly Microfabrication Process for Producing Micro-Basin Array for Single Cell Analysis [C] / / 2021 IEEE 34th International Conference on Micro Electro Mechanical Systems (MEMS). IEEE, 2021. DOI: 10.1109 / MEMS51782.2021.9375448.]. For the preparation flow chart, see Figure 1 .

[0075] The specific steps are as follows:

[0076] First, a photoresist is spin-coated onto a hydrophobic glass substrate. Then, standard ultraviolet lithography and development are performed to produce a specific micropore size and the desired micropore sidewall shape on the photoresist. Next, a silicon dioxide film is deposited by physical sputtering, and then stripped off with acetone to remove the photoresist, leaving behind a micro-basin composed of silicon dioxide in contact with the hydrophobic glass substrate and silicon dioxide in contact with the micropore sidewalls of the photoresist.

[0077] The size of the micro-pot array chip is: 10mm*10mm, containing 1 million micro-pots; single hydrophilic micro-pot (round): diameter: 4.5μm; spacing 4.5μm; micro-pot depth: 1μm. For the electron microscope photo of the micro-pot array chip, see Figure 2 .

[0078] 2. Antigen capture

[0079] The key components and operation flow chart of the test are shown in Figure 3 .

[0080] Lung cancer tumor antigens to be tested: CYFRA21-1 (cytokeratin 19 fragment), CEA (carcinoembryonic antigen), NSE (neuron-specific enolase), SCC (squamous cell carcinoma antigen), CA125 (carbohydrate antigen 125).

[0081] Source of capture antibody: Company: Elabscience, CYFRA21-1 antibody catalog number: E-KAB-0282; CEA antibody catalog number: E-KAB-0016; NSE antibody catalog number: E-KAB-0059; SCC antibody catalog number: E-KAB-0286; CA125 antibody catalog number: E-KAB-0139. The magnetic beads used are micron-sized magnetic beads with acetate groups on the surface, purchased from Variant. The source of the detection antibody coupled with biotin is Elabscience, the catalog number is the same as the capture antibody, and the antibody pair is purchased.

[0082] The magnetic beads are pre-coated with an antibody against the selected lung cancer tumor antigen - the capture antibody. The specific steps are:

[0083] Prepare capture antibody solutions respectively; the solvent of the capture antibody solution is 0.1M sodium phosphate buffer (pH7.4); the concentration of each capture antibody in the capture antibody solution is 1nM. The volume ratio of the number of magnetic beads to the capture antibody solution is 6×10 4 ~6×10 6 Pieces: 1pM.

[0084] The magnetic beads were soaked in the capture antibody solution for 45 min, and each magnetic bead was coated with about 250,000 antibodies;

[0085] Add magnetic beads and test samples: Mix the coated magnetic beads with 50 μl of serum sample collected from fingertip blood and incubate at 23°C for 2 hours;

[0086] Washing: The magnetic beads were then separated using a magnetic separator and washed three times with a PBS solution containing 0.1% Tween-20;

[0087] The magnetic beads were then mixed with a 1 nM concentration of biotin-coupled detection antibody and incubated at 23°C for 45 min. The magnetic beads were then separated using a magnetic method and washed three times with a PBS solution containing 0.1% Tween-20 by volume. 6 The magnetic beads were incubated with streptavidin-coupled galactosidase (50pM) at 23°C for 30 minutes, and then washed three times with PBS solution containing 0.1% Tween-20. The magnetic beads were then resuspended in 100μl of PBS solution, and the magnetic bead solution was spread on the micro-pot array chip, and then the excess solution was aspirated. The number of magnetic beads is generally only 1,000 to 100,000. According to Poisson statistical distribution, most micro-pots have no magnetic beads or one magnetic bead, ensuring that each micro-pot contains or does not contain one magnetic bead.

[0088] 3. Enzymatic reaction of antibody coupling

[0089] 100 μl of the fluorescent substrate of β-galactosidase, resorufin-β-D-galactopyranoside (RGP), was added to start the reaction, and the micropot array chip was oil-sealed.

[0090] In the experiment, the control sample was subjected to enzymatic reaction for different time periods, and the test results were compared to find the optimal time. The fluorescence intensity was detected using a fluorescence detector as the reaction time changed. The optimal time should be the earliest time when the fluorescence brightness exceeded 3 times the background brightness (noise).

[0091] 4. Detection of fluorescent substrates

[0092] After the fluorescence reaction occurs, the micro-pot array chip is placed under a fluorescence microscope to take a picture (fluorescence detection wavelength 514nm excitation, 577nm emission), and the number of fluorescence signals is recorded. The data is then analyzed in combination with artificial intelligence to detect tumor antigens. Specifically: prepare antigens of known concentrations, the gradients are: 0.1pM, 1pM, 10pM, 100pM, record the number of fluorescence corresponding to each gradient, and obtain the corresponding relationship. For the detection of antigens of unknown concentration, refer to the above corresponding relationship, measure the number of luminous micro-pots, and then obtain the concentration of the antigen to be tested.

[0093] Test Example 1 Comparative Test of Micropores and Micro Basins

[0094] The micropores have a width of 2.7 μm and a height of 2 μm. In this test example, the ratio and time of micron-sized magnetic beads (microspheres) entering the micropores were counted by optical microscope. It was observed that it took 300 seconds for about 50% of the microspheres to enter the micropores, while it only took 10 seconds for 50% of the microspheres to enter the microbowls. It was observed that it took 1200 seconds for 90% of the microspheres to enter the micropores, while it only took 20 seconds for 90% of the microspheres to enter the microbowls. This proves that the use of microbowls can reduce the time for microspheres to enter and shorten the entire detection time.

[0095] The test results can be found in Figure 4 .

[0096] The experimental results show that the time required to disperse the magnetic beads into individual microwells or micropots is reduced by more than an order of magnitude when using micropots compared to using microwells.

[0097] Test Example 2 Comparative test of different concentrations

[0098] In the experiment, tumor antigens with concentrations ranging from 0.01 pg / mL, 0.1, 1, 10, 100, 1000, 10000, and 100000 pg / mL were prepared, and then the test was performed according to the method of Example 1, and the number of luminescent micro-pots was counted.

[0099] The test results can be found in Figure 5 and Table 1.

[0100] The test results show that the method of the present invention can detect 0.01pg / mL, and at this concentration, there are 9 luminescent micro-pots.

[0101] Table 1 Correspondence between the actual measured antibody concentration and the number of luminescent microtubules

[0102] Concentration (pg / mL) Number of luminous micro-pots 100000 9000154 10000 2002311 1000 965207 100 95421 10 9982 1 986 0.1 92 0.01 9

[0103] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Claims

1. A detection system for early tumor screening, characterized in that: It includes antigen capture system, antibody-coupled enzymatic reaction system and fluorescence detection system; The antigen capture system comprises micron-sized magnetic beads coated with capture antibodies; the capture antibodies are used to capture tumor antigens; The antibody-coupled enzymatic reaction system includes a micro-pot array chip and ELISA detection reagents; The micro-pot array chip has a hydrophobic material as a substrate, and a plurality of micro-pots with openings facing upward are arranged on the substrate; the diameter of each micro-pot is 1.2 to 1.8 times the diameter of the magnetic bead; the inner wall of the micro-pot is a hydrophilic material; The size of the micro basin is micrometer level; The ELISA detection reagents include: a biotin-labeled detection antibody, β-galactosidase coupled with streptavidin, and a fluorescent substrate of β-galactosidase; The micro-basin is used as a reaction carrier for ELISA detection; the micron-sized magnetic beads are coated with capture antibodies; the capture antibodies are used to capture tumor antigens; Each micron-sized magnetic bead is coated with 1×10 4 ~1×10 6 The diameter of the micron-sized magnetic beads is 2.7 μm; The shape of the micro-pot array chip is square; the specification of the micro-pot array chip is 10mm×10mm; the micro-pots on the micro-pot array chip are arranged in a matrix; the number of micro-pots on each micro-pot array chip is 1×10 6 indivual; The opening of each micro-pot is a circular opening; the diameter of the opening of each micro-pot is 4.5 μm; the distance between two adjacent micro-pots is 4.5 μm; the depth of the micro-pot is 1 μm; The ratio of the number of micron-sized magnetic beads coated with capture antibodies to the volume of the serum sample to be tested is 2×10 6 ~2×10 8 Piece: 20μl.

2. Use of the detection system according to claim 1 in the preparation of tumor antigen detection products.

3. The use according to claim 2, characterized in that: The tumor includes lung cancer.

4. The use according to claim 3, characterized in that: The tumor antigens include one or more of CYFRA21-1, CEA, NSE, SCC and CA125.

5. A method for detecting tumor antigens for non-diagnostic purposes based on the detection system of claim 1, characterized in that: The following steps are involved: Mixing the micron-sized magnetic beads coated with the capture antibody and the sample to be tested, performing a first incubation, and obtaining a first incubation product; Separating the reacted magnetic beads from the first incubation product, washing them, mixing them with the biotin-labeled detection antibody, and performing a second incubation to obtain a second incubation product; The reacted magnetic beads are separated from the second incubation product, washed, mixed with β-galactosidase coupled with streptavidin, and incubated for the third time to obtain a third incubation product; The reacted magnetic beads are separated from the third incubation product, and the beads are washed and resuspended to obtain a resuspended solution; the resuspended solution is added to the micro-pot array chip, and after absorbing the excess solution, a fluorescent substrate of β-galactosidase is added to the micro-pot array chip to perform a fluorescent reaction, and then the micro-pot array chip is oil-sealed; The fluorescence detection system is used to perform fluorescence detection on the product after the fluorescence reaction, and the number of fluorescence signals is recorded; The concentration of the tumor antigen in the sample to be tested is calculated according to the number of the fluorescent signals.

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