CN111273000A - Digital ELISA detection device and detection method - Google Patents

Abstract

The invention discloses a digital Elisa detection device and a detection method; the detection device comprises an optical fiber image transmission bundle integrating a detection cavity, an image sensor and an image acquisition circuit; the detection cavity is located on the upper end surface of the optical fiber image transmission bundle; the image sensor and the image The acquisition circuit is connected, the lower end face of the optical fiber image transmission bundle is seamlessly coupled with the image sensor, and the upper surface of the optical fiber image transmission bundle is provided with a micropore array; the detection method is specifically: modifying the surface of the magnetic microsphere with the antibody to be tested, and using a double antibody sandwich The antigen is detected by the method, and the washed magnetic microspheres are dispersed in the micropores on the upper surface of the optical fiber image transmission beam. , shoot the image at this time, and the software calculates the ratio of light and dark of the micro-hole; the invention adopts the method of coupling the optical fiber image transmission beam and the image sensor to form the image, the detection volume is greatly reduced, and the portable application can be realized.

Description

A digital ELISA detection device and detection method

technical field

The invention relates to enzyme-linked immunosorbent assay, in particular to a digital ELISA detection device and a detection method.

Background technique

Enzyme Linked Immunosorbent Assay (ELISA for short) is a diagnostic technique used to detect the presence of viral or tumor markers. It can be used to detect any antigen in serum, plasma and urine, such as viral or tumor-associated antigens. There are many ELISA detection methods, and the double-antibody sandwich detection is a typical one. First, the targeted biomarker is specifically captured by the capture antibody (covalently attached to the surface of the reaction chamber) through the antigen-antibody reaction. The unbound antibody is washed away by washing, and the bioisomerization detection antibody that binds to the antigen is added, and the enzyme-conjugated antibody that can undergo a color reaction is added, and then undergoes a fluorescence reaction. The enzyme enhances the fluorescence reaction to produce a fluorescent product in an aqueous solution of a fluorescent matrix, and the concentration of the biomarker is determined by the fluorescence intensity.

Digital ELISA can achieve higher sensitivity. In a digital ELISA, fluorescent reactions are performed in microchambers in a series of microliter volumes. The concentration of the target biomarker is determined by the ratio of light and dark microchambers detected by the CMOS image sensor. Therefore, enzymes can be replaced with corresponding fluorescent dyes, and this compact medical diagnostic device can realize low-power, portable, and routine biomarker detection, and has been widely used in practice. In resource-limited countries, it can be a powerful tool for suppressing infectious epidemics and, in home diagnostics, for early detection and effective treatment.

SUMMARY OF THE INVENTION

Purpose of the invention: In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a lens-less miniaturized digital ELISA detection device based on an image sensor, and another purpose of the present invention is to provide a low-cost, A detection method of a digital ELISA detection device with a large detection area.

Technical scheme: A digital ELISA detection device according to the present invention includes an optical fiber image transmission bundle, an image sensor, an image acquisition circuit, a detection microchamber, a magnetic microsphere and a coil; the upper end face of the optical fiber image transmission bundle is provided with at least 2 detection microchambers; an image sensor is provided at the lower end of the optical fiber image transmission bundle; the image sensor is coupled with the optical fiber image transmission bundle; the image sensor is connected with an image acquisition circuit; a coil is provided on the outside of the optical fiber image transmission bundle, The magnetic field is generated by the coil, and the magnetic microspheres enter the detection microchamber, and the image acquisition circuit is connected with the PC.

A few turns of coils are wound outside the optical fiber image beam to generate a magnetic field to accelerate the magnetic microspheres into the detection microchamber.

The diameter of the detection microchamber (4) is less than 100 micrometers, and the depth of the detection microchamber (4) is less than 100 micrometers. The size of the magnetic microspheres matches the size of the detection microchamber, and each detection microchamber has and can only place one magnetic microsphere; the magnetic microspheres are modified with specific antibodies.

The specific antibody includes an antibody, an antigen and a labeled secondary antibody arranged in sequence to form a sandwich structure. The labeling of the labeled secondary antibody is enzymatic labeling or fluorescent labeling. The enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is fluorescent dye label, fluorescent quantum dot label or up-conversion luminescence label.

The general ELISA procedure of the double-antibody sandwich method with the specifically labeled magnetic microspheres (adding the antibody to be tested, washing and incubating, adding another antibody specific for the antigen, washing and incubating, adding two antibodies with enzymes or fluorescent dyes) (secondary antibody, incubation and washing) are flattened into the detection microchamber, and the upper surface of the optical fiber image beam is illuminated with LED light. The software controls the picture at this time. If there are no magnetic microspheres in the detection microchamber, the detection microchamber will be bright during the process. If the enzyme is marked, a color developing solution will be added to the detection microchamber during the detection process, and the software controls to capture the fluorescence image at this time. , run the analysis software to compare and analyze the two images collected to calculate the light-dark ratio of the micro-holes in the images. If the labeled fluorescent dye is used, the excitation wavelength of the fluorescent dye is used to illuminate the upper surface of the optical fiber imaging beam, and the software controls to take the picture at this time. In this process, the detection micro-chamber is bright, and the number of bright detection micro-chambers in this process is counted as _N1 . If there are magnetic microspheres in the detection micro-chamber and the magnetic microspheres are not combined with the antibody to be tested, there is no fluorescence. During the process, the detection micro- _chamber is dark; the computer compares and analyzes the two images collected through the image collection PCB board (3), and calculates the quantity of the substance to be tested in the template through _Na and Nl.

The image acquisition circuit is connected with the computer, and the image acquisition circuit includes a FLASH module, an FPGA module, a voltage control module and an interface module;

Described FLASH module, is used for the short-term storage of video or image;

The FPGA module is used as a control core for image processing;

The voltage control module provides a stable power supply for the circuit board;

The interface module is used to transmit the collected image to the computer, so as to realize the communication with the external device.

The image sensor is a CCD image sensor or a CMOS image sensor. The pixel size of the image sensor matches the diameter of the image-transmitting optical fiber on the lower end face of the optical fiber image-transmitting bundle, and each optical fiber corresponds to a complete pixel.

The detection method of the above-mentioned digital ELISA detection device, comprises the following steps:

If the secondary antibody is labeled with enzymes such as horseradish peroxidase, alkaline phosphatase, etc. that can catalyze the luminescence reaction, then:

(a), modify the specific antibody to be detected on the surface of the magnetic microsphere, and carry out the general ELISA operation steps of the double antibody sandwich method (adding the antigen to be tested, washing and incubating, adding another antibody specific for the antigen, Wash incubation, add secondary antibodies with enzymes or fluorescent dyes, incubate and wash);

(b) Lay the magnetic microspheres after the operation in the previous step into the detection microchamber, the coil is energized to control the magnetic field to accelerate the magnetic microspheres to fall into the detection microchamber, and use the cardboard to scrape the magnetic microspheres into the detection microchamber ;

(c) Inject the chromogenic solution into the detection micro-chamber and then coat with a layer of fluorinated oil to isolate the micro-area; and wash away the excess magnetic microspheres;

(d) Use the LED to illuminate the upper surface of the optical fiber image beam, and the software controls to take the picture at this time. If there are magnetic microspheres in the detection microchamber, the detection microchamber will be dark during the process. If there is no magnetic microsphere in the detection microchamber If the ball is in the process, the detection micro-chamber is bright, which is convenient for deducting the number of empty detection micro-chambers in the next step;

(e), add a color developing solution to the detection microchamber, and the software controls to take the picture at this time;

(f), the computer compares and analyzes the two images collected by the image acquisition circuit, and calculates the quantity of the substance to be tested in the template.

If the secondary antibody is labeled with fluorescent labels such as fluorescent dye labels, fluorescent quantum dot labels, upconversion luminescence labels, etc., then:

(a), modify the specific antibody to be detected on the surface of the magnetic microsphere, and perform the general ELISA operation steps of the double antibody sandwich method (adding the antibody to be tested, washing and incubating, adding another antibody specific for the antigen, Wash and incubate, add secondary antibody with fluorescent dye, incubate and wash);

(b) Pour the magnetic microspheres operated in the previous step into the detection optical fiber image transmission beam, the coil is energized to control the magnetic field to accelerate the magnetic microspheres to fall into the detection microchamber, and the magnetic microspheres are scraped with cardboard to make them enter the detection microchamber. chamber; apply another layer of fluorinated oil to isolate the micro-area; and wash away the excess magnetic microspheres;

(c) Use the LED to illuminate the upper surface of the optical fiber image beam, and the software controls to take the picture at this time. If there are magnetic microspheres in the detection microchamber, the detection microchamber is dark in the process, and the total number of dark detection microchambers is N _a; if there are no magnetic microspheres in the detection microchamber, the detection microchamber is bright in the process, which is convenient for deducting the number of empty detection microchambers in the next step;

(d) Use the upper surface of the optical fiber imaging beam with the excitation wavelength of the fluorescent dye, and the software controls to take the picture at this time. If there are magnetic microspheres in the detection microchamber and the magnetic microspheres are bound with the antigen to be tested, the During the process, the detection micro-chamber is bright, and the number of bright detection micro-chambers in this process is counted as _N1 . If there is a magnetic microsphere in the detection micro-chamber and the magnetic microsphere is not bound with the antibody to be tested, there is no fluorescence, then in the process. The detection chamber is dark;

(e), the computer compares and analyzes the two images collected by the image acquisition circuit, and calculates the quantity of the substance to be tested in the template through _{Na and N l .}

Working principle: We introduce a detection device that combines fluorescence detection with a microwell array. The device makes small holes of the microarray on the optical fiber panel by photolithography, and the small holes are matched with the size of the magnetic microspheres. Each detection microchamber can only drop one magnetic microsphere, and the optical fiber panel is seamlessly coupled with each pixel of the CMOS image sensor. The enzyme that emits light from the color solution, if there is no magnetic microsphere bound to the antigen, there is no enzyme on the surface. The magnetic microsphere is loaded into the micropore by the magnetic field controlled by the coil, and the color solution is added. The luminescence is enhanced by the enzyme, and the solution without the enzyme does not emit light. In addition, the conjugated secondary antibody can also be detected by combining fluorescent dyes with laser excitation and emission fluorescence detection, and then use fluorocarbon oil to remove excess beads on the surface of the array, and seal and isolate For each detection microchamber, during image acquisition, the bright field and fluorescence images of an array block are continuously obtained, the bright field is illuminated with white light, and the CMOS imaging under the chip is used. The number of holes of the magnetic microsphere beads and the percentage of fluorescent micropores were calculated from the ratio of fluorescent wells to magnetic microsphere wells.

Beneficial effect: Compared with the prior art, the present invention has the following remarkable features:

1. The lensless imaging design is adopted, so the size of the detection device is greatly reduced, and it can be made into a portable device, which is convenient for outdoor or portable applications;

2. The spatial bandwidth product is not limited by traditional lens imaging, and can form large-area high-resolution images. Large-area imaging of micro-droplets of digital ELISA micron size greatly reduces the cost of scanning and imaging with traditional lenses;

3. Using a large target image sensor, it can perform imaging analysis on millions or even tens of millions and hundreds of millions of microdroplets, and a high number of liquids can reduce sampling errors and dispensing errors, thereby further improving detection accuracy.

Description of drawings

Fig. 1 is the front view of the detection device of the present invention;

Fig. 2 is the top view of the optical fiber panel of the present invention;

Fig. 3 is the structural representation of the magnetic microsphere of the present invention;

In the figure: 1. Optical fiber image beam, 2. Image sensor, 3. Image acquisition circuit, 4. Detection microchamber, 5. Magnetic microsphere, 6. Specific antibody, 7. Coil.

Detailed ways

Example 1

A digital ELISA detection device includes an optical fiber image transmission bundle 1, an image sensor 2, an image acquisition circuit 3, a detection microchamber 4, a magnetic microsphere 5 and a coil 7; the upper end face of the optical fiber image transmission bundle 1 is provided with at least 2 There are detection microchambers 4; the lower end of the optical fiber image transmission bundle 1 is provided with an image sensor 2, and the image sensor 2 is connected with the image acquisition circuit 3; , and make the magnetic microspheres 5 enter the detection microchamber 4 .

The diameter of the detection microchamber 4 is less than 100 micrometers, and the depth of the detection microchamber 4 is less than 100 micrometers.

A magnetic microsphere 5 is placed in each detection microchamber 4 ; the magnetic microsphere 5 is modified with a specific antibody 6 .

The specific antibody 5 includes an antibody, an antigen and a labeled secondary antibody arranged in sequence.

The labeling of the labeled secondary antibody is enzymatic labeling or fluorescent labeling. The enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is fluorescent dye label, fluorescent quantum dot label or up-conversion luminescence label.

The image acquisition circuit includes a FLASH module, an FPGA module, a voltage control module and an interface module;

Described FLASH module, is used for the short-term storage of video or image;

The FPGA module is used as a control core for image processing;

The voltage control module provides a stable power supply for the circuit board

The interface module is used to transmit the collected images to the computer to realize communication with external devices

The image sensor 2 is a CCD image sensor or a CMOS image sensor.

Example 2

The CMOS image sensor 2 is a 1.2-inch black-and-white CMOS with a pixel count of 2048(H) x 2048(V) and a pixel size of 6.5 x6.5(μm). The internal integrated cooling module is adopted to reduce the interference of dark noise. The diameter of each fiber of the detection fiber image transmission bundle 1 panel is 6.5 microns and corresponds to the pixels one by one. A 2mm micro-groove is opened above the optical fiber panel to hold the fluorinated oil solution, and the size of the micro-hole is 4 microns in diameter and 4 microns in depth. , the center distance is 6.5 microns, and the micro-hole is placed in the center of the fiber to correspond to the pixel below. During detection, a white light source and a green laser light source are placed on the device.

The human interleukin IL-8 kit was used for the ELISA reaction. The IL-8 antibody was immobilized on the magnetic microspheres 5 with a diameter of 3.7um, and the ELISA reaction was carried out in a test tube. After adding the antigen to be tested, washing and incubating, biotin antibody , washing and incubation, adding streptavidin with HRP label, incubation and washing, etc., inject the magnetic microsphere 5 suspension after the reaction into the detection microchamber 4 on the upper end face of the optical fiber imaging bundle 1 panel through a pipette gun , deposit for 2 minutes, wait for the magnetic microspheres 5 to fall into the micropores by gravity, slowly add fluorinated oil from one side, and the magnetic microspheres 5 that stay on the surface of the panel under the action of the fluorinated oil are pushed into the micropores , the excess magnetic microspheres 5 are taken away to prevent interference detection. During detection, the brightfield image at this time is now photographed with white light illumination, and the image is processed by imageJ software to set appropriate threshold binarization processing, and the holes with magnetic microspheres 5 can be distinguished from those without magnetic microspheres 5 . The white light was turned off again, and the fluorescent image was taken under the dark condition for 2000ms exposure, and then the number of fluorescent wells and the wells containing magnetic microspheres 5 were counted, and the percentage of fluorescent micropores was calculated by the ratio of fluorescent wells and magnetic microspheres 5 wells.

The prepared magnetic microspheres 5 are combined with the anti-human IL-8 capture antibody in advance, and the detection process is completed through the steps of sampling, reaction, and imaging.

The sampling operation steps are as follows:

1. Take the serum of the sample to be tested, place the whole blood at room temperature for 1 hour, and let it stand. After the whole blood is naturally coagulated and the serum is precipitated, centrifuge at 1500g for 10 minutes. Refrigerate in degrees Celsius for later use;

2. Dilute serum and sample analysis buffer at a ratio of 1:5;

3. Equilibrate the purchased biotin antibody reagents at room temperature for 20 minutes;

4. To prepare a washing solution, dilute the washing solution (20X) to 1X with deionized water.

Detection operation steps:

1. Add sample: Add 50ul of biotin-labeled antibody and the sample to be tested to the anti-human IL-8-bound magnetic microspheres 5 test tube, cover with the lid and shake gently for 1 min to mix, and incubate at room temperature for 2 hours;

2. Washing: wash 3 times with 300ul of washing liquid, 30 seconds each time, and then pat dry on absorbent paper after each wash. If washing with a plate washer, increase the number of washes by one;

3. SA-HRP: Add SA-HRP 100ul, cover with lid, shake gently to mix, and incubate at room temperature for 30 minutes;

4. Washing: pour the liquid in the test tube, wash 5 times with 300ul of washing solution, 30 seconds each time, and pat dry on absorbent paper after each wash.

Imaging steps:

1. Pour the magnetic microspheres 5 after the operation in the previous step into the detection optical fiber image transmission beam 1, the coil 7 is energized to control the magnetic field to accelerate the magnetic microspheres 5 to fall into the detection microchamber 4, and the magnetic microspheres 5 are scraped with cardboard to make the magnetic microspheres 5 fall into the detection microchamber 4. It enters the detection microchamber 4;

2. Use the LED to illuminate the upper surface of the optical fiber image beam 1, and the software controls to take the picture at this time. If there are magnetic microspheres 5 in the detection microchamber 4, the detection microchamber 4 is dark during the process. Without magnetic microspheres 5, the detection microchamber 4 is bright in this process, which is convenient for deducting the number of empty detection microchambers 4 in the next step;

3. Inject 100ul of the color developing solution into the detection microchamber 4, and then apply a layer of fluorinated oil to isolate the microregion, and wash away the excess magnetic microspheres 5;

4. The computer compares and analyzes the two images collected by the image collection PCB board (image collection circuit 3), and calculates the quantity of the substance to be tested in the template.

Example 3

The difference from Example 2 is that in this example, the enzyme bound to the secondary antibody was replaced with the fluorescent dye CY3. Imaging using laser excitation.

The CMOS image sensor 2 is a 1.2-inch black and white CMOS with a pixel count of 2048(H) x 2048(V) and a pixel size of 6.5x 6.5(μm). The internal integrated cooling module is adopted to reduce the interference of dark noise. The diameter of each fiber of the detection fiber image transmission bundle 1 panel is 6.5 microns and corresponds to the pixels one by one. A 2mm micro-groove is opened above the optical fiber panel to hold the fluorinated oil solution, and the size of the micro-hole is 4 microns in diameter and 4 microns in depth. , the center distance is 6.5 microns, and the micro-hole is placed in the center of the fiber to correspond to the pixel below. A white light source is placed on the device during detection.

Human interleukin IL-8 kit was used for ELISA reaction, IL-8 antibody was immobilized on magnetic microspheres 5 with a diameter of 3.7um, and ELISA reaction was carried out in a test tube. After adding the antigen to be tested, washing and incubating, adding biotin antibody , washing and incubating, adding streptavidin with CY3 label, incubation and washing, etc., inject the magnetic microsphere 5 suspension after reaction into the detection microchamber 4 on the upper end face of the optical fiber panel through a pipette, and deposit 2 Wait for the magnetic microspheres 5 to fall into the micropores by gravity, and slowly add fluorinated oil from one side. The microspheres 5 are carried away to prevent interference with the detection. During detection, the brightfield image at this time is now photographed with white light illumination, and the image is processed by imageJ software to set appropriate threshold binarization processing, and the holes with magnetic microspheres 5 can be distinguished from those without magnetic microspheres 5 . Then turn off the white light, and then excite cy3 with laser to take a fluorescent image, expose for 2000ms to take a fluorescent image, and then count the number of fluorescent wells and the number of wells containing magnetic microspheres 5, and the percentage of fluorescent micropores is calculated by the ratio of fluorescent wells and magnetic microspheres 5 wells .

The prepared magnetic microspheres 5 are combined with the anti-human IL-8 capture antibody in advance, and the detection process is completed through the steps of sampling, reaction, and imaging.

The sampling operation steps are as follows:

1. Take the serum of the sample to be tested, place the whole blood at room temperature for 1 hour, and let it stand. After the whole blood is naturally coagulated and the serum is precipitated, centrifuge at 1500g for 10 minutes. Refrigerate in degrees Celsius for later use;

2. Dilute serum and sample analysis buffer at a ratio of 1:5;

3. Equilibrate the purchased biotin antibody reagents at room temperature for 20 minutes;

4. To prepare a washing solution, dilute the washing solution (20X) to 1X with deionized water.

Detection operation steps:

1. Add sample: Add 50ul of biotin-labeled antibody and the sample to be tested to the anti-human IL-8-bound magnetic microspheres 5 test tube, cover with the lid and shake gently for 1 min to mix, and incubate at room temperature for 2 hours;

2. Washing: wash 3 times with 300ul of washing liquid, 30 seconds each time, and then pat dry on absorbent paper after each wash. If washing with a plate washer, increase the number of washes by one;

3. SA-CY3: Add SA-CY3100ul, cover with lid, shake gently to mix, and incubate at room temperature for 30 minutes;

4. Washing: pour the liquid in the test tube, wash 5 times with 300ul of washing solution, 30 seconds each time, and pat dry on absorbent paper after each wash.

Imaging steps:

1. Pour the magnetic microspheres 5 after the operation in the previous step into the detection optical fiber image transmission beam 1, the coil 7 is energized to control the magnetic field to accelerate the magnetic microspheres 5 to fall into the detection microchamber 4, and the magnetic microspheres 5 are scraped with cardboard to make the magnetic microspheres 5 fall into the detection microchamber 4. It enters the detection microchamber 4;

2. Use the LED to illuminate the upper surface of the optical fiber image beam 1, and the software controls to take the picture at this time. If there are magnetic microspheres 5 in the detection microchamber 4, the detection microchamber 4 is dark during the process. If the detection microchamber 4 If there is no magnetic microsphere 5 in the middle, the detection microchamber 4 is bright in this process, which is convenient for deducting the number of empty detection microchambers 4 in the next step;

3. Use a green light source to illuminate the upper surface of the optical fiber image transmission beam 1, and the software controls to take the picture at this time. If there is a magnetic microsphere 5 in the detection microchamber 4 and the magnetic microsphere 5 is bound with the antigen to be tested, then during the process The detection microchamber 4 is bright, and if there is a magnetic microsphere 5 in the detection microchamber 4 and the magnetic microsphere 5 is not bound to the antigen to be tested, then the detection microchamber 4 is dark in the process without CY3;

4. The computer compares and analyzes the two images collected by the image collection PCB board (image collection circuit 3), and calculates the quantity of the substance to be tested in the template.

The above-mentioned embodiments are only the preferred technical solutions of the present invention, and should not be regarded as limitations of the present invention. The protection scope of the present invention should be based on the technical solutions recorded in the claims, including the equivalent replacement of the technical features in the technical solutions recorded in the claims. The scheme is the protection scope, that is, the equivalent replacement and improvement within this scope are also within the protection scope of the present invention.

Claims (10)

1. A digital ELISA detection device, characterized in that it comprises an optical fiber image transmission beam (1), an image sensor (2), an image acquisition circuit (3), a detection microchamber (4), a magnetic microsphere (5) and a coil (7); the upper end face of the optical fiber image transmission bundle (1) is provided with at least two detection microchambers (4); the lower end of the optical fiber image transmission bundle (1) is provided with an image sensor (2), the image sensor (2) Connected to the image acquisition circuit (3); a coil (7) is provided on the outside of the optical fiber image transmission bundle (1), a magnetic field is generated by the coil (7), and the magnetic microspheres (5) enter the detection microchamber ( 4) inside. 2. A digital ELISA detection device according to claim 1, characterized in that: the diameter of the detection microchamber (4) is less than 100 microns, and the depth of the detection microchamber (4) is less than 100 microns. 3. A digital ELISA detection device according to claim 1, characterized in that: a magnetic microsphere (5) is placed in each detection microchamber (4); the magnetic microspheres (5) are modified with specific Sexual antibodies (6). 4 . The digital ELISA detection device according to claim 3 , wherein the specific antibody ( 5 ) comprises an antibody, an antigen and a labeled secondary antibody arranged in sequence. 5 . 5 . The digital ELISA detection device according to claim 4 , wherein the label of the labeled secondary antibody is an enzyme label or a fluorescent label. 6 . 6. A digital ELISA detection device according to claim 5, characterized in that: the enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is a fluorescent dye label, a fluorescent quantum dot label Or up-converting luminous markers. 7. A digital ELISA detection device according to claim 1, characterized in that: the image acquisition circuit (3) comprises a FLASH module, an FPGA module, a voltage control module and an interface module; Described FLASH module, is used for the short-term storage of video or image; The FPGA module is used as a control core for image processing; The voltage control module provides a stable power supply for the circuit board; The interface module is used to transmit the collected image to the computer, so as to realize the communication with the external device. 8 . The digital ELISA detection device according to claim 1 , wherein the image sensor ( 2 ) is a CCD image sensor or a CMOS image sensor. 9 . 9. A method for detecting using the digital ELISA detection device according to claim 1-8, characterized in that: when the label of the labeled secondary antibody is an enzyme label, the detection method steps are as follows: (a) The specific antibody (5) to be detected is modified on the surface of the magnetic microsphere (5); (b) laying the magnetic microspheres (5) into the detection microchamber (4), the coil (7) is energized to control the magnetic field, and the magnetic microspheres (5) fall into the detection microchamber (4); (c) adding a color-developing solution to the micropore array on the surface of the optical fiber imaging bundle (1), and then applying a layer of fluorinated oil to isolate the micro-area, and washing away the excess magnetic microspheres (5); (d) The brightfield light source illuminates the upper surface of the optical fiber image transmission beam (1), and the software controls to take pictures. If there are magnetic microspheres (5) in the detection microchamber (4), the detection microchamber (4) is dark. If there is no magnetic microsphere (5) in the detection microchamber (4), the detection microchamber (4) is bright, and a suitable threshold is set to distinguish the number of bright and dark microchambers, and the total number of dark microchambers is _Na ; (e) Turn off the light source, the computer controls the long exposure through the image acquisition circuit (3), and takes the picture at this time, and the number of bright micro-chambers in this process is counted as N _l; (f) Compare and analyze the first two images, and calculate the amount of the template substance to be tested by _{Na and N l .} 10. A method for detection using the digital ELISA detection device according to claim 1-8, characterized in that: when the label of the labeled secondary antibody is a fluorescent dye label, the method comprises the following steps: (a) The specific antibody (5) to be detected is modified on the surface of the magnetic microsphere (5); (b) Pour the magnetic microspheres (5) into the detection optical fiber image transmission bundle (1), the coil is energized to control the magnetic field, and the magnetic microspheres (5) fall into the detection microchamber (4), and use a cardboard to scrape the magnetic field. The microspheres (5) are put into the detection microchamber (4); a layer of fluorinated oil is then applied to isolate the micro-area, and the excess magnetic microspheres (5) are washed away; (c) The light source illuminates the upper surface of the optical fiber image transmission beam (1), and the software controls to take pictures. If there are magnetic microspheres (5) in the detection microchamber (4), the detection microchamber (4) is dark, and the dark count is dark. The total number of chambers is _Na ; if there is no magnetic microsphere (5) in the microchamber, the detection microchamber (4) is bright; (d) The upper surface of the optical fiber imaging beam (1) is irradiated with the light of the excitation wavelength of the fluorescent dye, and the picture is taken under the control of the software. If there is a magnetic microsphere (5) in the detection microchamber (4) and the magnetic microsphere (5) If the antigen to be tested is bound, the detection microchamber (4) is bright, and the number of bright microchambers is counted as N _l . If there is a magnetic microsphere (5) in the detection microchamber (4) and the magnetic microsphere ( 5) If the antigen to be tested is not bound, there is no fluorescence, and the detection microchamber (4) is dark; (e) The computer compares and analyzes the two images collected by the image collection circuit (3), and calculates the quantity of the substance to be tested in the template through N _a and N _l .

Images