CN111273000A - Digital ELISA detection device and detection method - Google Patents
Digital ELISA detection device and detection method Download PDFInfo
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- CN111273000A CN111273000A CN202010108352.4A CN202010108352A CN111273000A CN 111273000 A CN111273000 A CN 111273000A CN 202010108352 A CN202010108352 A CN 202010108352A CN 111273000 A CN111273000 A CN 111273000A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention discloses a digital Elisa detection device and a detection method; the detection device comprises an optical fiber image transmission bundle integrated with a detection cavity, an image sensor and an image acquisition circuit; the detection cavity is positioned on the surface of the upper end face of the optical fiber image transmission bundle; the image sensor is connected with the image acquisition circuit, 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 specifically comprises the following steps: modifying the surface of a magnetic microsphere with an antibody to be detected, detecting an antigen by using a double-antibody sandwich method, dispersing the washed magnetic microsphere in micropores on the upper surface of an optical fiber image transmission bundle, setting the size of each micropore as that each micropore can only be used for accommodating the next magnetic microsphere at most, then adding a developing solution for reaction, shooting an image at the moment, and calculating the brightness-dark ratio of the micropore by software; the invention adopts the mode of coupling the optical fiber image transmission bundle and the image sensor for imaging, greatly reduces the detection volume and can realize portable application.
Description
Technical Field
The invention relates to enzyme-linked immunosorbent assay, in particular to a digital ELISA detection device and a detection method.
Background
Enzyme Linked Immunosorbent Assay (ELISA) is a diagnostic technique for detecting the presence of viral or tumor markers. Can be used to detect any antigen in serum, plasma and urine, such as a virus or tumor associated antigen. There are many ELISA detection formats, of which the two-antibody sandwich detection is typical, the targeted biomarker is first captured exclusively by a capture antibody (covalently attached to the inner surface of the reaction chamber) via an antigen-antibody reaction. The unbound antibody is washed away by washing, and a biosimilar detection antibody that binds to the antigen is added, and then an enzyme-bound antibody that can undergo a color reaction is subjected to a fluorescence reaction. The enzyme enhances the fluorescence reaction to generate a fluorescence product in the fluorescent substrate aqueous solution, and the concentration of the biomarker is determined by the fluorescence intensity.
Whereas digital ELISA can achieve higher sensitivity. In digital ELISA, the fluorescence reaction is performed in a series of microliter volumes of microcavities. The concentration of the target biomarker is determined by the proportion of light and dark microchambers detected by the CMOS image sensor. Therefore, the enzyme can be replaced by the corresponding fluorescent dye, and the compact medical diagnostic equipment can realize low-power consumption, portable and daily biomarker detection and is widely applied in practice. In countries with limited resources, it can be a powerful tool for inhibiting infectious epidemics, and also helps to find diseases early and treat them effectively in home diagnosis.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention aims to provide a lens-free miniaturized digital ELISA detection device based on an image sensor, and the invention also aims to provide a detection method of the digital ELISA detection device which is low in cost, free of lenses and large in detection area.
The technical scheme is as follows: the invention relates to a digital ELISA detection device, which comprises an optical fiber image transmission bundle, an image sensor, an image acquisition circuit, a detection microchamber, magnetic microspheres and a coil, wherein the optical fiber image transmission bundle is arranged on the detection microchamber; the upper end surface of the optical fiber image transmission bundle is provided with at least 2 detection microchambers; the lower end of the optical fiber image transmission bundle is provided with an image sensor; the image sensor is coupled with the optical fiber image transmission bundle; the image sensor is connected with the image acquisition circuit; the outside of the optical fiber image transmission bundle is provided with a coil, a magnetic field is generated through the coil, the magnetic microspheres enter the detection micro-chamber, and the image acquisition circuit is connected with the PC.
The optical fiber image transmission bundle is wound by a plurality of turns of coils to generate a magnetic field, so that the magnetic microspheres are accelerated to enter the detection microchamber.
The diameter of the detection micro-chamber (4) is less than 100 microns, and the depth of the detection micro-chamber (4) is less than 100 microns. The size of the magnetic microspheres is matched with that of the detection micro-chamber, and only one magnetic microsphere can be placed in each detection micro-chamber; the magnetic microspheres are modified with specific antibodies.
The specific antibody comprises an antibody, an antigen and a labeled secondary antibody which are sequentially arranged to form a sandwich structure. The label of the labeled secondary antibody is an enzyme label or a fluorescent label. The enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is a fluorescent dye label, a fluorescent quantum dot label or an up-conversion luminescent label.
Specifically marked magnetic microspheres are subjected to general ELISA operation steps of a double-antibody sandwich method (adding an antibody to be detected, washing and incubating, adding another antigen-specific antibody, washing and incubating, adding a secondary antibody with enzyme or fluorescent dye, incubating and washing), the two antibodies are tiled in a detection micro-chamber, an LED is used for illuminating the upper surface of an optical fiber image transmission beam, software controls to shoot a picture at the moment, if the magnetic microspheres exist in the detection micro-chamber, the detection micro-chamber is dark in the process, if the magnetic microspheres exist in the detection micro-chamber, the detection micro-chamber is bright in the process, if the magnetic microspheres exist in the detection micro-chamber, a chromogenic solution is added in the detection micro-chamber in the detection process, the software controls to shoot a fluorescence image at the moment, and analysis software is operated to compare the collected images twice to calculate the brightness and darkness ratio of the image micro-chamber. If the labeled fluorochrome is used, the upper surface of an optical fiber image transmission bundle is illuminated by the excitation wavelength of the fluorochrome, the software controls to shoot the picture at the moment, if the magnetic microsphere is in the detection microchamber and is combined with the antigen to be detected, the detection microchamber is bright in the process, and the number of the bright detection microchambers is N in the processlIf the magnetic microsphere exists in the detection microchamber and the magnetic microsphere is not combined with the antibody to be detected and has no fluorescence, the detection microchamber is dark in the process; the computer compares and analyzes the two images collected by the image collecting PCB (3) through NaAnd NlAnd calculating to obtain the quantity of the substance to be detected of the template.
The image acquisition circuit is connected with the computer and comprises a FLASH module, an FPGA module, a voltage control module and an interface module;
the FLASH module is used for temporarily storing videos or images;
the FPGA module is used as a control core for processing the image;
the voltage control module is used for providing a stable power supply for the circuit board;
and the interface module is used for transmitting the acquired image to a computer to realize communication with external equipment.
The image sensor is a CCD image sensor or a CMOS image sensor. The size of the image element of the image sensor is matched with the diameter of the image transmission optical fiber on the lower end face of the optical fiber image transmission bundle, and each optical fiber corresponds to a complete image element.
The detection method of the digital ELISA detection device comprises the following steps:
if the secondary antibody is labeled with an enzyme label such as horseradish peroxidase, alkaline phosphatase, etc., which can catalyze the luminescence reaction, then:
(a) modifying the surface of the magnetic microsphere with a specific antibody to be detected, and carrying out the general ELISA operation steps of a double antibody sandwich method (adding an antigen to be detected, washing and incubating, adding another antibody specific to the antigen, washing and incubating, adding a secondary antibody with enzyme or fluorescent dye, incubating and washing);
(b) flatly paving the magnetic microspheres operated in the previous step into a detection micro-chamber, electrifying a coil to control a magnetic field to accelerate the magnetic microspheres to fall into the detection micro-chamber, and scraping the magnetic microspheres by using a paper board to enter the detection micro-chamber;
(c) injecting a color developing solution into the detection micro chamber and coating a layer of fluorinated oil to isolate the micro area; and washing away the redundant magnetic microspheres;
(d) using an LED to illuminate the upper surface of the optical fiber image transmission bundle, and controlling by software to shoot a picture at the moment, wherein if magnetic microspheres exist in the detection microchamber, the detection microchamber is dark in the process, and if no magnetic microspheres exist in the detection microchamber, the detection microchamber is bright in the process, so that the number of empty detection microchambers can be conveniently deducted in the next step;
(e) adding a color developing solution into the detection micro-chamber, and controlling by software to shoot a picture at the moment;
(f) and the computer compares and analyzes the two images acquired by the image acquisition circuit, and calculates to obtain the quantity of the substance to be detected of the template.
If the secondary antibody is marked by a fluorescent marker such as a fluorescent dye marker, a fluorescent quantum dot marker, an up-conversion luminescent marker and the like, then:
(a) modifying the surface of the magnetic microsphere with a specific antibody to be detected, and performing the general ELISA operation steps of a double antibody sandwich method (adding the antibody to be detected, washing and incubating, adding another antibody with antigen specificity, washing and incubating, adding a secondary antibody with fluorescent dye, incubating and washing);
(b) pouring the magnetic microspheres operated in the previous step into a detection optical fiber image transmission beam, electrifying a coil to control a magnetic field to accelerate the magnetic microspheres to fall into a detection micro-chamber, and scraping the magnetic microspheres by using a paper board to enter the detection micro-chamber; coating a layer of fluorinated oil to isolate micro-areas; and washing away the redundant magnetic microspheres;
(c) illuminating the upper surface of the image transmission bundle by using an LED, controlling by software to shoot a picture at the moment, if the magnetic microspheres exist in the detection microchamber, the detection microchamber is dark in the process, and the total number of the dark detection microchambers is counted to be Na；If the detection microchamber is free of the magnetic microspheres, the detection microchamber is bright in the process, and the number of empty detection microchambers is conveniently deducted in the next step;
(d) using the excitation wavelength of the fluorescent dye to illuminate the upper surface of the optical fiber image transmission bundle, controlling by software to shoot a picture at the moment, if a magnetic microsphere is arranged in the detection microchamber and the magnetic microsphere is combined with an antigen to be detected, the detection microchamber is bright in the process, and the number of the bright detection microchambers is N in the processlIf the magnetic microsphere exists in the detection microchamber and the magnetic microsphere is not combined with the antibody to be detected and has no fluorescence, the detection microchamber is dark in the process;
(e) the computer compares and analyzes the two images acquired by the image acquisition circuit, and the images are acquired by the image acquisition circuit through NaAnd NlAnd calculating to obtain the quantity of the substance to be detected of the template.
The working principle is as follows: we have introduced a kind of detection device which combines the fluorescence detection with the micropore array, the device makes the micropore of the microarray on the optical fiber panel by photolithography, the micropore matches with the magnetic microsphere size to make each detection microchamber only fall one magnetic microsphere, the optical fiber panel couples with each pixel of the CMOS image sensor seamlessly, if the magnetic microsphere surface which completes the ELISA procedure has antigen, then combines with the enzyme which can enhance the luminescence of the color developing solution, if the magnetic microsphere which does not combine with the antigen does not have enzyme, the magnetic microsphere is loaded into the micropore by the magnetic field controlled by the coil, adds the color developing solution, if the magnetic microsphere has enzyme, then obviously the solution is enhanced by the enzyme to luminesce, if the magnetic microsphere does not combine with the enzyme, the solution does not luminesce, in addition, the combined secondary antibody can also be excited by the same light laser by combining with the fluorochrome to emit the fluorescence detection, then the fluorocarbon oil is used to remove the surplus on the array surface, and sealing and isolating each detection microchamber, continuously obtaining a bright field and a fluorescence image of an array block during image acquisition, wherein the bright field is illuminated by white light, a CMOS (complementary metal oxide semiconductor) below the chip is used for imaging, the fluorescence image is obtained after the white light image is obtained, then the number of fluorescence holes and the number of holes containing magnetic microsphere beads are calculated, and the percentage of fluorescence micropores is calculated by the ratio of the fluorescence holes to the magnetic microsphere holes.
Has the advantages that: compared with the prior art, the invention has the following remarkable characteristics:
1. the design of lens-free imaging is adopted, so that the volume of the detection device is greatly reduced, and the detection device can be made into a portable device, thereby being convenient for outdoor or portable application;
2. the space bandwidth product can not be limited by the imaging of the traditional lens, a large-area high-resolution image can be formed, and the scanning imaging cost of the traditional lens is greatly reduced by carrying out large-area imaging on the digital ELISA micron-sized micro-droplets;
3. by adopting the large target surface image sensor, millions of micro-droplets, even millions and billions of micro-droplets can be subjected to imaging analysis, and the high liquid separation quantity can reduce sampling errors and liquid separation errors, so that the detection precision is further improved.
Drawings
FIG. 1 is a front view of the inspection device of the present invention;
FIG. 2 is a top view of a fiber optic faceplate of the present invention;
FIG. 3 is a schematic structural diagram of a magnetic microsphere of the present invention;
in the figure: 1. the system comprises an optical fiber image transmission bundle 2, an image sensor 3, an image acquisition circuit 4, a detection micro chamber 5, magnetic microspheres 6, a specific antibody 7 and a coil.
Detailed Description
Example 1
A digital ELISA detection device comprises an optical fiber image transmission bundle 1, an image sensor 2, an image acquisition circuit 3, a detection microchamber 4, magnetic microspheres 5 and a coil 7; the upper end surface of the optical fiber image transmission bundle 1 is provided with at least 2 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 an image acquisition circuit 3; the outside of the optical fiber image transmission bundle 1 is provided with a coil 7, a magnetic field is generated through the coil 7, and the magnetic microspheres 5 enter the detection microchamber 4.
The diameter of the detection micro-chamber 4 is less than 100 microns, and the depth of the detection micro-chamber 4 is less than 100 microns.
Each detection micro-chamber 4 is internally provided with a magnetic microsphere 5; the magnetic microsphere 5 is modified with a specific antibody 6.
The specific antibody 5 comprises an antibody, an antigen and a labeled secondary antibody which are sequentially arranged.
The label of the labeled secondary antibody is an enzyme label or a fluorescent label. The enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is a fluorescent dye label, a fluorescent quantum dot label or an up-conversion luminescent label.
The image acquisition circuit comprises a FLASH module, an FPGA module, a voltage control module and an interface module;
the FLASH module is used for temporarily storing videos or images;
the FPGA module is used as a control core for processing the image;
the voltage control module provides a stable power supply for the circuit board
The interface module is used for transmitting the collected image to a computer to realize communication with external equipment
The image sensor 2 is a CCD image sensor or a CMOS image sensor.
Example 2
The CMOS image sensor 2 has a 1.2-inch black-and-white CMOS pixel count of 2048(H) x 2048(V) and a pixel size of 6.5x 6.5(μm). And the internal integrated refrigeration module is adopted, so that the interference of dark noise is reduced. The diameter of each optical fiber of the panel of the detection optical fiber image transmission bundle 1 is 6.5 micrometers, the diameter of each optical fiber corresponds to the pixels one by one, a 2mm micro-groove is formed above the optical fiber panel and used for containing a fluorinated oil solution, the size of each micro-hole is 4 micrometers in diameter and 4 micrometers in depth, the center distance is 6.5 micrometers, and the micro-holes are arranged in the center of the optical fibers and correspond to the pixels below the micro-holes. A white light source and a green laser source are arranged above the device during detection.
Adopting a human interleukin IL-8 kit to carry out ELISA reaction, fixing an IL-8 antibody on a magnetic microsphere 5 with the diameter of 3.7um, carrying out ELISA reaction in a test tube, adding an antigen to be detected, washing and incubating, a biotin antibody, washing and incubating, adding streptavidin marked by HRP, incubating and washing and the like, injecting a suspension of the reacted magnetic microsphere 5 into a detection microchamber 4 on the upper end surface of a panel of an optical fiber image transmission bundle 1 through a pipette gun, depositing for 2 minutes, waiting for the magnetic microsphere 5 to fall into a micropore by virtue of gravity, slowly adding fluorinated oil from one side, pushing the magnetic microsphere 5 staying on the surface of the panel into the micropore under the action of the fluorinated oil, and taking away the redundant magnetic microsphere 5 to prevent interference detection. During detection, a bright field image at the moment is shot by using white light illumination, the image is processed through imageJ software, appropriate threshold binarization processing is set, and holes with magnetic microspheres 5 and holes without the magnetic microspheres 5 can be distinguished. And then closing the white light, exposing for 2000ms under dark conditions to shoot a fluorescence image, and then calculating the number of fluorescence holes and the number of holes containing the magnetic microspheres 5, wherein the percentage of fluorescence micropores is calculated by the ratio of the fluorescence holes to the magnetic microspheres 5.
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, imaging and the like.
The sampling operation steps are as follows:
1. taking serum of a sample to be detected, placing whole blood for 1 hour at room temperature, standing, centrifuging at 1500g for 10 minutes after the whole blood is naturally coagulated and the serum is separated out, taking supernatant fluid, namely the serum, and refrigerating the prepared serum at 4 ℃ for later use;
2. serum was mixed with sample assay buffer at 1: 5, dilution in proportion;
3. equilibrating purchased different biotin antibody reagents for 20 minutes at room temperature;
4. preparing a washing solution, and diluting the washing solution (20X) to 1X with deionized water.
A detection operation step:
1. sample adding: adding 50ul of biotin labeled antibody and a sample to be detected into a magnetic microsphere 5 test tube combined with anti-human IL-8, slightly shaking for 1min by covering a cover, uniformly mixing, and incubating at room temperature for 2 h;
2. washing: washing with 300ul of washing solution for 3 times, each time for 30 seconds, and then patting the paper on absorbent paper to be dry after each time of washing. If the plate washing machine is used for washing, the washing times are increased once;
3. SA-HRP: adding SA-HRP100ul, covering with a cover, gently shaking, mixing, and incubating at room temperature for 30 min;
4. washing: the liquid in the test tube is poured, the washing liquid is washed for 5 times by 300ul, each time is 30 seconds, the water-absorbent paper is dried after the washing is finished, and the washing times are increased once if a plate washing machine is used for washing.
An imaging operation step:
1. pouring the magnetic microspheres 5 operated in the previous step into the detection optical fiber image transmission bundle 1, electrifying a coil 7 to control a magnetic field to accelerate the magnetic microspheres 5 to fall into the detection micro-chamber 4, and scraping the magnetic microspheres 5 by using a paper board to enable the magnetic microspheres 5 to enter the detection micro-chamber 4;
2. using an LED to illuminate the upper surface of the optical fiber image transmission bundle 1, controlling by software to shoot a picture at the moment, wherein if the magnetic microspheres 5 exist in the detection microchamber 4, the detection microchamber 4 is dark in the process, and if the magnetic microspheres 5 do not exist in the detection microchamber 4, the detection microchamber 4 is bright in the process, so that the number of empty detection microchambers 4 can be conveniently deducted in the next step;
3. injecting 100ul of developing solution into the detection micro-chamber 4, coating a layer of fluorinated oil to isolate micro-areas, and flushing away redundant magnetic microspheres 5;
4. and the computer compares and analyzes the two images acquired by the image acquisition PCB (image acquisition circuit 3) and calculates to obtain the quantity of the substances to be detected of the template.
Example 3
Unlike example 2, this example replaces the enzyme bound to the secondary antibody with the fluorescent dye CY 3. Laser excitation imaging is used.
The CMOS image sensor 2 has a 1.2-inch black-and-white CMOS pixel count of 2048(H) x 2048(V) and a pixel size of 6.5x 6.5(μm). And the internal integrated refrigeration module is adopted, so that the interference of dark noise is reduced. The diameter of each optical fiber of the panel of the detection optical fiber image transmission bundle 1 is 6.5 micrometers, the diameter of each optical fiber corresponds to the pixels one by one, a 2mm micro-groove is formed above the optical fiber panel and used for containing a fluorinated oil solution, the size of each micro-hole is 4 micrometers in diameter and 4 micrometers in depth, the center distance is 6.5 micrometers, and the micro-holes are arranged in the center of the optical fibers and correspond to the pixels below the micro-holes. A white light source is arranged above the device during detection.
Adopting a human interleukin IL-8 kit to carry out ELISA reaction, fixing an IL-8 antibody on a magnetic microsphere 5 with the diameter of 3.7um, carrying out ELISA reaction in a test tube, adding an antigen to be detected, washing and incubating, adding a biotin antibody, washing and incubating, adding streptavidin marked with CY3, incubating and washing, injecting a suspension of the magnetic microsphere 5 after reaction into a detection microchamber 4 on the upper end surface of an optical fiber panel through a pipette gun, depositing for 2 minutes, waiting for the magnetic microsphere 5 to fall into a micropore by virtue of gravity, slowly adding fluorinated oil from one side, pushing the magnetic microsphere 5 staying on the surface of the panel into the micropore under the action of the fluorinated oil, and taking away the redundant magnetic microsphere 5 to prevent interference detection. During detection, a bright field image at the moment is shot by using white light illumination, the image is processed through imageJ software, appropriate threshold binarization processing is set, and holes with magnetic microspheres 5 and holes without the magnetic microspheres 5 can be distinguished. Then the white light is turned off, the laser excitation cy3 is used for shooting a fluorescence image, the fluorescence image is shot after exposure for 2000ms, then the number of the fluorescence holes and the holes containing the magnetic microspheres 5 is calculated, and the percentage of the fluorescence micropores is calculated by the ratio of the fluorescence holes and the holes of the magnetic microspheres 5.
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, imaging and the like.
The sampling operation steps are as follows:
1. taking serum of a sample to be detected, placing whole blood for 1 hour at room temperature, standing, centrifuging at 1500g for 10 minutes after the whole blood is naturally coagulated and the serum is separated out, taking supernatant fluid, namely the serum, and refrigerating the prepared serum at 4 ℃ for later use;
2. serum was mixed with sample assay buffer at 1: 5, dilution in proportion;
3. equilibrating purchased different biotin antibody reagents for 20 minutes at room temperature;
4. preparing a washing solution, and diluting the washing solution (20X) to 1X with deionized water.
A detection operation step:
1. sample adding: adding 50ul of biotin labeled antibody and a sample to be detected into a magnetic microsphere 5 test tube combined with anti-human IL-8, slightly shaking for 1min by covering a cover, uniformly mixing, and incubating at room temperature for 2 h;
2. washing: washing with 300ul of washing solution for 3 times, each time for 30 seconds, and then patting the paper on absorbent paper to be dry after each time of washing. If the plate washing machine is used for washing, the washing times are increased once;
3. SA-CY 3: adding SA-CY3100ul, covering with a cover, gently shaking, mixing, and incubating at room temperature for 30 min;
4. washing: the liquid in the test tube is poured, the washing liquid is washed for 5 times by 300ul, each time is 30 seconds, the water-absorbent paper is dried after the washing is finished, and the washing times are increased once if a plate washing machine is used for washing.
An imaging operation step:
1. pouring the magnetic microspheres 5 operated in the previous step into the detection optical fiber image transmission bundle 1, electrifying a coil 7 to control a magnetic field to accelerate the magnetic microspheres 5 to fall into the detection micro-chamber 4, and scraping the magnetic microspheres 5 by using a paper board to enable the magnetic microspheres 5 to enter the detection micro-chamber 4;
2. using an LED to illuminate the upper surface of the optical fiber image transmission bundle 1, controlling by software to shoot a picture at the moment, wherein if the magnetic microspheres 5 exist in the detection microchamber 4, the detection microchamber 4 is dark in the process, and if the magnetic microspheres 5 do not exist in the detection microchamber 4, the detection microchamber 4 is bright in the process, so that the number of empty detection microchambers 4 can be conveniently deducted in the next step;
3. illuminating the upper surface of the optical fiber image transmission bundle 1 by using a green light source, controlling by software to shoot a picture at the moment, wherein if the magnetic microsphere 5 exists in the detection microchamber 4 and the magnetic microsphere 5 is combined with an antigen to be detected, the detection microchamber 4 is bright in the process, and if the magnetic microsphere 5 exists in the detection microchamber 4 and the magnetic microsphere 5 is not combined with the antigen to be detected, the detection microchamber 4 is dark in the process without CY 3;
4. and the computer compares and analyzes the two images acquired by the image acquisition PCB (image acquisition circuit 3) and calculates to obtain the quantity of the substances to be detected of the template.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.
Claims (10)
1. A digital ELISA detection device, characterized in that: the device comprises an optical fiber image transmission bundle (1), an image sensor (2), an image acquisition circuit (3), a detection microchamber (4), magnetic microspheres (5) and a coil (7); the upper end surface of the optical fiber image transmission bundle (1) is provided with at least 2 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 an image acquisition circuit (3); the outer side of the optical fiber image transmission bundle (1) is provided with a coil (7), a magnetic field is generated through the coil (7), and the magnetic microspheres (5) enter the detection microchamber (4).
2. The digital ELISA detection device of claim 1, wherein: the diameter of the detection micro-chamber (4) is less than 100 microns, and the depth of the detection micro-chamber (4) is less than 100 microns.
3. The digital ELISA detection device of claim 1, wherein: each detection microchamber (4) is internally provided with a magnetic microsphere (5); the magnetic microspheres (5) are modified with specific antibodies (6).
4. The digital ELISA detection device of claim 3, wherein: the specific antibody (5) comprises an antibody, an antigen and a labeled secondary antibody which are sequentially arranged.
5. The digital ELISA detection device of claim 4, wherein: the label of the labeled secondary antibody is an enzyme label or a fluorescent label.
6. The digital ELISA detection device of claim 5, wherein: the enzyme label is horseradish peroxidase or alkaline phosphatase; the fluorescent label is a fluorescent dye label, a fluorescent quantum dot label or an up-conversion luminescent label.
7. The digital ELISA detection device of claim 1, wherein: the image acquisition circuit (3) comprises a FLASH module, an FPGA module, a voltage control module and an interface module;
the FLASH module is used for temporarily storing videos or images;
the FPGA module is used as a control core for processing the image;
the voltage control module is used for providing a stable power supply for the circuit board;
and the interface module is used for transmitting the acquired image to a computer to realize communication with external equipment.
8. The digital ELISA detection device of claim 1, wherein: the image sensor (2) is a CCD image sensor or a CMOS image sensor.
9. A method for detecting by using the digital ELISA detection device of claims 1-8, wherein: when the label of the labeled secondary antibody is an enzyme label, the detection method comprises the following steps:
(a) modifying the surface of the magnetic microsphere (5) with a specific antibody (5) to be detected;
(b) the magnetic microspheres (5) are flatly laid in the detection microchamber (4), the coil (7) is electrified to control the magnetic field, and the magnetic microspheres (5) fall into the detection microchamber (4);
(c) adding a color developing solution into the micropore array on the surface of the optical fiber image transmission bundle (1), coating a layer of fluorinated oil for micro-area isolation, and flushing away redundant magnetic microspheres (5);
(d) a bright field light source irradiates the upper surface of the optical fiber image transmission beam (1), a picture is shot under the control of software, if magnetic microspheres (5) exist in the detection microchamber (4), the detection microchamber (4) is dark, if no magnetic microspheres (5) exist in the detection microchamber (4), the detection microchamber (4) is bright, a proper threshold value is set to distinguish the number of bright and dark microchambers, and the total number of the dark microchambers is counted as Na；
(e) The light source is turned off, the computer controls the long exposure through the image acquisition circuit (3), the picture at the moment is shot, and the number of the bright microchambers is counted as N in the processl；
(f) Comparing and analyzing the first two images through NaAnd NlAnd calculating to obtain the quantity of the substance to be detected of the template.
10. A method for detecting by using the digital ELISA detection device of claims 1-8, wherein: when the label of the labeled secondary antibody is a fluorescent dye label, the method comprises the following steps:
(a) modifying the surface of the magnetic microsphere (5) with a specific antibody (5) to be detected;
(b) pouring the magnetic microspheres (5) into the detection optical fiber image transmission bundle (1), electrifying a coil to control a magnetic field, enabling the magnetic microspheres (5) to fall into the detection micro-chamber (4), and scraping the magnetic microspheres (5) by using a paper board to enable the magnetic microspheres to enter the detection micro-chamber (4); coating a layer of fluorinated oil to isolate micro-areas, and flushing away redundant magnetic microspheres (5);
(c) the light source irradiates the upper surface of the optical fiber image transmission beam (1), the software controls the shooting of pictures, if the magnetic microspheres (5) exist in the detection microchamber (4), the detection microchamber (4) is dark, and the total number of the dark microchambers is counted to be Na(ii) a If there is no magnetic microsphere (5) in the microchamber, the detection microchamber (4) is bright;
(d) irradiating the upper surface of the optical fiber image transmission beam (1) by using light with a fluorescent dye excitation wavelength, controlling by software to shoot a picture at the moment, wherein if a magnetic microsphere (5) is arranged in the detection microchamber (4) and the magnetic microsphere (5) is combined with an antigen to be detected, the detection microchamber (4) is bright, and the number of the bright microchambers is NlIf the magnetic microsphere (5) is arranged in the detection micro-chamber (4) and the antigen to be detected is not combined with the magnetic microsphere (5), no fluorescence exists, and the detection micro-chamber (4) is dark;
(e) the computer compares and analyzes the two images acquired by the image acquisition circuit (3) through NaAnd NlAnd calculating to obtain the quantity of the substance to be detected of the template.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117388227A (en) * | 2023-10-18 | 2024-01-12 | 博瑞生物医药（苏州）股份有限公司 | Method, computer readable medium and analysis device for determining concentration of target molecules in sample using detection microspheres |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080160639A1 (en) * | 2006-12-29 | 2008-07-03 | Xina Su | Programmable electromagnetic array for molecule transport |
| CN108479875A (en) * | 2018-06-01 | 2018-09-04 | 东莞东阳光科研发有限公司 | A kind of digital microcurrent-controlled chip report control platform |
| CN108993621A (en) * | 2018-07-09 | 2018-12-14 | 浙江大学 | A kind of small room array micro-fluidic chip and method for digital enzyme linked immunosorbent detection |
| CN109661580A (en) * | 2016-07-29 | 2019-04-19 | 塞尔玛生物技术有限责任公司 | The improvement of digital counting method |
| CN109718873A (en) * | 2017-10-31 | 2019-05-07 | 中国科学院大连化学物理研究所 | The polynary immune response system of micro- magnetic bead based on digital drop micro-fluidic chip |
-
2020
- 2020-02-21 CN CN202010108352.4A patent/CN111273000A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080160639A1 (en) * | 2006-12-29 | 2008-07-03 | Xina Su | Programmable electromagnetic array for molecule transport |
| CN109661580A (en) * | 2016-07-29 | 2019-04-19 | 塞尔玛生物技术有限责任公司 | The improvement of digital counting method |
| CN109718873A (en) * | 2017-10-31 | 2019-05-07 | 中国科学院大连化学物理研究所 | The polynary immune response system of micro- magnetic bead based on digital drop micro-fluidic chip |
| CN108479875A (en) * | 2018-06-01 | 2018-09-04 | 东莞东阳光科研发有限公司 | A kind of digital microcurrent-controlled chip report control platform |
| CN108993621A (en) * | 2018-07-09 | 2018-12-14 | 浙江大学 | A kind of small room array micro-fluidic chip and method for digital enzyme linked immunosorbent detection |
Non-Patent Citations (3)
| Title |
|---|
| HUAIBIN ZHANG, ET AL: "Oil-sealed femtoliter fiber-optic arrays for single molecule analysis", 《 LAB CHIP》 * |
| KAREN LEIRS, ET AL: "Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA", 《ANAL. CHEM.》 * |
| KENJI AKAMA,ET AL: "Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis", 《ACS NANO》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117388227A (en) * | 2023-10-18 | 2024-01-12 | 博瑞生物医药（苏州）股份有限公司 | Method, computer readable medium and analysis device for determining concentration of target molecules in sample using detection microspheres |
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