CN118858632B - Detection system for tumor early screening and application of detection system in preparation of tumor antigen detection products - Google Patents
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention provides a detection system for tumor early screening and application thereof in preparation of tumor antigen detection products, and belongs to the technical field of tumor early screening. The invention provides an application of a micro-basin array chip combined with micron-sized magnetic beads in preparing ELISA detection products for tumor early screening. The invention takes a micron-sized hydrophilic micropipe as a reaction carrier for ELISA detection, adopts a micron-sized magnetic bead to coat a capture antibody for capturing tumor antigens, and ensures high local concentration of fluorescent product molecules by limiting fluorescent groups generated by a single enzyme catalytic substrate in a very small volume so as to generate a sufficiently strong optical signal to be detected, thereby realizing detection of tumor antigens with very low concentration. Meanwhile, the invention can reduce the sample collection amount, such as blood collection amount, and the blood serum amount required to be detected can be achieved by adopting fingertip blood sampling, thereby greatly reducing the trauma degree of patients.
Description
Technical Field
The invention belongs to the technical field of tumor early screening, and particularly relates to a detection system for tumor early screening and application of the detection system in preparation of tumor antigen detection products.
Background
Malignant tumors are one of the major diseases threatening human health. Tumor screening refers to a series of targeted medical examinations conducted under healthy conditions that help detect tumors that are already present in the body at an early or treatable stage. The current methods for early screening of tumors generally include tumor marker examination, imaging examination, endoscopy, pathology examination and the like. Among them, tumor marker examinations generally include serum carcinoembryonic antigen, alpha fetoprotein, prostate specific antigen, chorionic gonadotrophin, and the like. However, at present, the tumor marker detection method has certain limitations, such as large blood consumption and low detection sensitivity.
Disclosure of Invention
The invention aims to provide a detection system for tumor early screening and application thereof in preparation of tumor antigen detection products. The detection system has the advantages of small blood consumption and high detection sensitivity.
The invention provides an application of a micro-basin array chip combined with micron-sized magnetic beads in preparation of ELISA detection products for early tumor screening, wherein the micro-basin array chip takes a hydrophobic material as a substrate, a plurality of micro-basins with upward openings are arranged on the substrate, the caliber of each micro-basin is 1.2-1.8 times of the diameter of each magnetic bead, the inner wall of each micro-basin is made of a hydrophilic material, the size of each micro-basin is micron-sized, the micro-basin is used as a reaction carrier for ELISA detection, and capture antibodies are coated on the micron-sized magnetic beads and are used for capturing tumor antigens.
The invention also provides a detection system for the tumor early screening, which comprises an antigen capture system, an antibody coupled enzymatic reaction system and a fluorescence detection system, wherein the antigen capture system comprises micron-sized magnetic beads coated with capture antibodies, the capture antibodies are used for capturing tumor antigens, the antibody coupled enzymatic reaction system comprises a micro-basin array chip and an ELISA detection reagent, the micro-basin array chip is made of a hydrophobic material, a plurality of micro-basins with upward openings are arranged on the substrate, the caliber of each micro-basin is 1.2-1.8 times of the diameter of the magnetic beads, the inner wall of each micro-basin is made of a hydrophilic material, the size of each micro-basin is in a micron-sized state, and the ELISA detection reagent comprises a biotin-labeled detection antibody, and fluorescent substrates of beta-galactosidase and beta-galactosidase coupled with streptavidin.
Preferably, each micron-sized magnetic bead is coated with 1X 10 4～1×106 capture antibodies, and the diameter of the micron-sized magnetic bead is 2.7 μm.
Preferably, the shape of the micro-basin array chip is square, the specification of the micro-basin array chip is 10mm multiplied by 10mm, the micro-basins on the micro-basin array chip are arranged in a matrix, and the number of the micro-basins on each micro-basin array chip is 1 multiplied by 10 6.
Preferably, the opening of each micro basin is a circular opening, the diameter of the opening of each micro basin is 4.5 mu m, the distance between two adjacent micro basins is 4.5 mu m, and the depth of the micro disk is 1 mu m.
Preferably, the ratio of the number of the micron-sized magnetic beads coated with the capture antibody to the volume of the serum sample to be tested is 2×10 6～2×108 pieces per 20 μl.
The invention also provides application of the detection system in preparing tumor antigen detection products.
Preferably, the tumor comprises lung cancer.
Preferably, the tumor antigen comprises one or more of CYFRA21-1, CEA, NSE, SCC and CA 125.
The invention also provides a tumor antigen detection method for non-diagnostic purposes based on the detection system according to the scheme, which comprises the following steps:
Mixing the micron-sized magnetic beads coated with the capture antibody with a sample to be tested, and performing first incubation to obtain a first incubation product;
separating the reacted magnetic beads from the first incubation product, washing, mixing with the biotin-labeled detection antibody, and performing second incubation to obtain a second incubation product;
separating the reacted magnetic beads from the second incubation product, cleaning, mixing with beta-galactosidase coupled with streptavidin, and performing third incubation to obtain a third incubation product;
adding the heavy suspension to a micro-basin array chip, absorbing redundant solution, adding a fluorescent substrate of beta-galactosidase to the micro-basin array chip for fluorescence reaction, and then carrying out oil sealing on the micro-basin array chip;
Carrying out fluorescence detection on the product after fluorescence reaction by using a fluorescence detection system, and recording the number of fluorescence signals;
And calculating the concentration of the tumor antigen in the sample to be detected according to the number of the fluorescent signals.
The invention provides an application of a micro-basin array chip combined with micro-scale magnetic beads in preparation of ELISA detection products for early tumor screening, wherein the micro-basin array chip takes a hydrophobic material as a substrate, a plurality of micro-basins with upward openings are arranged on the substrate, the caliber of each micro-basin is 1.2-1.8 times of the diameter of each micro-basin, the inner wall of each micro-basin is made of a hydrophilic material, the size of each micro-basin is in a micro-scale, the micro-basin is used as a reaction carrier for ELISA detection, the micro-scale magnetic beads are coated with capture antibodies, and the capture antibodies are used for capturing tumor antigens.
The invention takes a micron-sized hydrophilic micropipe arranged on a micropipe array chip as a reaction carrier for ELISA detection, adopts a micron-sized magnetic bead to coat a capture antibody for capturing tumor antigens, ensures high local concentration of fluorescent product molecules by limiting fluorescent groups generated by a single enzyme catalytic substrate in a very small volume so as to generate a sufficiently strong optical signal to be detected, thereby realizing detection of the tumor antigens with very low concentration. Meanwhile, the invention adopts the micron-sized hydrophilic micropipe as a reaction carrier for ELISA detection, can reduce the sample collection amount, such as blood collection amount, and can achieve the serum amount required to be detected by fingertip blood sampling, thereby greatly reducing the trauma degree of patients. The conjugate of streptavidin and enzyme is adopted, and can react with a biotin-marked detection antibody (secondary antibody) combined with antigen, so that steric hindrance is overcome. In addition, with the increase of streptavidin-enzyme conjugate, the signal intensity can be improved, and thus the measurement sensitivity can also be improved. In summary, the invention combines the micro-basin array chip with ELISA detection for the first time and is used for early screening of tumor antigens. Compared with the traditional ELISA detection of tumor antigens, the invention can realize fingertip blood sampling and can be used for detecting ultra-low concentration antigens.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a flow chart of the preparation of a micro-tub array chip;
FIG. 2 is an electron micrograph of a micro-tub array chip;
FIG. 3 is a flow chart of the key components and operations of the present invention; wherein a represents coating the micron-sized magnetic beads with a capture antibody, wherein the microspheres with captured antigen molecules can be further coupled with a biotin-labeled detection antibody, and then are mixed with beta-galactosidase coupled with streptavidin, and then a third incubation product is obtained; b represents the rapid and efficient separation of magnetic beads (microspheres) into a micropipe on a micropipe chip, c represents an electron micrograph of the magnetic beads dispersed into the micropipe, d represents the magnetic beads capturing antigen molecules, and the magnetic beads capturing the antigen molecules can convert substrates into fluorescent products through enzyme linking, so that luminescence is realized;
FIG. 4 is a graph showing the comparison of the time required for separation of magnetic beads (microspheres) using both micropipettes and microwells;
Fig. 5 is a graph of calibration test results.
Detailed Description
The invention provides an application of a micro-basin array chip combined with micron-sized magnetic beads in preparation of ELISA detection products for early tumor screening, wherein the micro-basin array chip takes a hydrophobic material as a substrate, a plurality of micro-basins with upward openings are arranged on the substrate, the caliber of each micro-basin is 1.2-1.8 times of the diameter of each magnetic bead, the inner wall of each micro-basin is made of a hydrophilic material, the size of each micro-basin is micron-sized, the micro-basin is used as a reaction carrier for ELISA detection, and capture antibodies are coated on the micron-sized magnetic beads and are used for capturing tumor antigens.
In the present invention, each of the micrometer-sized magnetic beads is preferably coated with 1X 10 4～1×106 capture antibodies, and the micrometer-sized magnetic beads preferably have a diameter of 2.7. Mu.m.
In the invention, the shape of the micro-basin array chip is preferably square, the specification of the micro-basin array chip is preferably 10mm multiplied by 10mm, the micro-basins on the micro-basin array chip are preferably arranged in a matrix, the number of the micro-basins on each micro-basin array chip is preferably 1 multiplied by 10 6, the micro-basin array chip is preferably a glass micro-basin array chip, and the diameter of the glass micro-basin array chip is preferably equal to that of the glass micro-basin array chip.
In the invention, the opening of each micro-basin is a circular opening, the diameter of the opening of each micro-basin is 4.5 mu m, the distance between two adjacent micro-basins is 4.5 mu m, and the depth of the micro-basin is 1 mu m.
In the present invention, the preparation method of the micro-tub array chip is preferably performed with reference to the prior art, particularly with reference to 【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]//2021IEEE 34th International Conference on Micro Electro Mechanical Systems(MEMS).IEEE,2021.DOI:10.1109/MEMS51782.2021.9375448.】
In the specific implementation process of the invention, the preparation method of the micro-basin array chip comprises the following steps:
Spin-coating photoresist on hydrophobic glass substrate, making specific micropore size and required micropore side wall shape on photoresist by standard ultraviolet lithography and development, depositing silicon dioxide film by physical sputtering, stripping by acetone, removing photoresist, and leaving micro basin composed of silicon dioxide contacted with hydrophobic glass substrate and silicon dioxide contacted with photoresist micropore side wall.
In the present invention, the height of the micro-tub is determined by the thickness of the photoresist, and can be controlled by selecting a specific type of photoresist and spin-coating rotational speed. The preparation method of the micro-basin array chip is that the photoresist is developed to form a unique side wall, and the side wall with radian is not a traditional vertical side wall. After sputtering the silicon dioxide film, there is a discontinuity of the film on the undercut region, creating an opening for acetone to penetrate to completely dissolve the photoresist for successful lift-off. The micron-scale hydrophilic micro-basin array chip is manufactured by the method. The method has the greatest characteristics that the method can realize a three-dimensional micro-basin structure without etching toxic hydrofluoric acid of silicon dioxide and only by using acetone for stripping. Since silica is hydrophilic and the substrate is a hydrophobicized glass, utilizing the difference between hydrophilic and hydrophobic properties, millions of individual droplets are formed in a few seconds upon contact with an aqueous solution, completing the physical separation of the target solution.
The micro-basin array chip can be produced in a large scale in a low cost manner, can reduce the cost of detection equipment, and is beneficial to clinical popularization.
The invention further provides a detection system for the tumor early screening, which comprises an antigen capture system, an antibody coupled enzymatic reaction system and a fluorescence detection system, wherein the antigen capture system comprises micron-sized magnetic beads coated with capture antibodies, the capture antibodies are used for capturing tumor antigens, the antibody coupled enzymatic reaction system comprises a micro-basin array chip and an ELISA detection reagent, the micro-basin array chip is made of a hydrophobic material, a plurality of micro-basins with upward openings are arranged on the substrate, the caliber of each micro-basin is 1.2-1.8 times of the diameter of the magnetic beads, the inner wall of each micro-basin is made of a hydrophilic material, the size of each micro-basin is in a micron-sized state, and the ELISA detection reagent comprises a biotin (biotin) marked detection antibody, and fluorescent substrates of beta-galactosidase (SβG) and beta-galactosidase coupled with streptavidin (streptoavidin).
In the present invention, each of the micrometer-sized magnetic beads is preferably coated with 1×10 4～1×106 capture antibodies, more preferably 2.5× 10 4 capture antibodies, and the micrometer-sized magnetic beads preferably have a diameter of 2.7 μm. In the present invention, the micron-sized magnetic beads are preferably purchased from Variant corporation, and the surface of the micron-sized magnetic beads is acetate-modified (carboxyl-terminal).
In the present invention, the fluorogenic substrate of the beta-galactosidase is preferably resorufin-beta-D-galactopyranoside (Resorufin-beta-D-galactopyranoside, RGP).
In the present invention, the working concentration of the biotin-labeled detection antibody is preferably 1nM.
In the invention, the working concentration of the streptavidin-coupled beta-galactosidase is preferably 1-50 pM.
In the invention, the fluorescence detection system is preferably a fluorescence microscope photographing system, and the fluorescence microscope photographing system preferably comprises a mercury lamp, an optical fiber, a microscope and a CCD camera. The fluorescent microscope photographing system is not particularly limited, and the fluorescent microscope photographing system conventional in the art can be adopted.
In one embodiment of the invention, the tumor is lung cancer, the tumor antigens are CYFRA21-1, CEA, NSE, SCC and CA125, and in the invention the capture antibodies are purchased from Elabscience. Wherein, the CYFRA21-1 antibody product number is E-KAB-0282, the CEA antibody product number is E-KAB-0016, the NSE antibody product number is E-KAB-0059, the SCC antibody product number is E-KAB-0286, and the CA125 antibody product number is E-KAB-0139.
In the invention, the shape of the micro-basin array chip is preferably square, the specification of the micro-basin array chip is preferably 10mm multiplied by 10mm, the micro-basins on the micro-basin array chip are preferably arranged in a matrix, and the number of the micro-basins on each micro-basin array chip is preferably 1 multiplied by 10 6.
In the invention, the opening of each micro-basin is a circular opening, the diameter of the opening of each micro-basin is 4.5 mu m, the distance between two adjacent micro-basins is 4.5 mu m, and the depth of the micro-basin is 1 mu m.
The invention also provides application of the detection system in preparing tumor antigen detection products.
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).
The detection system is used for detecting tumor antigens, can realize early discovery and early treatment, and improves the cure rate of cancers. The sensitivity of the invention is greatly improved, and the concentration of the disease factors lower than 1pg/ml can be detected. For certain people with high risk, early tumor screening can help them to be treated and diagnosed before disease discovery, shorten treatment time, reduce side effects of treatment, reduce treatment cost, and avoid expensive treatment cost required for advanced cancers through early discovery and treatment.
The invention also provides a tumor antigen detection method for non-diagnostic purposes based on the detection system according to the scheme, which comprises the following steps:
Mixing the micron-sized magnetic beads coated with the capture antibody with a sample to be tested, and performing first incubation to obtain a first incubation product;
separating the reacted magnetic beads from the first incubation product, washing, mixing with the biotin-labeled detection antibody, and performing second incubation to obtain a second incubation product;
separating the reacted magnetic beads from the second incubation product, cleaning, mixing with beta-galactosidase coupled with streptavidin, and performing third incubation to obtain a third incubation product;
adding the heavy suspension to a micro-basin array chip, absorbing redundant solution, adding a fluorescent substrate of beta-galactosidase to the micro-basin array chip for fluorescence reaction, and then carrying out oil sealing on the micro-basin array chip;
and (3) carrying out fluorescence detection on the product after fluorescence reaction by using a fluorescence detection system, recording the number of fluorescent signals, and calculating the concentration of tumor antigens in the sample to be detected according to the number of fluorescent signals.
The detection principle of the invention is that the micron-sized magnetic beads coated with the capture antibody are mixed with a sample to be detected, and if the sample to be detected contains a disease factor (tumor antigen), the antigen on the surface of the magnetic beads can be specifically combined with the disease factor (biomarker). Mixing the magnetic beads with the biotin-labeled detection antibody, wherein only the magnetic beads combined with the disease factors can be combined with the biotin-labeled detection antibody; and mixing the magnetic beads with a fluorescent substrate of the beta-galactosidase, wherein the fluorescent substrate of the beta-galactosidase is combined with the streptavidin, the fluorescent reaction can be carried out on the magnetic beads and the fluorescent substrate of the beta-galactosidase, the subsequent substrate is continuously converted into a fluorescent product, and the fluorescent detection is carried out to obtain a detection result.
Firstly, mixing the micron-sized magnetic beads coated with the capture antibody with a sample to be tested, and performing first incubation to obtain a first incubation product. In the present invention, the time of the first incubation is preferably 2 hours.
In the invention, the sample to be detected is preferably a serum sample obtained by centrifugation after fingertip blood sampling, the dosage of the sample to be detected is preferably 20-50 mu l, and the ratio of the number of the micron-sized magnetic beads coated with the capture antibody to the volume of the sample to be detected is preferably 2X 10 6～2×108 and 20 mu l.
In the invention, the preparation method of the micron-sized magnetic beads coated with the capture antibody preferably comprises the following steps of immersing the magnetic beads in a capture antibody solution for coating to obtain the micron-sized magnetic beads coated with the capture antibody. The invention controls the number of the surfaces of the micrometer magnetic beads by adjusting the concentration of the capture antibody.
After a first incubation product is obtained, the reacted magnetic beads are separated from the first incubation product, and the magnetic beads are mixed with a biotin-marked detection antibody after washing, and then a second incubation is carried out to obtain a second incubation product. In the present invention, the time of the second incubation is preferably 10 to 60min, more preferably 45min.
In the present invention, the working concentration of the biotin-labeled detection antibody is preferably 1nM, and the ratio of the concentration of the biotin-labeled detection antibody to the number of the capturing antibody-coated micrometer-sized magnetic beads is preferably 1 nM:6X10 7～6×109.
After the second incubation product is obtained, the reacted magnetic beads are separated from the second incubation product, and the magnetic beads are mixed with beta-galactosidase coupled with streptavidin after being washed, and then the third incubation is carried out, so that a third incubation product is obtained. In the present invention, the time of the third incubation is preferably 30min.
In the invention, the working concentration of the streptavidin-coupled beta-galactosidase is preferably 1-50 pM. In the present invention, the ratio of the concentration of streptavidin-coupled β -galactosidase to the number of capture antibody-coated micrometer-sized magnetic beads is preferably 1pm to 6×10 4～6×106.
In the present invention, the temperatures of the first incubation, the second incubation, and the third incubation are each preferably 23 ℃.
In the present invention, the reagent used for the washing is preferably PBS solution containing Tween-20 in a volume concentration of 0.1%, and the number of times of washing in each step is preferably 3.
After a third incubation product is obtained, the reacted magnetic beads are separated from the third incubation product, the magnetic beads are washed and resuspended to obtain a resuspension, the resuspension is added to a micro-basin array chip, after redundant solution is absorbed, a fluorescent substrate of beta-galactosidase is added to the micro-basin array chip to carry out fluorescence reaction, then the micro-basin array chip is subjected to oil sealing, a fluorescence detection system is used for carrying out fluorescence detection on the products after the fluorescence reaction, the number of fluorescent signals is recorded, and the concentration of tumor antigens in a sample to be detected is calculated according to the number of the fluorescent signals.
In the present invention, the solvent used for the resuspension is preferably a PBS solution.
In the present invention, the fluorogenic substrate of the beta-galactosidase is preferably used in an amount based on immersion of the microtanks.
In the invention, the excitation wavelength of the fluorescence detection is preferably 514nm or 558nm, and the emission wavelength of the fluorescence detection is preferably 577nm.
In the invention, after the heavy suspension is added to the micro basin array chip, the separation of the magnetic beads can be realized in a few seconds by utilizing the micron-sized micro basin, so that the capturing efficiency can be greatly improved, and the processing time is shortened.
The method is relatively simple to operate, easy to standardize and automate and more convenient to operate manually.
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1
1. Preparation of micro basin array chip
The preparation method of the micro-basin array chip is carried out according to the prior art, and particularly, the preparation flow chart of 【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]//2021IEEE 34th International Conference on Micro Electro Mechanical Systems(MEMS).IEEE,2021.DOI:10.1109/MEMS51782.2021.9375448.】. is shown in fig. 1.
The method comprises the following specific steps:
First, a photoresist is spin-coated on a hydrophobic glass substrate. Then, standard ultraviolet lithography and development are performed to produce specific pore sizes and desired pore sidewall shapes on the photoresist. Next, a thin film of silicon dioxide is deposited by physical sputtering and then stripped with acetone, removing the photoresist, leaving behind a micropipe made of silicon dioxide in contact with the hydrophobic glass substrate and silicon dioxide in contact with the sidewalls of the photoresist micropores.
The micro-basin array chip has the size of 10mm and 10mm, 100 ten thousand micro-basins, a single hydrophilic micro-basin (round) with the diameter of 4.5 mu m, the interval of 4.5 mu m and the depth of 1 mu m. An electron micrograph of the micro-tub array chip is shown in FIG. 2.
2. Antigen capture
The key components detected and the operational flow chart are shown in fig. 3.
The lung cancer tumor antigens to be tested are CYFRA21-1 (cytokeratin 19 fragment), CEA (carcinoembryonic antigen), NSE (neuron specific enolase), SCC (squamous cell carcinoma antigen), and CA125 (carbohydrate antigen 125).
The sources of capture antibodies were Elabscience, CYFRA21-1 antibody accession number E-KAB-0282, CEA antibody accession number E-KAB-0016, NSE antibody accession number E-KAB-0059, SCC antibody accession number E-KAB-0286, and CA125 antibody accession number E-KAB-0139. The magnetic beads used were micron-sized magnetic beads with acetate modifications on the surface, available from Variant corporation. The source of biotin-conjugated detection antibody was Elabscience, the same as the capture antibody, and an antibody pair was purchased.
The magnetic beads are pre-coated with an antibody-capture antibody directed against the selected lung cancer tumor antigen. The method comprises the following specific steps:
The capture antibody solutions were prepared separately, the solvent of the capture antibody solution was 0.1M sodium phosphate buffer (pH 7.4), and the concentration of each capture antibody in the capture antibody solution was 1nM. The volume ratio of the number of magnetic beads to the capture antibody solution was 6X 10 4～6×106 pieces per 1pM.
Immersing magnetic beads in the capture antibody solution for 45min, wherein each magnetic bead is coated with about 250000 antibodies;
Adding magnetic beads and a sample to be tested, namely mixing the coated magnetic beads with a serum sample obtained by blood sampling of 50 mu l of fingertips, and incubating for 2 hours at 23 ℃;
Washing, namely separating magnetic beads by utilizing magnetism, and washing 3 times by using PBS solution containing 0.1% Tween-20 by volume concentration;
The beads were then mixed with a biotin-conjugated detection antibody at a concentration of 1nM, incubated at 23℃for 45min, magnetically separated, washed 3 times with PBS containing 0.1% Tween-20 by volume, and 10 6 beads were incubated with streptavidin-conjugated galactosidase (50 pM) at 23℃for 30min, followed by washing the beads 3 times with PBS containing 0.1% Tween-20 by volume. Then, the magnetic beads are resuspended in 100 μl of PBS solution, and the magnetic bead solution is spread on the micro-basin array chip, then the redundant solution is sucked, the number of the magnetic beads is generally only 1000 to 10 ten thousand, and according to poisson statistical distribution, most of the micro-basins are free of magnetic beads or have one magnetic bead, so that each micro-basin is ensured to contain or not contain one magnetic bead.
3. Antibody-coupled enzymatic reactions
The reaction was started with the fluorogenic substrate resorufin-beta-D-galactopyranoside (RGP) added to 100. Mu.l of beta-galactosidase, and the micro-tub array chip was oil-sealed.
In the experiment, enzymatic reactions are carried out on control samples at different time, test results are compared, the optimal time is found, the change of fluorescence intensity along with the reaction time is detected by using a fluorescence detector, and the optimal time is required to be the earliest background brightness (noise) for realizing the fluorescence brightness exceeding 3 times.
4. Detection of fluorogenic substrates
And the micro-basin array chip after fluorescence reaction is placed under a fluorescence microscope to shoot (excitation with fluorescence detection wavelength of 514nm and emission with 577 nm), and the number of fluorescence signals is recorded. And then combining artificial intelligence to analyze the data and detect tumor antigens. Specifically, antigens with known concentrations are prepared, gradients are 0.1pM,1pM,10pM and 100pM, and the corresponding fluorescence number of each gradient is recorded to obtain a corresponding relation. And for the detection of the antigen with unknown concentration, referring to the corresponding relation, measuring to obtain the number of the luminous micro-basin, and further obtaining the concentration of the antigen to be detected.
Test example 1 comparative test of microwells and micropipettes
The specification of the microwells is 2.7 μm in width and 2 μm in height, and the proportion and time of micron-sized magnetic beads (microspheres) entering the microwells are counted through an optical microscope in the test example, wherein about 50% of the time for entering the microwells is observed to take 300s, while about 50% of the time for entering the microwells is observed to take 10s, while about 90% of the time for entering the microwells is observed to take 1200s, and about 20s. This demonstrates that the use of a micropipe can reduce the time for microspheres to enter and shorten the overall detection time.
The test results are shown in FIG. 4.
The test results show that the time required for using the microplates is reduced by more than an order of magnitude from that required for using the microwells at the step required for dispersing the magnetic beads into individual microwells or microplates.
Test example 2 comparative tests at different concentrations
In the experiment, tumor antigens were prepared at concentrations of 0.01pg/mL,0.1,1,10,100,1000,10000,100000pg/mL, and then tested in the same manner as in example 1 to count the number of luminescent micropipes.
The test results are shown in FIG. 5 and Table 1.
The test results demonstrate that the method of the present invention is capable of measuring 0.01pg/mL, at which concentration there are 9 luminescent micropipes.
TABLE 1 correspondence between actually measured antibody concentration and number of luminescent micropipes
| Concentration (pg/mL) | Number of luminous micro-basins |
| 100000 | 9000154 |
| 10000 | 2002311 |
| 1000 | 965207 |
| 100 | 95421 |
| 10 | 9982 |
| 1 | 986 |
| 0.1 | 92 |
| 0.01 | 9 |
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Claims (5)
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