CN111323595A - Multi-element visual pathological section examination method based on colloidal crystal microspheres - Google Patents
Multi-element visual pathological section examination method based on colloidal crystal microspheres Download PDFInfo
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Abstract
The invention provides a colloidal crystal microsphere-based multi-element visual pathological section examination method, which comprises the following steps: 1) coupling the colloidal crystal microspheres with antibodies; 2) dispersing microspheres coupled with an antibody in a phosphate buffer solution to obtain a microsphere phosphate mixed solution, then soaking pathological sections in the microsphere phosphate mixed solution, carrying out specific binding and incubation on an antigen to be detected, taking out the pathological sections, and washing to remove redundant microspheres; 3) and (5) drying the washed section, and observing the distribution condition of the microspheres on the pathological section by naked eyes. The visual pathological section examination method based on the colloidal crystal microspheres has the advantages of rapid real-time detection, improvement of detection efficiency and completion of diversified detection by one pathological section.
Description
Technical Field
The invention relates to a colloidal crystal microsphere-based multi-element visual pathological section examination method, and belongs to the technical field of biomedical research and clinical detection and diagnosis.
Background
In recent years, the incidence and mortality of tumors have been on the rise year by year. According to the report of the World Health Organization (WHO), more than 1200 ten thousand new tumor cases in 2010 and 760 ten thousand cancer deaths, cancer becomes the first cause of death of young and strong human beings. At present, the main treatment modes of tumors comprise operations, chemotherapy, radiotherapy, targeted therapy and immunotherapy, and various medicaments are applied to tumor treatment. However, the above treatment methods have various problems of postoperative recurrence, various complications, high treatment price and the like. The most effective means for tumor diagnosis and timely treatment still exist at present, and various deaths caused by cancer can be avoided. However, currently, a single tumor marker is often used in clinical diagnosis, and has the problems of low sensitivity or specificity, high false positive rate or false negative rate and the like, and the detection of a single tumor marker can cause over-diagnosis or missed diagnosis. Therefore, the development of a multiplex visualization pathological section examination method is of great significance in tumor diagnosis.
The current multiplex detection methods are mainly divided into fixed identification based on biomolecules and fluorescent dye coded identification based on flow carriers. In the former, different coded biomolecules are placed in porous plates isolated from each other by a microarray biochip or fixed on a glass substrate, and are coded by two-dimensional coordinates of the substrate. Compared with the wide and efficient detection capability, the detection speed is limited by the diffusion speed of the target molecules, the reaction speed of the probe and the detection target molecules is greatly reduced, and the detection time is usually longer. The latter can accomplish high-throughput rapid detection, can greatly shorten reaction time, improve sensitivity and reduce the amount of samples required for detection. However, fluorescent dyes have the problems of difficult purification, high price, unstable molecules, mutual interference among fluorescence and the like, and false negative detection results are often caused by fluorescence quenching.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a colloidal crystal microsphere-based multi-element visual pathological section examination method, which solves the defects of overlong detection time and false negative caused by extraction and extinction of fluorescent dye based on a fixed microarray and flow carrier multi-element detection method.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a colloidal crystal microsphere-based multi-element visual pathological section examination method, which comprises the following steps:
1) coupling the colloidal crystal microspheres with antibodies: preparing nanoparticles into colored microspheres by a microfluidic technology, coupling antibody molecules on the surfaces of the colored microspheres, and sealing redundant sites on the surfaces of the microspheres to obtain the microspheres coupled with the antibodies;
2) dispersing microspheres coupled with an antibody in a phosphate buffer solution, then soaking pathological sections in the phosphate buffer solution, carrying out specific binding and incubation on an antigen to be detected, taking out the pathological sections, and washing to remove redundant microspheres;
3) and drying the washed section, observing the distribution condition of the detection target on the pathological section by naked eyes, and observing the finer distribution under a microscope, wherein the part displaying the color of the microsphere represents that the antibody-antigen binding reaction of the antigen to be detected and the corresponding antibody molecule occurs, and determining the type of the antigen carried by the pathological section.
Wherein:
the nano particles in the step 1) are solid silica nano particles, mesoporous silica nano particles or polystyrene nano particles.
The diameters of the three nano particles in the step 1) are 100-300 nm, and the diameter of the colored microsphere is 10-60 mu m.
The antibody molecules are coupled on the surface of the colored microspheres, the types of the antibody molecules correspond to the colors of the microspheres one by one, namely, the antibody molecules of different types are coupled with the microspheres of different colors.
Coupling antibody molecules to the surface of the colored microspheres in the step 1), and then sealing redundant sites on the surface of the microspheres, namely coupling the antibody molecules to the microspheres by a chemical coupling method, and then cleaning the microspheres by phosphate buffer solution and sealing the redundant sites by bovine serum albumin, specifically comprising the following steps: sequentially soaking colored microspheres in 3-5 wt% 3-aminopropyltriethoxysilane alcohol solution for 3-6 hours and 3-5 wt% glutaraldehyde aqueous solution for 2-5 hours, washing the microspheres with phosphate Tween 20 buffer solution for multiple times to remove redundant glutaraldehyde, soaking the microspheres in 100-200 mu g/ml antibody phosphate buffer solution for overnight at 2-4 ℃, blocking the microspheres with 0.8-1 wt% bovine serum albumin, and removing redundant bovine serum albumin molecules with the phosphate buffer solution to obtain the antibody-coupled microspheres.
The chemical coupling method is condensation reaction of amino and aldehyde group.
Dispersing the microspheres of the coupled antibody prepared in the step 1) in a phosphate buffer solution at the temperature of 2-6 ℃ for later use.
Dispersing the microspheres of the coupled antibody in the phosphate buffer solution to obtain a microsphere phosphate mixed solution, wherein the microspheres of the coupled antibody have the same or different colors, only one antigen to be detected can be detected if the microspheres of the coupled antibody have the same color, and a plurality of antigens to be detected can be detected if the microspheres of the coupled antibody have different colors.
After the target to be detected in the step 2) is specifically combined and incubated, taking out the pathological section, washing to remove redundant microspheres, namely incubating for 30-60 min at the temperature of 36-38 ℃, washing the pathological section for 2-3 times by using a phosphate buffer solution, and washing to remove redundant microspheres.
The detection target in the step 2) is a biomolecule and comprises one or more of small molecule compounds, hormones, enzymes, antigen antibodies, neurotransmitters, cytokines, growth factors or tumor markers.
The step 3) of drying the washed slices refers to drying at room temperature.
In the detection process of the step 3), what color microspheres remained on the pathological section after the antigen-antibody reaction can be observed by naked eyes, so that the type of the antigen contained in the pathological section can be determined qualitatively.
Has the advantages that: the invention uses the optical coding colloidal crystal microsphere as the carrier to realize the examination of the multi-element visual pathological section, compared with the prior art, the invention has the following advantages:
1. due to the use of the colloidal crystal microspheres with different colors, the problem of color quenching can not occur, and color difference can be identified under the assistance of naked eyes or a microscope, so that biomolecules contained in pathological sections can be rapidly evaluated, and the purpose of pathological diagnosis can be achieved;
2. the colloidal crystal microspheres with various colors can be used for realizing the detection of multiple indexes of a single pathological section, so that the steps of dyeing, dewatering and sealing in the traditional pathological section process are omitted, and the detection efficiency is improved;
3. the detection method is simple and easy to implement, accurate and quick in detection, and capable of reducing operation errors of operators.
Drawings
FIG. 1 colloidal crystal microspheres under a microscope;
fig. 2 is a schematic diagram of a multi-element visual pathological section examination based on colloidal crystal microspheres.
Detailed Description
The invention provides a colloidal crystal microsphere-based multi-element visual pathological section examination method, which comprises the following steps: 1) coupling the colloidal crystal microspheres with an antibody to obtain antibody-bound colloidal crystal microspheres; 2) the colloidal crystal microsphere combined with the antibody is fully contacted with the surface of the pathological section, so that the antibody on the surface of the microsphere is combined with the antigen in the section tissue, and the microsphere with the color is remained at the corresponding position. The visual pathological section examination method based on the colloidal crystal microspheres has the advantages of rapid real-time detection, improvement of detection efficiency and realization of diversified detection by one pathological section.
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples, in which FIG. 1 is colloidal crystalline microspheres under a microscope; fig. 2 is a visual pathological section detection method, which mainly comprises the following steps: 1) preparing a target pathological paraffin section, selecting optical coding colloidal crystal microspheres coupled with corresponding antibodies according to a detection target, and soaking the pathological section in phosphate buffer solution of the microspheres for specific binding reaction; 2) observing the color types of the microspheres remained on the pathological section, and qualitatively determining the antigen types on the pathological section.
Example 1
A multi-element visual pathological section examination method (prostate cancer tumor index detection) based on colloidal crystal microspheres comprises the following specific steps:
1) coupling the colloidal crystal microspheres with antibodies: solid silica nanoparticles with the diameters of 140nm, 220nm and 260nm are adopted to prepare colloid crystal microspheres with the diameters of 10 microns and respectively presenting blue, orange and red colors by a microfluidic device, wherein the surface of the blue microsphere is coupled and coated with a prostate cancer specific antigen antibody (PSA), the surface of the orange microsphere is coupled and coated with a prostate cancer surface antigen antibody (PSMA), and the surface of the red microsphere is coupled and coated with a novel prostate cancer antigen 3(PCA3) antibody (the coupling and coating process is as follows, the colloid crystal microspheres are sequentially soaked in 3 wt% 3-aminopropyl triethoxysilane alcohol solution for 6h and 5 wt% glutaraldehyde aqueous solution for 2h, then washed for many times by phosphate Tween 20 buffer solution to remove redundant glutaraldehyde, then the microspheres are soaked in 100 μ g/ml antibody phosphate buffer solution and are kept overnight at the temperature of 2 ℃); finally, blocking redundant aldehyde group sites on the surface of the microspheres by using 1 wt% of bovine serum albumin, cleaning the microspheres by using a phosphate buffer solution again, and then respectively soaking the microspheres in a PBS buffer solution at the temperature of 2 ℃ for later use;
2) dispersing microspheres coated with different antibodies in phosphate buffer solution with the temperature of 2 ℃ and the pH value of 7.2, then inserting the microspheres into pathological sections of the prostate cancer, incubating the pathological sections at the temperature of 36 ℃ for 30min, and then taking out the pathological sections and washing redundant microspheres by PBS;
3) and (4) drying the washed section, observing the color type of the residual microspheres on the pathological section by naked eyes, and further observing the color of the residual microspheres under a microscope. The presence of a large amount of blue, orange and red microspheres is found, so that the content of PSA, PSMA and PCA3 in the pathological section is judged to be high.
Example 2
A multi-element visual pathological section examination method (breast cancer tumor index detection) based on colloidal crystal microspheres comprises the following specific steps:
1) coupling the colloidal crystal microspheres with antibodies: mesoporous silica nano particles with the diameters of 100nm, 160nm, 210nm and 300nm are adopted to prepare colloid crystal microspheres with the diameters of 60 mu m and respectively presenting four colors of purple, green, orange and red through a microfluidic device, wherein the surfaces of the purple microspheres are coupled and coated with Estrogen Receptor (ER), the surfaces of the green microspheres are coupled and coated with Progestogen Receptor (PR), the surfaces of the orange microspheres are coupled and coated with human epidermal growth factor receptor 2(HER-2) and the surfaces of the red microspheres are coupled and coated with cell proliferation related nuclear antigen (Ki-67) (the coupling coating process is that the colloid crystal microspheres are sequentially soaked in 5 wt% 3-aminopropyl triethoxy silane alcohol solution for 3h and 3 wt% glutaraldehyde aqueous solution for 2h, then phosphate 20 buffer solution is used for washing to remove redundant glutaraldehyde, and then the microspheres are soaked in 200 mu g/ml corresponding antibody phosphate buffer solution, overnight at 4 ℃). Washing the microspheres coated with different antibodies twice by using Phosphate Buffer Solution (PBS), blocking redundant aldehyde sites on the surfaces by using 0.8 wt% Bovine Serum Albumin (BSA), washing by using Phosphate Buffer Solution (PBS) again, and then respectively soaking in the PBS at 4 ℃ for later use;
2) dispersing microspheres coated with different antibodies in phosphate buffer solution with the temperature of 4 ℃ and the pH of 7.6, then inserting the microspheres into pathological sections of breast cancer patients, incubating for 60min at the temperature of 37 ℃, and then washing away redundant microspheres by PBS;
3) and (4) drying the washed section, observing the color type of the residual microspheres on the pathological section by naked eyes, and further observing the color of the residual microspheres under a microscope. A large amount of purple, green and red microspheres exist, and only a small amount of orange microspheres remain, so that the pathological section is judged to contain more ER, PR and HER-2.
Example 3
A colloidal crystal microsphere-based multi-element visual pathological section examination method (colon cancer tumor index detection) comprises the following specific steps:
1) coupling the colloidal crystal microspheres with antibodies: polystyrene nano-particles with the diameters of 150nm and 250nm are adopted to prepare colloid crystal microspheres with the diameters of 20 microns and respectively showing blue and red colors through a microfluidic device, wherein carcinoembryonic antigen (CEA) is coupled and coated on the surfaces of the blue colloid crystal microspheres, carbohydrate antigen (CA-199) is coupled and coated on the surfaces of the red colloid crystal microspheres (the coupling and coating process is that the colloid crystal microspheres are sequentially soaked in 4 wt% 3-aminopropyl triethoxysilane alcohol solution for 5h and 4 wt% glutaraldehyde aqueous solution for 3h, then the excessive glutaraldehyde is removed by washing with phosphate Tween 20 buffer solution for a plurality of times, then the microspheres are soaked in 150 mu g/ml corresponding antibody phosphate buffer solution and are kept overnight at 3 ℃), the colloid crystal microglobulin coated with different antibodies is respectively washed twice with Phosphate Buffer Solution (PBS), blocking redundant aldehyde sites on the surface by using 0.9 wt% Bovine Serum Albumin (BSA), washing by using Phosphate Buffer Solution (PBS) again, and then respectively soaking in the PBS buffer solution at 6 ℃ for later use;
2) dispersing microspheres coated with different antibodies in phosphate buffer solution with the temperature of 6 ℃ and the pH of 7.4, then inserting the microspheres into pathological sections of a colon cancer patient, incubating the pathological sections at 37 ℃ for 40min, and then washing redundant microspheres by PBS;
3) and (3) drying the washed section, observing the color type of the residual microspheres on the pathological section by naked eyes, and finding that only the red microspheres are remained on the pathological section, thereby judging that the carcinoembryonic antigen exists in the pathological section of the colon cancer.
Example 4
A multi-element visual pathological section examination method (lung cancer tumor index detection) based on colloidal crystal microspheres comprises the following specific steps:
1) coupling the colloidal crystal microspheres with antibodies: silica nano-particles with the diameters of 120nm, 150nm, 220nm and 280nm are adopted to prepare colloid crystal microspheres with the diameters of 30 mu m and respectively showing four colors of purple, green, orange and red through a microfluidic device, wherein the surfaces of the purple colloid crystal microspheres are coupled and coated with TTF-1 (thyroid transcription factor-1), the surfaces of the green colloid crystal microspheres are coupled and coated with Napsin A (aspartic proteinase A), the surfaces of the orange colloid crystal microspheres are coupled and coated with CK5/6 and the surfaces of the red colloid crystal microspheres are coupled and coated with p63 (the coupling and coating process is that the colloid crystal microspheres are sequentially soaked in 3-aminopropyl triethoxy silane alcohol solution with the weight percent of 3 for 5 hours and glutaraldehyde aqueous solution with the weight percent of 4.5 hours, then phosphate Tween 20 buffer solution is used for washing off redundant glutaraldehyde, then the microspheres are soaked in corresponding antibody phosphate buffer solution with the weight of 180 mu g/ml, overnight at 4 ℃), washing the colloidal crystal microglobulin coated with different antibodies twice with Phosphate Buffer Solution (PBS), blocking redundant aldehyde sites on the surface with 1 wt% Bovine Serum Albumin (BSA), washing with Phosphate Buffer Solution (PBS), and then soaking in PBS buffer solution at 4 ℃ for later use;
2) dispersing microspheres coated with different antibodies in phosphate buffer solution with the temperature of 6 ℃ and the pH of 7.4, then inserting the microspheres into pathological sections of lung cancer patients, incubating for 50min at 37 ℃, and then washing away redundant microspheres by PBS;
3) and (4) drying the washed section, observing the color type of the residual microspheres on the pathological section by naked eyes, and further observing the color of the residual microspheres under a microscope. Purple and green microspheres were found to be present in large amounts, while orange and red microspheres were found to have only a small residue, thereby judging that the pathological section belongs to lung adenocarcinoma (TTF-1 and Napsin A belong to common immune markers for lung adenocarcinoma, and p63 and CK5/6 belong to common immune markers for lung squamous carcinoma).
Claims (10)
1. A multi-element visual pathological section examination method based on colloidal crystal microspheres is characterized in that: the method comprises the following steps:
1) coupling the colloidal crystal microspheres with antibodies: preparing nanoparticles into colored microspheres by a microfluidic technology, coupling antibody molecules on the surfaces of the colored microspheres, and sealing redundant sites on the surfaces of the microspheres to obtain the microspheres coupled with the antibodies;
2) dispersing microspheres coupled with an antibody in a phosphate buffer solution to obtain a microsphere phosphate mixed solution, then soaking pathological sections in the microsphere phosphate mixed solution, carrying out specific binding and incubation on an antigen to be detected, taking out the pathological sections, and washing to remove redundant microspheres;
3) and (3) drying the washed section, observing the distribution condition of the microspheres on the pathological section by naked eyes, and observing the finer distribution under a microscope, wherein the part displaying the color of the microspheres represents that the antibody-antigen binding reaction of the antigen to be detected and the corresponding antibody molecule occurs, so that the type of the antigen carried by the pathological section is determined.
2. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: the nano particles in the step 1) are solid silica nano particles, mesoporous silica nano particles or polystyrene nano particles.
3. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: the diameter of the nano particles in the step 1) is 100-300 nm, and the diameter of the colored microspheres is 10-60 mu m.
4. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: the antibody molecules are coupled on the surface of the colored microspheres, the types of the antibody molecules correspond to the colors of the microspheres one by one, namely, the antibody molecules of different types are coupled with the microspheres of different colors.
5. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: coupling antibody molecules to the surface of the colored microspheres in the step 1), and then sealing redundant sites on the surfaces of the colored microspheres, namely coupling the antibody molecules to the nanoparticles on the surfaces of the microspheres by a chemical coupling method, and then cleaning the coupling by phosphate buffer solution and sealing the redundant sites by bovine serum albumin, which specifically comprises the following steps: sequentially soaking colored microspheres in 3-5 wt% 3-aminopropyltriethoxysilane alcohol solution for 3-6 hours and 3-5 wt% glutaraldehyde aqueous solution for 2-5 hours, washing the microspheres for multiple times by phosphate Tween 20 buffer solution to remove redundant glutaraldehyde, soaking the microspheres in 100-200 mu g/ml antibody phosphate buffer solution for overnight at 2-4 ℃, blocking the microspheres by 0.8-1 wt% bovine serum albumin, and removing redundant bovine serum albumin molecules by using the phosphate buffer solution to obtain the antibody-coupled microspheres.
6. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: dispersing the microspheres of the coupled antibody prepared in the step 1) in a phosphate buffer solution at the temperature of 2-6 ℃ for later use.
7. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: dispersing the microspheres coupled with the antibody in a phosphate buffer solution to obtain a microsphere phosphate mixed solution, wherein the color of the microspheres coupled with the antibody is the same or different, the temperature of the phosphate buffer solution is 2-6 ℃, and the pH value is 7.2-7.6.
8. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: after the target to be detected in the step 2) is specifically combined and incubated, taking out the pathological section, washing to remove redundant microspheres, namely incubating for 30-60 min at the temperature of 36-38 ℃, washing the pathological section for 2-3 times by using a phosphate buffer solution, and washing to remove redundant microspheres.
9. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: the detection target in the step 2) is a biomolecule and comprises one or more of small molecule compounds, hormones, enzymes, antigen antibodies, neurotransmitters, cytokines, growth factors or tumor markers.
10. The method for examining the multivariate visual pathological section based on the colloidal crystal microspheres as claimed in claim 1, wherein the method comprises the following steps: the step 3) of drying the washed slices refers to drying at room temperature.
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