CN112083160A - Preparation and application of quantum dot immunofluorescence kit for detecting cervical cancer - Google Patents

Preparation and application of quantum dot immunofluorescence kit for detecting cervical cancer Download PDF

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CN112083160A
CN112083160A CN201910519258.5A CN201910519258A CN112083160A CN 112083160 A CN112083160 A CN 112083160A CN 201910519258 A CN201910519258 A CN 201910519258A CN 112083160 A CN112083160 A CN 112083160A
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冯颖淑
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57411—Specifically defined cancers of cervix

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Abstract

The invention discloses preparation and application of a quantum dot immunofluorescence kit for detecting cervical cancer. The kit comprises: specific recognition of p on cervical cancer cells16Monoclonal antibody p of antigen16INK4ABiotinylated rabbit anti-mouse IgG, and quantum dot-labeled streptavidin complex. The quantum dot labeled streptavidin complex can recognize biotin-p16Antigen-antibody complex, forming specific probe for target binding to cervical cancer cell. Unlinked monoclonal antibody p16INK4AThe quantum dots of (a) do not bind or bind very little to cervical cancer cells. The quantum dot immunofluorescence kit prepared by the method has wide application prospect in cervical cancer diagnosis.

Description

Preparation and application of quantum dot immunofluorescence kit for detecting cervical cancer
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a preparation method and application of a quantum dot immunofluorescence kit for detecting cervical cancer. Is suitable for accurate and quantitative detection of cervical cancer cells and tissue antigens and quality control in QD-SA complex application, and also relates to application of the kit in quantitative detection of the antigens in the cervical cancer cells and tissues.
Background
Cervical cancer is a gynecological malignant tumor which seriously threatens female health at present, and the incidence of the gynecological malignant tumor is gradually younger and the incidence rate of the gynecological malignant tumor is gradually increased year by year. With data statistics, about 50 ten thousand new cases of cervical cancer worldwide each year, 2313 ten thousand women die of the disease, 80 percent of the new cases of cervical cancer occur in developing countries, and the number of new cases of cervical cancer in China accounts for 1/3 of the worldwide morbidity each year. Therefore, the improvement of the early diagnosis level of the cervical cancer has important significance for the prognosis of the cervical cancer. The traditional cervical cancer diagnosis methods include imaging examination (such as colposcopy), laboratory examination (such as tumor marker determination, cytology examination and HPV detection) and pathological tissue biopsy (such as cervical conization and cervical tissue biopsy). Traditional examination methods are inherently practical but have drawbacks, and therefore, establishing new diagnostic and screening techniques is critical to reducing mortality from cervical cancer.
The quantum dot is a novel nano luminescent particle developed in recent years, and compared with the traditional dye, the quantum dot has the unique optical characteristics of high fluorescence intensity, long fluorescence service life, strong photobleaching resistance, wide excitation spectrum, narrow emission spectrum, capability of simultaneously exciting multiple fluorescence and the like. These characteristics make it very widely used in the biomedical field, especially as a new marker in the molecular pathology and in vitro imaging diagnosis of tumors. The detection of the QDs on the cervical cancer is realized by constructing a specific probe, namely the QDs is combined with a single antibody, polypeptide or other biological micromolecules to prepare a high-quality fluorescent QD probe which is combined with a single tumor cell in a targeted manner, so that the early diagnosis of the cervical cancer is realized.
p16The gene is a multiple tumor suppressor gene and is the first gene found to act directly on the cell cycle to suppress cell division. Studies have shown that p16The deletion, mutation and methylation of genes exist in various human tumor cells, and the close relation between the deletion, mutation and methylation of the genes and the occurrence and development of tumors is suggested. In pair p16In further studies, p was examined by immunohistochemistry16After the protein is discovered, p exists in more than 90 percent of cervical cancer cases16The protein is highly expressed, and shows a gradually rising trend along with the occurrence and the development of cervical cancer diseases, but is not expressed in normal cells. Thus, p16The probe can be used as a target for specific detection of cervical cancer, and the early detection of the cervical cancer is realized by constructing a specific target probe of a quantum dot-streptavidin system-biotin-antigen-antibody complex.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a quantum dot immunofluorescence kit for detecting cervical cancer and provides application of the quantum dot kit. The kit can be combined with a biotinylated antibody which is currently used for detecting cervical cancer cells and tissue antigens, and can achieve the aim of quickly, economically, sensitively, efficiently and accurately detecting tumor antigens.
The technical scheme adopted by the invention for solving the technical problems is as follows: a quantum dot immunofluorescence kit for detecting cervical cancer is characterized in that: the kit comprises a) a stationary liquid b) a penetrating liquid c) a washing liquid d) a blocking liquid e) a monoclonal antibody P16INK4AF) biotin-rabbit anti-mouse Ig G complex G) Quantum Dot (QDs) -labeled Streptavidin (SA) complex probes; the streptavidin compound marked by the water-soluble CdSe/ZnS core-shell quantum dots is taken as a fluorescent probe to be combined with the biotinylated antigen-antibody compound on the cervical cancer cell to indirectly mark and detect the P on the cervical cancer cell16An antigen. The fixing solution is 0.01M buffer solution with pH7.6 TBS, and is prepared with paraformaldehyde solution according to the volume percentage of 100: 4. The penetrating fluid is prepared by 0.01M pH7.6 TBS buffer solution and Tween-20 according to the volume percentage of 100: 0.1. The washing solution is 0.01M pH7.6 TBS buffer solution, and is prepared with bovine serum albumin BSA according to the mass percentage of 100: 1. The blocking solution was 0.02M pH7.6 TBS buffer, prepared with normal rabbit serum at 100: 10 volume percent and bovine serum albumin BSA at 100: 5 mass percent.
The monoclonal antibody P16INK4AThe corresponding receptors are highly expressed (up to 90% or more) on cervical cancer cells and are poorly expressed on other normal cells. The quantum dots are as follows: one or the combination of any several nano particles of CdSe, CdTe, CdSe/ZnS, CdTe/CdSe, InP, InAs, InGaAs, InGaP and InGaP/ZnS.
The preparation method of the biotin-rabbit anti-mouse Ig G compound comprises the following steps:
(1) 15mg of biotin, 3.6mg of N-hydroxysuccinimide (NHSS), 2.4mg of ethyl 3- (3-dimethylamino) carbodiimide hydrochloride (EDC) were dissolved in 2mL of 0.02M PBS buffer pH 6.5;
(2) adding 5.3mg rabbit anti-mouse Ig G antibody into the solution, placing on a mixing machine at room temperature, and stirring for 30 min;
(3) carrying out decompression spin-drying on the solution to remove the solvent, dissolving deionized water in PBS and deionized water, and dialyzing for 1 d; after dialysis, the resulting solution was stored at-20 ℃.
The QD-SA complex is prepared as follows:
(1) taking 200 mul quantum dots (the concentration is 8.3 mul), adopting an active ester method, respectively adding 0.166mg EDC and 0.166mg NHS into 300 mul borate buffer solution with pH5.5 according to the mol ratio of 1: 500, and reacting for 5-10min at room temperature;
(2) adjusting the pH value of the solution to 8-8.5, adding 4.38mg of SA according to the molar ratio of 40: 1, continuously mixing uniformly for 2 hours, adding glucosamine with the final concentration of 2 percent, reacting for 45min, and stopping the reaction;
(3) coupled quantum dot marked SA compound 10000 r.min-1After centrifugation for 5min to remove a small amount of precipitate, the pellet was washed with pH7 borate buffer in 100K ultrafiltration tubes (10 volume exchange) and finally resuspended in 0.2mL of pH7 borate buffer. Glucosamine in step 2 has a molecular formula of C6H13O5N can seal the residual carboxyl on the surface of the quantum dot in the coupling product through the amino on the N, so that the nonspecific adsorption of the coupling product to tumor tissues and cells is reduced.
The application of the quantum dot kit is provided. The method is used for detecting the cervical cancer cells and comprises the following specific steps: (1) obtaining HeLa cells growing in logarithmic phase, planting the HeLa cells in a 96-well plate at 1x104Cell/well at 37 ℃ 5% CO2Incubating in an incubator for 24 hours to ensure more than 90% of cells adhere to the wall;
(2) washing with washing solution for 3 times, fixing cells with fixing solution at room temperature for 15min, and immediately washing with washing solution for 3 times;
(3) penetrating the cells for 20min at normal temperature by using penetrating fluid, washing for 3 times by using washing liquid, and sealing non-specific binding sites on the cells for 1h by using sealing liquid;
(4) removing the blocking solution, adding appropriate amount of monoclonal antibody P16INK4ACo-incubating with cells at normal temperature for 2 h; (5) washing with washing solution for 3 times (5 min each time), adding biotinylated-rabbit anti-mouse Ig G complex, incubating with cells at room temperature for 1h, and washing with washing solution for 3 times (5 min each time);
(6) adding a proper amount of QD-SA compound, reacting for 30min, washing for 3 times, removing background color, immediately observing fluorescence signals under an inverted fluorescence microscope, and quantitatively analyzing by a fluorescence signal acquisition system.
The technical scheme of the invention has the following beneficial effects:
1. the invention amplifies the quantum dot fluorescence signal by times by means of the signal amplification effect of a biotin-streptavidin system, and can realize the detection of tumor cells under the condition of lower tumor antigen expression quantity;
2. the scheme is that antibody molecules are coupled with biotin, so that the defects of reduced antibody activity and large steric hindrance caused by coupling the antibody molecules on the surfaces of quantum dots in the conventional method are overcome;
3. compared with the traditional method for immunostaining tumor cell tissues, the quantum dots have high immunofluorescence signal intensity and long duration due to the unique optical properties of the quantum dots, and the result is easy to store and repeatedly observe;
4. in the process of preparing QD-SA, glucosamine is introduced, and the amino on the glucosamine seals the residual carboxyl on the surface of the quantum dot in the coupling product, so that on one hand, the charges on the surface of the coupling product are neutralized, on the other hand, the carboxyl is sealed to be converted into neutral hydroxyl, the surface zeta potential of the QD-SA coupling product is reduced, the recovery efficiency of the coupling product can be improved, meanwhile, the nonspecific adsorption of the coupling product to tumor tissues and cells is reduced, and the background effect is reduced;
5. the method has the advantages of simple operation, easy standardization, relatively small external influence factors, good repeatability and convenient result judgment, and can achieve quantitative analysis of fluorescence intensity by collecting fluorescence signals and indirectly reflect the expression condition of the antigen on tumor tissues or cells.
Drawings
FIG. 1 shows HeLa cell diagram of cervical cancer detected by quantum dot reagent kit in vitro; (A) imaging HeLa cell quantum dots by using a biotin-streptavidin signal amplification system; (B) a conventional HeLa cell quantum dot imaging graph of a biotin-streptavidin system is not used; (C) non-specific imaging plots of HeLa cells stained with untreated (i.e. not reacted with glucosamine) quantum dots without primary antibody treatment; (D) treating the non-specific staining pattern of the HeLa cells after the quantum dots are treated by glucosamine without adding primary antibody; (E) in vitro staining lung cancer A549 cell map by using a quantum dot kit; a scale: 20 μm;
Detailed Description
Example 1 application of a quantum dot immunofluorescence kit for detecting cervical cancer in vitro on detecting cervical cancer HeLa cells by using the technical scheme of the invention, a biotinylated antibody complex and a quantum dot labeled streptavidin complex are respectively prepared and used for specific targeted combination with antigens on tumor tissues or cells.
1. The preparation method of the biotin-rabbit anti-mouse Ig G complex comprises the following steps:
(1) 15mg of biotin (Waka chemical Co., Ltd.) 3.6mg of N-hydroxysuccinimide (NHSS)2.4mg of ethyl 3- (3-dimethylamino) carbodiimide hydrochloride (EDC) (Sigma-Aldrich, USA) was dissolved in 2ml of 0.02M PBS buffer pH 6.5;
(2) adding 5.3mg rabbit anti-mouse Ig G antibody (Boster Corp.) in the above solution, stirring for 30min at room temperature;
(3) carrying out decompression spin-drying on the solution to dissolve the solvent in deionized water, and dialyzing in PBS and deionized water for 1 d; after dialysis, the resulting solution was stored at-20 ℃.
2. QD-SA complex preparation method:
(1) taking 200 mul of 8.3 mul quantum dots, adopting an active ester method, respectively adding 0.166mg EDC and 0.166mg NHS according to a molar ratio of 1: 500, dissolving in 300 mul of borate buffer solution with pH of 5.5, and reacting for 5-10min at room temperature;
(2) adjusting the pH value of the solution to 8-8.5, adding 4.38mg of SA (Hualan chemical company) according to the molar ratio of 40: 1, continuously mixing for 2h, adding glucosamine with the final concentration of 2%, and reacting for 45min to terminate the reaction;
(3) coupled quantum dot marked SA compound 10000 r.min-1After centrifugation for 5min to remove a small amount of precipitate, the pellet was washed with pH7 borate buffer in a 100K ultrafiltration tube (10-fold volume exchange) and finally resuspended in 0.2mL of pH7 borate buffer.
3. Experimental groups: HeLa cells grown in logarithmic phase (donated to the experimental center of the first subsidiary hospital of Nanchang university, Jiangxi) were obtained,planted in 96-well plates at about 1X104Cell/well at 37 ℃ 5% CO2And incubating for 24 hours in the incubator to ensure more than 90 percent of cells to adhere to the wall. Washing with washing solution for 3 times, fixing cells with fixing solution at room temperature for 15min, and immediately washing with washing solution for 3 times. Penetrating the cells with penetrating fluid at normal temperature for 20min, washing with washing solution for 3 times, and blocking the non-specific binding sites on the cells with blocking solution for 1 h. Removing the blocking solution, adding appropriate amount of monoclonal antibody P16INK4AIncubated with cells for 2h at ambient temperature. After washing with washing solution 3 times (5 min each time), biotinylated-rabbit anti-mouse Ig G complex was added and incubated with cells for 1h at room temperature, and washed with washing solution 3 times (5 min each time). Adding a proper amount of QD-SA compound, reacting for 30min, washing for 3 times to remove background color, immediately observing a fluorescence signal under an inverted fluorescence microscope, and carrying out quantitative analysis through a fluorescence signal acquisition system. Each group was repeated 3 times.
4. Control group: making four groups of control groups, planting HeLa cells with the same amount in a 96-well plate, and carrying out early-stage treatment on the same experimental group, wherein the first group is a group which does not use a biotin-streptavidin system and directly dyes the HeLa cells incubated with primary antibodies by QD-Ig G; the second group is that no monoclonal antibody P is added into cells16INK4AReplacing with 1XPBS solution with the same volume and concentration, and simultaneously dyeing HeLa cells by QD-SA which is not treated by glucosamine, wherein the rest treatment is the same as the experimental group; the third group is that no monoclonal antibody P is added into cells16INK4A1XPBS liquid with the same volume and the same concentration is used for replacing, and QD-SA treated by glucosamine is used for dyeing HeLa cells, and the rest treatment is the same as the experimental group; in the fourth group, the same number of lung cancer a549 cells were used as another control group, and all treatments were the same as in the experimental group.
5. As a result: as a result, it was found that HeLa cells using the biotin-streptavidin signal amplification system of the present invention each had high-intensity green fluorescence (FIG. 1A) while HeLa cells not using the biotin-streptavidin signal amplification system had significantly darker fluorescence intensity (FIG. 1B) without addition of monoclonal antibody P16INK4AThe treated HeLa cells showed significantly less or no green fluorescence (FIGS. 1C, 1D) but were non-specifically stained using the glucosamine-treated groupThe color was significantly weaker (fig. 1D), lung cancer a549 cells had relatively little and dark green fluorescence, and most had no green fluorescence (fig. 1E). It can be seen that the quantum dot kit provided by the invention can well identify and express p16The cervical cancer cell has high application value in cervical cancer detection.
Example 2 specific implementation procedure for detecting whether an actual patient is a cervical cancer patient using the kit of the present invention. The invention relates to a quantum dot immunofluorescence kit for detecting cervical cancer. Comprising a) a stationary liquid b) a permeate c) a wash liquid d) a blocking liquid e) a monoclonal antibody P16INK4AF) biotin-rabbit anti-mouse Ig G complex G) Quantum Dot (QDs) -labeled Streptavidin (SA) complex probes, namely QD-SA; the stationary liquid is 0.01M trihydroxy aminomethane-hydrochloric acid (TBS) buffer solution with the pH value of 7.6, and is prepared with paraformaldehyde solution according to the volume percentage of 100: 4; the penetrating fluid is prepared by 0.01M pH7.6 TBS buffer solution and Tween-20 according to the volume percentage of 100: 0.1; the washing solution is prepared by 0.01M pH7.6 TBS buffer solution and bovine serum albumin BSA according to the mass percentage of 100: 1; the blocking solution is prepared by 0.02M pH7.6 TBS buffer solution and normal rabbit serum according to the volume percentage of 100: 10 and bovine serum albumin BSA according to the mass percentage of 100: 5; carboxy functionalized quantum dots QD550 (green fluorescence 9nm) was purchased from Ocean corporation, usa.
1. The biotin-rabbit anti-mouse Ig G complex was prepared as follows:
(1) dissolving biotin (15 mg), N-hydroxysuccinimide (NHSS) (3.6 mg), and ethyl 3- (3-dimethylamino) carbodiimide hydrochloride (EDC) (2.4 mg) in PBS buffer (2 ml, 0.02M, pH 6.5);
(2) adding 5.3mg rabbit anti-mouse Ig G antibody into the solution, placing on a mixing instrument at room temperature, and stirring for 30 min;
(3) dissolving the solution in deionized water, and dialyzing for 1d in PBS and deionized water; after dialysis, the resulting solution was stored at-20 ℃.
2. The QD-SA complex was prepared as follows:
(1) taking 200 mul of 8.3 mul quantum dots, adopting an active ester method, respectively adding 0.166mg of EDC and 0.166mg of NHS according to a molar ratio of 1: 500, dissolving in 300 mul of borate buffer solution with pH of 5.5, and reacting at room temperature for 5-10 min;
(2) adjusting the pH value of the solution to 8-8.5, adding 4.38mg of SA according to the molar ratio of 40: 1, continuously mixing for 2 hours, adding glucosamine with the final concentration of 2 percent, reacting for 45 minutes, and stopping the reaction;
(3) coupled quantum dot marked SA compound 10000 r.min-1After centrifugation for 5min, a small amount of precipitate was removed, washed with pH7 borate buffer in 100K ultrafiltration tubes (10-fold volume exchange) and finally resuspended in 0.2mL of pH7 borate buffer.
3. Experimental groups: clinical cervical cancer tissues 20 and normal cervical tissues 15 were collected and confirmed by an experienced pathologist in a hospital. Each tissue was individually sectioned by a pathologist into at least 3 sections of 4 μm thickness and stained in 3 ways, one for the quantum dot kit according to the invention, one not for the biotin-streptavidin signal amplification system, and the other for traditional immunohistochemical staining.
4. As a result: the detection of cervical cancer by adopting the biotin-streptavidin signal amplification system is obviously higher in sensitivity and specificity, and has the advantages of simplicity, rapidness, high sensitivity and the like compared with the traditional immunohistochemical quantum dot detection kit.

Claims (8)

1. A quantum dot immunofluorescence kit for detecting cervical cancer, which is characterized in that: the kit comprises a) a fixing solution, b) a penetrating fluid, c) a washing solution, d) a confining liquid, e) a monoclonal antibody P16INK4A, f) a biotin-rabbit anti-mouse IgG compound, and g) a Quantum Dot (QDs) labeled Streptavidin (SA) compound probe, namely QD-SA.
2. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the fixing solution is 0.01M trihydroxy aminomethane-hydrochloric acid (TBS) buffer solution with pH7.6, and is prepared with paraformaldehyde solution according to the volume percentage of 100: 4.
3. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the penetrating fluid is 0.01M pH7.6 TBS buffer solution, and is prepared with Tween-20 in the volume ratio of 100 to 0.1.
4. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the washing solution is 0.01M pH7.6 TBS buffer solution, and is prepared with bovine serum albumin BSA according to the mass percentage of 100: 1.
5. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the blocking solution is 0.02M buffer solution with pH7.6 TBS, is prepared with normal rabbit serum according to the volume percentage of 100: 10 and is prepared with bovine serum albumin BSA according to the mass percentage of 100: 5.
6. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the quantum dots are one or the combination of any several nano particles of CdSe, CdTe, CdSe/ZnS, CdTe/CdSe, InP, InAs, InGaAs, InGaP and InGaP/ZnS.
7. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the preparation method of the biotin-rabbit anti-mouse IgG compound comprises the following steps:
(1) 15mg of biotin, 3.6mg of N-hydroxysuccinimide (NHSS), 2.4mg of ethyl 3- (3-dimethylamino) carbodiimide hydrochloride (EDC) were dissolved in 2mL of 0.02M PBS buffer pH 6.5;
(2) adding 5.3mg rabbit anti-mouse Ig G antibody into the solution, placing on a mixing machine at room temperature, and stirring for 30 min;
(3) carrying out decompression spin-drying on the solution to dissolve the solution in deionized water, and dialyzing the solution in PBS and deionized water for 1 d; after dialysis, the resulting solution was stored at-20 ℃.
8. The quantum dot immunofluorescence kit for detecting cervical cancer according to claim 1, wherein: the QD-SA complex is prepared as follows:
(1) taking 200 mul of 8.3 mul quantum dots, adopting an active ester method, respectively adding 0.166mg EDC and 0.166mg NHS according to a molar ratio of 1: 500, dissolving in 300 mul of borate buffer solution with pH of 5.5, and reacting for 5-10min at room temperature;
(2) adjusting the pH value of the solution to 8-8.5, adding 4.38mg of SA according to the molar ratio of 40: 1, continuously mixing uniformly for 2h, adding glucosamine with the final concentration of 2 percent, reacting for 45min, and stopping the reaction;
(3) coupled quantum dot marked SA compound 10000 r.min-1After centrifugation for 5min to remove a small amount of precipitate, the pellet was washed with pH7 borate buffer in a 100K ultrafiltration tube and finally resuspended in 0.2mL of borate buffer pH7.
CN201910519258.5A 2019-06-14 2019-06-14 Preparation and application of quantum dot immunofluorescence kit for detecting cervical cancer Pending CN112083160A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112724960A (en) * 2021-01-13 2021-04-30 北京工业大学 Dual-core shell quantum dot CdTe @ CdSe @ ZnS suitable for targeted cancer photothermal therapy
CN115097129A (en) * 2022-08-24 2022-09-23 山东子峰生物技术有限公司 Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112724960A (en) * 2021-01-13 2021-04-30 北京工业大学 Dual-core shell quantum dot CdTe @ CdSe @ ZnS suitable for targeted cancer photothermal therapy
CN115097129A (en) * 2022-08-24 2022-09-23 山东子峰生物技术有限公司 Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1
CN115097129B (en) * 2022-08-24 2023-03-10 山东子峰生物技术有限公司 Detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1

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Application publication date: 20201215