CN105886596B - Cervical cancer cell detection kit - Google Patents

Cervical cancer cell detection kit Download PDF

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CN105886596B
CN105886596B CN201610266982.8A CN201610266982A CN105886596B CN 105886596 B CN105886596 B CN 105886596B CN 201610266982 A CN201610266982 A CN 201610266982A CN 105886596 B CN105886596 B CN 105886596B
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reagent
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cervical cancer
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CN105886596A (en
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吴萍
陈力
蔡称心
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Nanjing Normal University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

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Abstract

The invention discloses a cervical cancer cell detection kit based on boron-doped graphene quantum dots, and belongs to the field of biological detection. The cervical cancer cell detection kit comprises reagents A, B and C, wherein the reagent A is a boron-doped graphene quantum dot solution, the reagent B is a cerium nitrate solution, and the reagent C is an adenosine triphosphate solution. The kit selects alkaline phosphatase as a target for detection, adopts boron-doped graphene quantum dots as a fluorescent probe, realizes the rapid and efficient detection of the alkaline phosphatase on the surface of the cervical cancer cells by a quantum dot fluorescence analysis method, can be used for detecting the cervical cancer cells and the concentration thereof in the field of medicine, and has the advantages of easy operation, high accuracy of detection results, high sensitivity, low cost and the like.

Description

Cervical cancer cell detection kit
Technical Field
The invention relates to a detection kit, in particular to a cervical cancer cell detection kit, and belongs to the technical field of biological detection.
Background
Cervical cancer is a malignant tumor disease which seriously threatens the health of women at present, and the incidence rate of the malignant tumor disease is on a trend of increasing year by year. According to data statistics, about 50 ten thousand new cases of cervical cancer occur worldwide each year, and about 26 thousand women die of the disease. 85% of new cases occur in developing countries, where each year cervical cancer accounts for about 1/3 of the total number of worldwide cases in our country. Therefore, the development of a high-efficiency, convenient and high-sensitivity cervical cancer cell detection method has important significance for early diagnosis of cervical cancer.
Conventional methods for diagnosing cervical cancer include cervical iodine test, colposcopy, cervical photography, cytological examination of cervical shedding, HPV detection, conization of the cervix, and biopsy of cervical canal (20319Liy et al, prevention and early diagnosis of cervical cancer Chinese and Foreign Medical Research 2011,09(1), 112-. The traditional inspection method has practical applicability, but has disadvantages such as complex operation, low accuracy of detection result, large-scale instrument requirement and high detection cost. The spectroscopic technique is widely used in the field of disease detection due to its high sensitivity. Among them, the fluorescence spectroscopy has the advantages of large information amount, fast response speed, high sensitivity, simple operation, low cost, strong optical anti-interference capability, etc., and is receiving attention from people. Conventional fluorescent luminescent reagents are dye molecules, such as fluorescein isothiocyanate FITC (Xiuhan Yang, Xiaochuan Wang, Xiiaomin Zhang. Capillary zone electrophosphorescence separation of low concentration dyes in human urine with laser-induced fluorescence detection. Analytica Chimica Acta,2005,549(1-2),81-87.), cyanine dyes Cy3, Cy5, etc., which have some unavoidable disadvantages, such as: the photochemical stability is poor, and photobleaching and photodegradation are easy to occur, so that the observation for a long time is not facilitated. In addition, the traditional dye molecule has narrow excitation spectrum and wide emission spectrum, and is easily interfered by the autofluorescence of biomacromolecules in a complex life system. Therefore, designing and developing new fluorescence detection reagents becomes a hot spot of research.
With the research of quantum dots, the excellent optical properties (such as narrow bandwidth, high quantum yield, stable fluorescence signal, long fluorescence lifetime and the like) of the quantum dots lead the quantum dots to be applied to the research of cell and protein imaging more and more, and good results are obtained. The carbon material quantum dots are common fluorescent detection materials in cell detection due to the characteristics of excellent optical performance, good biocompatibility, easy regulation and control of emission wavelength and the like.
Disclosure of Invention
The invention aims to provide a cervical cancer cell detection kit based on boron-doped graphene quantum dots (B-GQDs), which can realize the rapid, sensitive and efficient detection of cervical cancer cells and the concentration thereof through the change of fluorescence signals of the B-GQDs.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
the cervical cancer cell detection kit is characterized by comprising reagents A, B and C, wherein the reagent A is a boron-doped graphene quantum dot (B-GQDs) solution, the reagent B is a cerium nitrate solution, and the reagent C is an Adenosine Triphosphate (ATP) solution.
The detection object of the kit is alkaline phosphatase on the surface of a cervical cancer cell membrane, which is an enzyme widely distributed in tissues of liver, bone, intestine, kidney, placenta and the like of a human body, and the abnormal expression of the enzyme has close relation with the occurrence and development of tumors. For example, alkaline phosphatase is expressed at a high level in cervical cancer cells, and the expression level thereof is correlated with cervical cancerThe disease progresses and shows a gradually rising trend, but shows low expression on the surface of normal cervical cells. The applicant has found that Ce (NO)3)3Quenching of the fluorescence signal induced by B-GQDs, but reacting B-GQDs with Ce (NO)3)3The mixed system of (1) and the cervical cancer cells act, the fluorescence signals of the B-GQDs are recovered in the presence of ATP, and the recovery degree of the fluorescence signals is in direct proportion to the concentration of the cervical cancer cells, so that the detection of the cervical cancer cells is realized.
Therefore, the invention selects alkaline phosphatase as a target for cervical cancer cell detection, adopts boron-doped graphene quantum dots as a fluorescent probe, and realizes high-sensitivity detection of the cervical cancer cells by constructing a boron-doped graphene quantum dot-quencher-adenosine triphosphate composite reagent. Compared with graphene quantum dots, the boron-doped graphene quantum dots (B-GQDs) have higher quantum yield and are beneficial to improving the detection sensitivity.
The detection principle of the kit provided by the invention is as follows: the boron-doped graphene quantum dots (B-GQDs) have strong fluorescence signals in cerium ion Ce3+When present, due to Ce3+And carboxyl (-COOH) on the surface of the B-GQDs quantum dot to cause fluorescence signal quenching; in a cervical cancer cell system, Adenosine Triphosphate (ATP) is added to stimulate alkaline phosphatase on the cell surface to hydrolyze and release phosphate radical (PO)4 3-) Free PO4 3-substituted-COOH with Ce3+The ions combine to cause fluorescence recovery of B-GQDs. The cervical cancer cells are detected according to the recovery of the fluorescence signals of the B-GQDs, and the detection of the concentration of the cervical cancer cells can be realized according to the fluorescence intensity.
The reagent A also comprises trihydroxyaminomethane-hydrochloric acid (TBS) buffer (preferably, the concentration is 0.1 mol/L, and the pH is 7.4).
The boron-doped graphene quantum dots can be synthesized by a method known in the prior art, such as a potentiostatic amperometric method in the examples, and the synthesis of the boron-doped graphene quantum dots can be found in the literature (Zetan Fan, Yunchao L i, Xiaohong L i, L ouzhen Fan, Shixin Zhou, Decai Fan, and Shihe Yang Surrouding media sensitive phosphor luminescent center of boron-doped graphene crystals, chemical sensing and bioiogicing 2014,70, 149-.
Preferably, in the detection kit, the reagent A is a quantum dot solution obtained by dispersing the reagent B-GQDs in trihydroxyaminomethane-hydrochloric acid (TBS) buffer solution, the concentration of the buffer solution is 15 mu g/m L, the concentration of the buffer solution is 0.1 mol/L, the pH value is 7.4, the concentration of cerium nitrate in the reagent B is 20 mmol/L, and the concentration of adenosine triphosphate in the reagent C is 20 mmol/L.
The detection kit is used for detecting the cervical cancer cells (He L a), and the detection steps are as follows:
1. preparing a reagent A: dispersing boron-doped graphene quantum dots (B-GQDs) in a TBS buffer solution to obtain a quantum dot solution;
2. preparing a reagent B: a certain amount of Ce (NO)3)3Dissolving in secondary water to obtain solution;
3. preparing a reagent C: dissolving a certain amount of ATP in secondary water to prepare a solution;
4. adding reagent B to reagent A, and recording fluorescence signal F of B-GQDs0；
5. Mixing reagent A and reagent B, adding reagent C to obtain mixed reagent, and mixing the mixed reagent with cervical cancer cell He L a (concentration 10-10)6M L) at 37 ℃ with 5% CO2Incubating for 2h in incubator, washing off residual reagent with TBS buffer solution, digesting He L a cells with pancreatin, and recording fluorescence signal F of the systemi；
6. Establishing a working curve for detecting the concentration of He L a cells by B-GQDs, namely changing the concentration of He L a cells and recording the fluorescence response signal flux F under different known He L a cell concentrations in step 5i(ii) a Calculating a difference Δ F (Δ F ═ F) of the response signalsi-F0) Drawing the concentration of the delta F and He L a cells into a delta F-c working curve, and establishing a He L a cell concentration detection working curve based on the fluorescence response of B-GQDs;
7. and (3) detecting the concentration of He L a in the sample to be detected, namely taking the He L a cell to be detected, reacting with the reagent in the kit according to the method same as the step 6), detecting the fluorescence signal of the cell, and calculating the concentration of the He L a cell to be detected according to the intensity of the fluorescence signal and the standard curve obtained in the step 6).
The linear range of the method for detecting the He L a cells is 10-106cell/m L, and detection limit of 10cell/m L.
Further, in the above-mentioned method,
in the reagent A prepared in the step 1), the concentration of boron-doped graphene quantum dots is 15 mu g/m L;
reagent B, Ce (NO3) prepared in step 2)3Has a molar concentration of 20 mmol/L;
and 3) preparing a reagent C with the ATP molar concentration of 20 mmol/L.
In the method, the excitation wavelength of the fluorescence spectrometer is 380nm, and the emission wavelength is 530 nm.
The technical scheme of the invention has the following beneficial effects:
according to the cervical cancer cell detection kit, alkaline phosphatase is selected as a target for detection, boron-doped graphene quantum dots are adopted as fluorescent probes, and high-sensitivity detection of cervical cancer cells is realized by constructing a boron-doped graphene quantum dot-quencher-adenosine triphosphate composite reagent. Compared with graphene quantum dots, the boron-doped graphene quantum dots (B-GQDs) have higher quantum yield and are beneficial to improving the detection sensitivity. The detection kit overcomes the defects of the traditional cervical cancer detection method and the fluorescent luminescent reagent, realizes the rapid and efficient detection of the cell surface alkaline phosphatase by the quantum dot fluorescence analysis method, and has the advantages of large information amount, high response speed, high sensitivity, low cost, strong optical anti-interference capability and the like. The boron-doped graphene quantum dots do not need to be additionally modified, and do not need to be pretreated by organic or biochemical materials which are expensive and have complicated connection steps, so the operation is simple.
Drawings
FIG. 1 is a transmission electron microscope image of a reagent A boron-doped graphene quantum dot B-GQDs in the invention.
FIG. 2 shows an XPS spectrum of a boron-doped graphene quantum dot B-GQDs as a reagent A in the invention.
FIG. 3 is a schematic diagram of the detection of the kit of the present invention. Wherein: curve a is the fluorescence signal response of B-GQDs; b is addition of reagent Ce (NO)3)3C is the fluorescence response signal after the mixed solution A, B and C in the kit are reacted with He L a cells.
FIG. 4 shows fluorescence intensity of B-GQDs in accordance with He L a cell concentration (0-10) in the present invention6cell/m L).
FIG. 5 is a working curve of He L a cell concentration detection in the present invention.
Detailed Description
EXAMPLE 1 preparation of the kit
(1) Preparation of B-GQDs
Synthesizing boron-doped graphene quantum dots (B-GQDs) by electrochemical workstation (CHI 660B) by constant potential chronoamperometry, specifically, preparing a 0.1 mol/L borax water solution, taking 20m L as an electrolyte, taking a high-purity graphite rod as a working electrode, a platinum wire electrode as a counter electrode, a calomel electrode as a reference electrode, reacting for 2 hours at a working potential of 3V, filtering the solution through a 0.22 mu m filter membrane, removing a graphene sheet layer, dialyzing the solution for 24 hours by a 3500Da dialysis bag, and removing Na in the solution+And B4O7 2-Thus obtaining B-GQDs. The obtained B-GQDS has uniform distribution, and the particle size is 3-8nm (figure 1); the elemental analysis of the product revealed that the product contained B element in an amount of 3.2% (FIG. 2).
(2) Preparing a reagent A, namely dispersing the synthesized B-GQDs in TBS buffer solution (the concentration is 0.1 mol/L, and the pH value is 7.4) to obtain quantum dot solution, wherein the concentration of the B-GQDs is 15 mu g/m L;
(3) preparing reagent B by adding a certain amount of Ce (NO)3)3Dissolving in secondary water to obtain cerium nitrate solution with the concentration of 20 mmol/L;
(4) preparation of reagent C A certain amount of ATP was dissolved in secondary water to prepare an ATP solution having a concentration of 20 mmol/L. example 2 evaluation of fluorescence Property
Detecting the B-GQDs solution by a fluorescence spectrometer to study the fluorescence property of the B-GQDs solution; wherein the excitation wavelength is 380nm and the emission wavelength is 530 nm.
Evaluation of fluorescence Properties of B-GQDs the quantum dot solution prepared in example 1 was dispersed in TBS buffer (0.1 mol/L, pH 7.4) at a concentration of 15. mu.g/m L, and the fluorescence spectrum of the solution was measured (curve a in FIG. 3), and the fluorescence signal was denoted as F0。
Fluorescence quenching of B-GQDs by reagent B to which 20. mu. L of Ce (NO) was added (15. mu.g/m L)3)3(20 mmol/L), the fluorescence signal F of B-GQDs was recorded (curve B in FIG. 3), indicating that the signal was quenched by a large amount.
Recovering B-GQDs fluorescence signal by He L a cell by mixing reagent A1.0 m L and reagent B20.0 μ L, adding reagent C10.0 μ L to obtain mixed reagent, and mixing the mixed reagent with cervical cancer cell He L a (10 a)6cell/m L) at 37 ℃ with 5% CO2And (3) incubating for 2h in the incubator, washing 3 times by using TBS buffer solution to wash out residual reagent, digesting He L a cells by using pancreatin, and recording the fluorescence signal of the system (curve c in figure 3), wherein the result shows that the fluorescence signal of B-GQDS is greatly enhanced, and the He L a can be detected according to the enhanced signal.
Example 3 set up a working curve for measuring He L a cell concentration
Mixing reagent A1.0 m L and reagent B20.0 μ L, adding reagent C10.0 μ L to obtain mixed reagent, and mixing the mixed reagent with cervical cancer cell He L a (10-10) with different concentrations6cell/m L) at 37 ℃ with 5% CO2Respectively incubating for 2h in the incubator; recording the fluorescence signal F of B-GQDs by a fluorescence spectrometeri(FIG. 4), in FIG. 4, a-g represent cell concentrations of 0, 10 and 10, respectively2、103、104、105、106cell/m L. calculate the difference Δ F of the response signals (Δ F ═ F)i-F0) The concentration of the delta F and the He L a cell is plotted into a delta F-c working curve (figure 5), so that a working curve for detecting the concentration of the He L a cell based on the fluorescence response of B-GQDs is established, and the linear range of the working curve is 10-106cell/mL。
EXAMPLE 4 detection of He L a cells in test sample
Taking He L a cells to be detected, acting with reagents in the kit according to the same method as that in the example 3 and detecting fluorescence signals of the He L a cells, wherein experimental results show that the fluorescence signals of quantum dots are obviously recovered when the He L a cells exist, and further obtaining the concentration of the He L a cells to be detected according to the standard curve obtained in the example 3 and the change degree of the He L a cell fluorescence.

Claims (2)

1. A cervical cancer cell detection kit is characterized by comprising reagents A, B and C, wherein the reagent A is a quantum dot solution obtained by dispersing boron-doped graphene quantum in trihydroxyaminomethane-hydrochloric acid buffer solution, the concentration of the quantum dot solution is 15 mu g/m L, the concentration of the buffer solution is 0.1 mol/L, the pH value is 7.4, the concentration of a cerium nitrate solution in the reagent B is 20 mmol/L, and the concentration of an adenosine triphosphate solution in the reagent C is 20 mmol/L;
the detection kit is used for detecting the cervical cancer cell He L a, and the detection steps are as follows:
1) preparing a reagent A: dispersing boron-doped graphene quantum dots (B-GQDs) in a TBS buffer solution to obtain a quantum dot solution;
2) preparing a reagent B: a certain amount of Ce (NO)3)3Dissolving in secondary water to obtain solution;
3) preparing a reagent C: dissolving a certain amount of ATP in secondary water to prepare a solution;
4) adding reagent B to reagent A, and recording fluorescence signal F of B-GQDs0；
5) Mixing the reagent A and the reagent B uniformly, adding the reagent C to prepare a mixed reagent, and mixing the mixed reagent with the concentration of 10-106/m L cervical carcinoma cell He L a at 37 deg.C and 5% CO2Incubating for 2h in incubator, washing off residual reagent with TBS buffer solution, digesting He L a cells with pancreatin, and recording fluorescence signal F of the systemi；
6) Establishing a working curve for detecting the concentration of He L a cells by B-GQDs by changing He L a cells in step 5)Recording the fluorescence response signal, pass F, at different known He L a cell concentrationsi(ii) a Calculating the difference value of response signals, wherein the difference value is Fi-F0Drawing the concentration of the delta F and He L a cells into a delta F-c working curve, and establishing a He L a cell concentration detection working curve based on the fluorescence response of B-GQDs;
7) and (3) detecting the concentration of He L a in the sample to be detected, namely taking the He L a cell to be detected, reacting with the reagent in the kit according to the method same as the step 6), detecting the fluorescence signal of the cell, and calculating the concentration of the He L a cell to be detected according to the intensity of the fluorescence signal and the standard curve obtained in the step 6).
2. The cervical cancer cell detection kit according to claim 1, characterized in that: the particle size of the boron-doped graphene quantum dots in the reagent A is 3-8 nm.
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CN108588178B (en) * 2018-04-03 2021-10-19 山东师范大学 Kit and method for detecting alkaline phosphatase
CN108559497B (en) * 2018-04-28 2021-03-30 华南师范大学 Doped graphene quantum dot for generating second harmonic and preparation method and application thereof
CN108896506B (en) * 2018-07-16 2020-10-27 济南大学 Method for detecting alkaline phosphatase activity and concentration of alkaline phosphatase inhibitor
CN112540097A (en) * 2020-12-01 2021-03-23 中国科学院上海微***与信息技术研究所 Nuclear magnetic resonance detection method and kit for detecting biomarkers based on biosensor

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