CN114113582B - Metal organic framework nanoenzyme biological probe and ELISA kit - Google Patents
Metal organic framework nanoenzyme biological probe and ELISA kit Download PDFInfo
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Abstract
The invention provides a metal organic framework nanoenzyme biological probe and an ELISA kit, and relates to the technical field of biological immunosensing. The invention adopts a metal organic framework nano composite material ZIF-67/Cu 0.76 Co 2.24 O 4 The biological probe formed by the biological probe and a sulfhydryl-containing biological material for specifically recognizing breast cancer cells has good peroxidase and laccase catalytic activity and stable structure under non-physiological conditions, and can catalyze chromogenic substrates to rapidly develop color, so that rapid visual detection of the breast cancer cells is realized. The ELISA kit for specifically detecting the breast cancer cells provided by the invention has the advantages of high detection accuracy, high sensitivity, wide detection range and low cost for the breast cancer cells, and has important scientific significance and clinical application value.
Description
Technical Field
The invention relates to the technical field of biological immunosensing, in particular to a metal organic framework nanoenzyme biological probe and an ELISA kit.
Background
Cancer is considered one of the most major public health problems in the world. Among them, breast cancer is often referred to as "pink killer", and its incidence is the first place of female malignancy. The greatest reason why the mortality rate of cancer has been high is that early diagnosis is difficult. Therefore, the development of a rapid, simple and low-cost detection method for early diagnosis of cancer is of great significance in terms of late-stage therapeutic effect, patient survival rate and the like.
At present, enzyme-linked immunosorbent assay (ELISA) is a common breast cancer detection method and has the advantages of high speed, high flux, low cost and the like. The reported ELISA detection method for breast cancer at home and abroad is mainly based on antigen-antibody reaction, and realizes detection by using the substrate color development of horseradish peroxidase (HRP) coupled on a secondary antibody. However, the accuracy of breast cancer detection is low due to the instability of the native enzyme under non-physiological conditions.
Disclosure of Invention
In view of the above, the present invention aims to provide a metal organic framework nanoenzyme bioprobe and an ELISA kit. The metal organic framework nanoenzyme biological probe provided by the invention has specificity for specifically detecting breast cancer cells, and has high detection precision and high sensitivity for the breast cancer cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a metal organic framework nano composite material in preparation of a biological probe for specifically detecting breast cancer cells or an ELISA kit for specifically detecting breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 。
The invention provides a metal organic framework nanoenzyme biological probe, which comprises a sulfhydryl-containing biological material and a metal organic framework nano composite material, wherein the sulfhydryl-containing biological material is used for specifically identifying breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 。
Preferably, the sulfhydryl-containing biomaterial comprises one or more of sulfhydryl-modified aptamer, recognition polypeptide and antibody.
Preferably, the mass ratio of the metal organic framework nano composite material to the mercapto-containing biological material is 1: 10 -5 ～10 -4 。
The invention provides application of the metal organic framework nanoenzyme bioprobe in preparing an ELISA kit for specifically detecting breast cancer cells.
The invention provides an ELISA kit for specifically detecting breast cancer cells, which comprises the metal organic framework nanoenzyme biological probe in the technical scheme.
Preferably, the kit also comprises an ELISA plate coated with a breast cancer antibody, nonspecific protein, sample diluent, a washing solution and a chromogenic substrate.
Preferably, the non-specific protein comprises one or more of bovine serum albumin, casein and ovalbumin.
Preferably, the chromogenic substrate comprises a peroxidase-active chromogenic substrate and/or a laccase-active chromogenic substrate.
Preferably, the peroxidase active chromogenic substrate comprises 3,3',5,5' -tetramethylbenzidine and H 2 O 2 ；
The laccase active chromogenic substrate comprises 2, 4-dichlorophenol.
The invention provides application of a metal organic framework nano composite material in preparation of a biological probe for specifically detecting breast cancer cells or an ELISA kit for specifically detecting breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 . ZIF-67/Cu adopted by the invention 0.76 Co 2.24 O 4 Has good adsorbability, and can be dissolved in water, ZIF-67/Cu 0.76 Co 2.24 O 4 The copper in the composition can be combined with sulfhydryl in sulfhydryl-containing biomaterials which have the specificity of identifying breast cancer cells, such as antibodies, aptamers, polypeptides and the like; the composite material has good peroxidase activity and laccase activity, has a stable structure, and provides a material for the subsequent preparation of products for detecting breast cancer.
The invention provides a metal organic framework nanoenzyme biological probe, which comprises a sulfhydryl-containing biological material and a metal organic framework nano composite material, wherein the sulfhydryl-containing biological material is used for specifically identifying breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 . The invention relates to a sulfhydryl-containing biological material for specifically identifying breast cancer cellsZIF-67/Cu nanocomposite material with metal organic framework through coordination bond (mercapto-copper) 0.76 Co 2.24 O 4 And the metal organic framework nano composite material has stable structure under non-physiological conditions, and the purpose of specifically identifying the sulfhydryl-containing biological material of the breast cancer cells by coupling the surface of the metal organic framework nano composite material is realized. The metal organic framework nanoenzyme biological probe provided by the invention not only has high catalytic activity of peroxidase and laccase, but also has the effect of specifically identifying breast cancer cells, has high sensitivity and accuracy for detecting the breast cancer cells, and provides a material for subsequently preparing products for detecting the breast cancer cells.
Furthermore, the metal organic framework nanoenzyme biological probe provided by the invention also specifically defines the thiol-containing biological material for specifically recognizing the breast cancer cells, including the aptamer of the breast cancer cells, the breast cancer cell recognition polypeptide and the antibody of the breast cancer cells. The invention utilizes the characteristic that the recognition polypeptide, the antibody and the aptamer can specifically recognize and combine breast cancer cells, and the invention is combined with the metal organic framework nano composite material ZIF-67/Cu 0.76 Co 2.24 O 4 The metal organic framework nanoenzyme biological probe for detecting breast cancer cells is constructed by combining the two methods, and has the advantages of convenience in detection, low cost, high sensitivity, high accuracy, high flux and the like.
The invention provides an ELISA kit for specifically detecting breast cancer cells, which comprises the metal organic framework nanoenzyme biological probe in the technical scheme. In the ELISA kit for specifically detecting the breast cancer cells, the metal organic framework nanoenzyme biological probe has the effect of specifically identifying the breast cancer cells, has high peroxidase and laccase catalytic activity and stable structure under non-physiological conditions, and can catalyze a chromogenic substrate to carry out rapid color development, so that rapid visual detection on the breast cancer cells is realized. Moreover, the ELISA kit for specifically detecting the breast cancer cells provided by the invention has the advantages of high detection accuracy, high sensitivity, wide detection range, low detection limit and low cost for the breast cancer cells, and has important scientific significance and clinical application value. Test results as in the examplesThe linear detection range of the ELISA kit for specifically detecting the breast cancer cells provided by the invention on the breast cancer cells is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL, recovery rate of 98.0-105%, relative standard deviation of 1.3-4.5%.
Further, the chromogenic substrate comprises a peroxidase-active chromogenic substrate and/or a laccase-active chromogenic substrate. The chromogenic substrate adopted by the invention adopts two enzymatic colorimetric signal readings, so that the output signal has higher sensitivity, and the detection sensitivity of the ELISA kit for specifically detecting the breast cancer cells to the breast cancer cells is further improved.
Drawings
FIG. 1 shows ZIF-67 and ZIF-67/Cu 0.76 Co 2.24 O 4 In the TEM image of (A) to (C) are ZIF-67, and (D) to (F) are ZIF-67/Cu 0.76 Co 2.24 O 4 ；
FIG. 2 is a diagram showing an ultraviolet absorption spectrum and a standard curve of MCF-7 with different concentrations in examples 2-3, wherein (A) is the ultraviolet absorption spectrum of example 2, (B) is the standard curve of example 2, (C) is the ultraviolet absorption spectrum of example 3, and (D) is the standard curve of example 3;
FIG. 3 is the ZIF-67/Cu sequence of example 8 0.76 Co 2.24 O 4 Wherein (A) and (C) are optimum pH values and (B) and (D) are optimum reaction times;
FIG. 4 is a photograph showing a comparison of fluorescent staining by a microscope of MCF-10A and MCF-7 in example 9, wherein (A) to (C) are MCF-10A and (D) to (F) are MCF-7.
Detailed Description
The invention provides application of a metal organic framework nano composite material in preparation of a biological probe for specifically detecting breast cancer cells or an ELISA kit for specifically detecting breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 。
In the present invention, the metal-organic framework nanocomposite is preferably self-made. In the present invention, the method for preparing the metal-organic framework nanocomposite preferably comprises the steps of:
mixing cobalt acetate, 2-methylimidazole, triethylamine and water, and reacting to obtain a zeolitic imidazolate framework material (ZIF-67);
mixing the zeolite imidazole ester framework material, copper nitrate and ethanol, and carrying out a solvothermal reaction to obtain a metal organic framework nano composite material (ZIF-67/Cu) 0.76 Co 2.24 O 4 )。
According to the invention, cobalt acetate, 2-methylimidazole, triethylamine and water are mixed for coordination reaction to obtain the zeolitic imidazolate framework material (ZIF-67). In the present invention, the mass ratio of cobalt acetate to 2-methylimidazole is preferably 1: 1.2 to 5, more preferably 1: 1.5 to 4.5, and more preferably 1: 2 to 4. In the present invention, the molar ratio of the cobalt acetate to the triethylamine is preferably 1: 0.1 to 1, more preferably 1: 0.1 to 0.5, and more preferably 1: 0.2 to 0.3. In the invention, the cobalt acetate, the 2-methylimidazole, the triethylamine and the water are preferably mixed, and the cobalt acetate is preferably dissolved in partial water to obtain a cobalt acetate solution; dissolving 2-methylimidazole and triethylamine in the residual water to obtain methylimidazole-triethylamine solution; the cobalt acetate solution and the methylimidazole-triethylamine solution were mixed. In the invention, the concentration of the cobalt acetate solution is preferably 10-60 g/L, more preferably 20-50 g/L, and even more preferably 30-40 g/L. In the invention, the concentration of 2-methylimidazole in the methylimidazole-triethylamine solution is preferably 30-140 g/L, more preferably 50-120 g/L, and even more preferably 80-100 g/L. The mixing mode of the invention is not particularly limited, and the mixing mode known to those skilled in the art can be adopted, such as stirring and mixing; the speed and time for stirring and mixing are not particularly limited, and the raw materials can be uniformly mixed. In the invention, the reaction temperature is preferably room temperature, and the reaction time is preferably 10-30 min, more preferably 15-25 min, and further preferably 20 min; in the reaction process, the ligand and metal ions generate the zeolite imidazole ester framework material. After the reaction is finished, the method preferably further comprises the steps of carrying out solid-liquid separation on reaction liquid obtained by the reaction, and sequentially carrying out ethanol washing, water washing and drying on obtained solid products to obtain the zeolite imidazole ester framework structure material. The solid-liquid separation mode is not particularly limited, and a solid-liquid separation mode well known to those skilled in the art can be adopted, specifically, centrifugal separation is performed, the rotating speed of the centrifugal separation is preferably 5000-10000 rpm, more preferably 6000rpm, and the time of the centrifugal separation is preferably 5-15 min, more preferably 10 min. In the present invention, the number of times of the ethanol washing is preferably 1 to 5 times, and more preferably 2 to 3 times. In the present invention, the number of washing with water is preferably 1 to 5 times, and more preferably 2 to 3 times. In the invention, the drying temperature is preferably 40-60 ℃, more preferably 50 ℃, and the drying time is preferably 6-12 h, more preferably 10 h.
After the zeolite imidazole ester framework material is obtained, the zeolite imidazole ester framework material, copper nitrate and ethanol are mixed for solvothermal reaction to obtain the metal organic framework nano composite material. In the present invention, the mass ratio of the zeolitic imidazolate framework material to copper nitrate is preferably 1: 0.5 to 5, more preferably 1: 0.5 to 2, and more preferably 1: 0.5 to 1. In the present invention, the ratio of the mass of the zeolitic imidazolate framework material to the volume of ethanol is preferably 1 g: 10-40 mL, more preferably 1 g: 20-30 mL. The mixing mode of the invention is not particularly limited, and the mixing mode known to those skilled in the art can be adopted, such as stirring and mixing; the stirring and mixing speed and time are not particularly limited, and the raw materials can be uniformly mixed. In the invention, the temperature of the solvothermal reaction is preferably 100-150 ℃, more preferably 120 ℃, and the time of the solvothermal reaction is preferably 2-6 h, more preferably 4 h. After the solvothermal reaction is finished, the method preferably further comprises the steps of cooling the reaction liquid obtained by the solvothermal reaction to room temperature, carrying out solid-liquid separation, and sequentially washing, washing with ethanol and drying the obtained solid product to obtain the zeolite imidazole ester framework structure material. The cooling method of the present invention is not particularly limited, and a cooling method known to those skilled in the art may be used, specifically, natural cooling. The solid-liquid separation mode is not particularly limited, and a solid-liquid separation mode known to those skilled in the art can be adopted, specifically, centrifugal separation is adopted, the rotation speed of the centrifugal separation is preferably 5000-10000 rpm, more preferably 6000rpm, and the time of the centrifugal separation is preferably 5-15 min, more preferably 10 min. In the present invention, the number of times of the water washing is preferably 3 to 5 times. In the present invention, the number of times of the ethanol washing is preferably 3 to 5 times. In the invention, the drying temperature is preferably 40-60 ℃, more preferably 50 ℃, and the drying time is preferably 6-12 h, more preferably 10 h.
The invention provides a metal organic framework nanoenzyme biological probe, which comprises a sulfhydryl-containing biological material and a metal organic framework nano composite material, wherein the sulfhydryl-containing biological material is used for specifically identifying breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 。
In the invention, the mass ratio of the metal-organic framework nanocomposite to the thiol-group-containing biomaterial is preferably 1: 10 -5 ～10 -4 More preferably 1: 5X 10 -5 ～10 -4 。
In the present invention, the thiol-group-containing biomaterial preferably includes one or more of a thiol-modified aptamer, a recognition polypeptide, and an antibody; the thiol-modified aptamer preferably comprises a thiol-modified MUC1 aptamer; the sulfhydryl modified MUC1 aptamer is preferably synthesized by Kunming division of Biotechnology Limited of Beijing Optimalaceae, and the nucleotide sequence of the sulfhydryl modified MUC1 aptamer is shown as SEQ ID NO. 1: 5 '-tttttgcagttgatcctttggataccctgg-SH-3'; the antibody for breast cancer cells preferably comprises anti-CD44 (cell surface transmembrane glycoprotein antibody).
The invention provides a preparation method of a metal organic framework nanoenzyme bioprobe in the technical scheme, which comprises the following steps:
and mixing the metal organic framework nano composite material, the sulfhydryl-containing biological material for specifically identifying the breast cancer cells and water to obtain the metal organic framework nano enzyme biological probe.
In the invention, the gold is addedThe preparation method comprises the following steps of (1) mixing an organic framework nano composite material, a sulfhydryl-containing biological material for specifically identifying breast cancer cells and water, preferably dispersing the metal organic framework nano composite material in the water to obtain a metal organic framework nano composite material dispersion liquid; and mixing the metal organic framework nano composite material dispersion liquid with a sulfhydryl-containing biological material for specifically identifying breast cancer cells. In the invention, the sulfhydryl-containing biomaterial capable of specifically recognizing breast cancer cells is preferably used in the form of a sulfhydryl-containing biomaterial solution capable of specifically recognizing breast cancer cells, the solvent in the sulfhydryl-containing biomaterial solution capable of specifically recognizing breast cancer cells preferably comprises a PBS buffer solution, and the pH value of the PBS buffer solution is preferably 7.0-7.5, and more preferably 7.1-7.4; the concentration of the sulfhydryl-containing biomaterial solution for specifically recognizing the breast cancer cells is preferably 0.1-1.0 mug/mL, and more preferably 0.2 mug/mL. In the invention, the concentration of the metal organic framework nano composite material dispersion liquid is preferably 0.5-2 g/L, and more preferably 1 g/L. In the invention, the mass ratio of the metal organic framework nanocomposite to the thiol-containing biomaterial specifically recognizing breast cancer cells is preferably 1: 10 -5 ～10 -4 More preferably 1: 5X 10 -5 ～10 -4 . In the present invention, the mass ratio of the metal-organic framework nanocomposite to water is preferably 1: 2000 to 500, more preferably 1: 1000 to 500, and more preferably 1: 1000. in the invention, the mixing temperature is preferably room temperature, and the mixing time is preferably 6-10 h, more preferably 7-9 h, and further preferably 8 h; the mixing is stirring mixing, the stirring mixing speed is not particularly limited, and the raw materials can be uniformly mixed; during the mixing process, the mercapto group forms a coordination bond with copper.
The invention provides application of the metal organic framework nanoenzyme bioprobe in preparing an ELISA kit for specifically detecting breast cancer cells.
The invention provides an ELISA kit for specifically detecting breast cancer cells, which comprises the metal organic framework nanoenzyme biological probe in the technical scheme.
In the invention, the metal organic framework nanoenzyme biological probe is preferably used in the form of a metal organic framework nanoenzyme biological probe solution, and the concentration of the metal organic framework nanoenzyme biological probe solution is preferably 0.5-2 mg/mL, and more preferably 1-1.5 mg/mL; the solvent in the metal organic framework nanoenzyme bioprobe solution preferably comprises deionized water.
In the present invention, the ELISA kit for specifically detecting breast cancer cells preferably further comprises an ELISA plate coated with a breast cancer antibody, a non-specific protein, a washing solution and a chromogenic substrate.
In the present invention, the breast cancer antibody in the microplate coated with the breast cancer antibody preferably comprises Anti-CD44 antibody. In the present invention, the method for preparing the elisa plate coated with the breast cancer antibody preferably comprises the following steps: and adding the breast cancer antibody solution into the ELISA plate for incubation to obtain the ELISA plate coated with the breast cancer antibody. In the invention, the solvent in the breast cancer antibody solution preferably comprises a PBS buffer solution, and the pH value of the PBS buffer solution is preferably 7.0-7.5, and more preferably 7.1-7.4; the concentration of the breast cancer antibody solution is preferably 0.1-0.5 mu g/mL, and more preferably 0.2 mu g/mL. In the invention, the incubation temperature is preferably 2-8 ℃, more preferably 4-5 ℃, and the incubation time is preferably 6-10 h, more preferably 8 h; the incubation is preferably performed in a refrigerator. After the incubation is finished, the method preferably further comprises the steps of washing by using a washing solution and then drying to obtain an ELISA plate coated with the breast cancer antibody; the washing solution preferably comprises phosphate buffer solution (PBS solution), and the pH value of the phosphate buffer solution is preferably 7.0-7.5.
In the present invention, the non-specific protein preferably includes one or more of Bovine Serum Albumin (BSA), casein, and Ovalbumin (OVA), and more preferably bovine serum albumin.
In the present invention, the chromogenic substrate preferably comprises a peroxidase-active chromogenic substrate and/or a laccase-active chromogenic substrate. In the present invention, the peroxidase-active chromogenic substrate preferably comprises 3,3',5,5' -Tetramethylbenzidine (TMB) and H 2 O 2 (ii) a The 3,3',5,5' -tetramethylbenzidine is preferably used in the form of a 3,3',5,5' -tetramethylbenzidine solution, and the concentration of the 3,3',5,5' -tetramethylbenzidine solution is preferably 5 to 30mM (mmol/L), more preferably 10 to 25mM, and further preferably 15 to 20 mM; the solvent in the 3,3',5,5' -tetramethylbenzidine solution preferably comprises deionized water; said H 2 O 2 Preferably with H 2 O 2 In the form of an aqueous solution, said H 2 O 2 The concentration of the aqueous solution is preferably 5 to 30mM (mmol/L), more preferably 10 to 25 mM. In the invention, the laccase active chromogenic substrate preferably comprises 2, 4-dichlorophenol (2,4-DP), the 2, 4-dichlorophenol is preferably used in the form of a 2, 4-dichlorophenol solution, and the concentration of the 2, 4-dichlorophenol solution is preferably 5-30 mM, more preferably 10-25 mM, and further preferably 15-20 mM; the solvent in the 2, 4-dichlorophenol amine solution preferably comprises deionized water.
In the invention, the washing solution preferably comprises phosphate buffer solution (PBS solution), and the pH value of the phosphate buffer solution is preferably 7.0-7.5, and more preferably 7.1-7.4.
The metal organic framework nanoenzyme bioprobe and the ELISA kit for specifically detecting breast cancer cells provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The chemicals and solvents used in the following examples and comparative examples are of analytical grade. MCF-7 and MCF-10A cells were derived from the Kunming animal research institute cell bank, Chinese academy of sciences.
Example 1
(1) 0.4152g of Co (CH) 3 COO) 2 Dissolving in 15mL of deionized water, and stirring for 30min by using a magnetic stirrer to obtain a cobalt acetate solution; 1.3686g of 2-methylimidazole and 0.0141g of Triethylamine (TEA) are dissolved in 15mL of deionized water and stirred for 30min by a magnetic stirrer to obtain a methylimidazole-triethylamine solution; stirring and mixing cobalt acetate solution and methylimidazole-triethylamine solution with a magnetic stirrer, reacting at room temperature for 10min, filtering, sequentially washing the obtained solid product with ethanol, water and 60 DEG CDrying for 7h under the vacuum environment condition to obtain a zeolitic imidazolate framework material (ZIF-67, purple);
(2) mixing 0.5g ZIF-67, 0.15g Cu (NO) 3 ) 2 Mixing with 15mL of ethanol, transferring into a polytetrafluoroethylene high-pressure reaction kettle, carrying out solvothermal reaction for 4h at 120 ℃, naturally cooling to room temperature, filtering, washing the obtained solid product with water for 3 times, washing with ethanol for 3 times, and drying at 60 ℃ for 12h to obtain the metal organic framework nano composite material (ZIF-67/Cu) 0.76 Co 2.24 O 4 Dark purple powder);
(3) mixing ZIF-67/Cu 0.76 Co 2.24 O 4 The dark purple powder is evenly dispersed in deionized water to obtain ZIF-67/Cu with the concentration of 1mg/mL 0.76 Co 2.24 O 4 A dispersion liquid; in 1mL of ZIF-67/Cu 0.76 Co 2.24 O 4 Adding 100 μ L MUC1 aptamer solution into the dispersion, stirring for 10h, and storing at 4 deg.C to obtain metal organic framework nanoenzyme biological probe (Apt 2/ZIF-67/Cu) 0.76 Co 2.24 O 4 )。
FIG. 1 shows ZIF-67 and ZIF-67/Cu prepared in example 1 0.76 Co 2.24 O 4 In the TEM image of (A) to (C) are ZIF-67, and (D) to (F) are ZIF-67/Cu 0.76 Co 2.24 O 4 . As can be seen from FIG. 1, ZIF-67 is a regular rhombohedral structure; ZIF-67/Cu 0.76 Co 2.24 O 4 The medium Cu is uniformly distributed on the surface of the ZIF-67, and the structure of the ZIF-67 is kept complete, meanwhile, the ZIF-67/Cu is shown 0.76 Co 2.24 O 4 Nanocomposites were successfully prepared.
Example 2
Apt2/ZIF-67/Cu prepared in example 1 0.76 Co 2.24 O 4 Breast cancer cell (MCF-7) detection at 652nm and 510nm
(1) Breast cancer cell detection at 652nm
(1.1) 50. mu.L of Anti-CD44 antibody solution with concentration of 0.2. mu.g/mL was dropped into a 96-well plate and incubated in a refrigerator at 4 ℃ for 10 h; unbound Anti-CD44 antibody was then washed away with PBS solution;
(1.2) 50. mu.L of 0.5% bovine serum albumin was added to the 96-well plate treated in step (1.1), and the plate was incubated at 25 ℃ for 20min, and after completion of the incubation, the plate was washed with PBS and dried.
(1.3) 100. mu.L of the different concentrations (10) 1.3 cells/mL、10 1.5 cells/mL、10 2.0 cells/mL、10 2.5 cells/mL、10 3.0 cells/mL、10 3.5 cells/mL、10 4.0 cells/mL and 10 4.5 cells/mL) of breast cancer cells (MCF-7) were added dropwise to the step (1.2) treated 96-well plate at 37 ℃ with 5% CO 2 Incubate in incubator for 2h, then wash with PBS and dry.
(1.4) adding 50. mu.L of Apt2/ZIF-67/Cu at a concentration of 1.0mg/mL to the 96-well plate treated in step (1.3) 0.76 Co 2.24 O 4 5% CO at 37 ℃ 2 Incubate in incubator for 2h, wash with PBS to remove unbound Apt2/ZIF-67/Cu 0.76 Co 2.24 O 4 And (5) removing.
(1.5) 50. mu.L of a 5mmol/L aqueous TMB solution and 50. mu.L of 20mmol/L H 2 O 2 Quickly adding the aqueous solution into the 96-well plate treated in the step (1.4), measuring the absorbance values of the breast cancer cell dispersion liquid with different concentrations at a position of 652nm by using a microplate reader, drawing a working curve by using the absorbance value (Y) as a vertical coordinate and the concentration (X) of the breast cancer cell dispersion liquid as a horizontal coordinate, and obtaining a standard linear equation shown as a formula (1):
y is 0.02644X +0.27525 formula (1).
(2) Cancer cell detection at 510nm
(2.1) 50. mu.L of Anti-CD44 antibody solution with concentration of 0.2. mu.g/mL was dropped into a 96-well plate and incubated in a refrigerator at 4 ℃ for 10 h; unbound Anti-CD44 antibody was then washed away with PBS solution;
(2.2) 50. mu.L of 0.5% bovine serum albumin was added to the 96-well plate treated in step (2.1), and the plate was incubated at 25 ℃ for 20min, and after completion of the incubation, the plate was washed with PBS and dried.
(2.3) 100. mu.L of the different concentrations (10) 1.3 cells/mL、10 1.5 cells/mL、10 2.0 cells/mL、10 2.5 cells/mL、10 3.0 cells/mL、10 3.5 cells/mL、10 4.0 cells/mL and 10 4.5 cells/mL) was added dropwise to the step (2.2) treated 96-well plate at 37 ℃ with 5% CO 2 Incubate in incubator for 2h, then wash with PBS and dry.
(2.4) adding 50. mu.L of Apt2/ZIF-67/Cu at a concentration of 1.0mg/mL to the 96-well plate treated in step (2.3) 0.76 Co 2.24 O 4 5% CO at 37 ℃ 2 Incubate in incubator for 2h, wash with PBS to remove unbound Apt2/ZIF-67/Cu 0.76 Co 2.24 O 4 And (5) removing.
(2.5) rapidly adding 50 μ L of 2,4-DP aqueous solution with the concentration of 5mmol/L and 50 μ L of 4-AP aqueous solution with the concentration of 20mmol/L into the 96-well plate treated in the step (2.4), measuring the absorbance values of the breast cancer cell dispersion liquid with different concentrations at 510nm by using a microplate reader, and drawing a working curve by using the absorbance value (Y) as a vertical coordinate and the concentration (X) of the breast cancer cell dispersion liquid as a horizontal coordinate to obtain a standard linear equation as shown in the formula (2):
y is 0.08702X +0.34102 formula (2).
Example 3
Apt2/ZIF-67/Cu prepared in example 1 was mixed in accordance with the method of example 2 0.76 Co 2.24 O 4 For detection of breast cancer cells at 652nm and 510nm (MCF-7), the difference from example 2 is that:
adding 1.0% bovine serum albumin in the step (1.2), and incubating for 40 min;
the concentration of the TMB aqueous solution in the step (1.5) is 10mmol/L, H 2 O 2 The concentration of the aqueous solution is 40 mmol/L;
adding 1.0% bovine serum albumin in the step (2.2), and incubating for 40 min;
in the step (1.5), the concentration of the 2,4-DP aqueous solution is 10mmol/L, and the concentration of the 4-AP aqueous solution is 40 mmol/L.
FIG. 2 is a graph showing the UV absorption spectrum and the standard curve of MCF-7 with different concentrations in examples 2-3, wherein (A) is the UV absorption spectrum of example 2, (B) is the standard curve of example 2, and (C) isExample 3 ultraviolet absorption spectrum, (D) is the standard curve of example 3. As can be seen from FIG. 2, the linear detection range of breast cancer cells is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL (S/N is 3), wide detection linear range, high sensitivity and low detection limit.
Example 4
Apt2/ZIF-67/Cu prepared in example 1 was mixed in accordance with the method of example 2 0.76 Co 2.24 O 4 For detection of breast cancer cells at 652nm and 510nm (MCF-7), the difference from example 2 is that:
adding 1.5% bovine serum albumin in the step (1.2), and incubating for 60 min;
the concentration of the TMB aqueous solution in the step (1.5) is 15mmol/L, H 2 O 2 The concentration of the aqueous solution is 60 mmol/L;
adding 1.5% bovine serum albumin in the step (2.2), and incubating for 60 min;
in the step (1.5), the concentration of the 2,4-DP aqueous solution is 15mmol/L, and the concentration of the 4-AP aqueous solution is 60 mmol/L.
The linear detection range of breast cancer cells in this example is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL, wide detection linear range, high sensitivity and low detection limit.
Example 5
Apt2/ZIF-67/Cu prepared in example 1 was mixed in accordance with the method of example 2 0.76 Co 2.24 O 4 For detection of breast cancer cells at 652nm and 510nm (MCF-7), the difference from example 2 is that:
adding 2.0% bovine serum albumin in the step (1.2), and incubating for 80 min;
the concentration of the TMB aqueous solution in the step (1.5) is 20mmol/L, H 2 O 2 The concentration of the aqueous solution is 80 mmol/L;
the concentration of the Anti-CD44 antibody solution in the step (2.1) is 0.2 mu g/mL;
adding 2.0% bovine serum albumin in the step (2.2), and incubating for 80 min;
in the step (1.5), the concentration of the 2,4-DP aqueous solution is 20mmol/L, and the concentration of the 4-AP aqueous solution is 80 mmol/L.
The linear detection range of breast cancer cells in this example is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL, wide detection linear range, high sensitivity and low detection limit.
Example 6
Apt2/ZIF-67/Cu prepared in example 1 was mixed according to the method of example 2 0.76 Co 2.24 O 4 For detection of breast cancer cells at 652nm and 510nm (MCF-7), the difference from example 2 is that:
adding 2.5% bovine serum albumin in the step (1.2), and incubating for 100 min;
the concentration of the TMB aqueous solution in the step (1.5) is 25mmol/L, H 2 O 2 The concentration of the aqueous solution is 100 mmol/L;
adding 2.5% bovine serum albumin in the step (2.2), and incubating for 100 min;
in the step (1.5), the concentration of the 2,4-DP aqueous solution is 25mmol/L, and the concentration of the 4-AP aqueous solution is 100 mmol/L.
The linear detection range of breast cancer cells in this example is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL, wide detection linear range, high sensitivity and low detection limit.
Example 7
Apt2/ZIF-67/Cu prepared in example 1 was mixed in accordance with the method of example 2 0.76 Co 2.24 O 4 For detection of breast cancer cells at 652nm and 510nm (MCF-7), the difference from example 2 is that:
adding 3.0% bovine serum albumin in the step (1.2), and incubating for 120 min;
the concentration of the aqueous solution of TMB in the step (1.5) is 30mmol/L, H 2 O 2 The concentration of the aqueous solution is 120 mmol/L;
adding 3.0% bovine serum albumin in the step (2.2), and incubating for 120 min;
in the step (1.5), the concentration of the 2,4-DP aqueous solution is 30mmol/L, and the concentration of the 4-AP aqueous solution is 120 mmol/L.
The linear detection range of breast cancer cells in this example is 10 1.3 ～10 4.5 cells/mL, detection limit of 10 1.3 cells/mL, wide detection linear range, high sensitivity and low detection limit.
Example 8
10 μ L of 1mg/mL ZIF-67/Cu 0.76 Co 2.24 O 4 The aqueous dispersion was placed in a 96-well plate, and 50. mu.L of an aqueous TMB solution having a concentration of 10 μm. and 50. mu.L of H having a concentration of 10 μm. were added 2 O 2 The aqueous solution was added to the treated 96-well plate, and finally acetic acid buffer solutions having 150. mu. LpH values of 3.5, 4.5, 5, 6, 6.5 and 7 were added thereto, and reacted at 37 ℃ for 30min, and the absorbance at 652nm was measured with a microplate reader, respectively, and the results of the measurement are shown in FIG. 3A. As shown in FIG. 3A, ZIF-67/Cu 0.76 Co 2.24 O 4 The optimum reaction pH for peroxidase activity of (2) was 4.5.
10 μ L of 1mg/mL ZIF-67/Cu 0.76 Co 2.24 O 4 The aqueous dispersion was placed in a 96-well plate, and 50. mu.L of an aqueous TMB solution having a concentration of 10 μm. and 50. mu.L of H having a concentration of 10 μm. were added 2 O 2 Adding the aqueous solution into a treated 96-well plate, finally adding an acetic acid buffer solution with the value of 150 mu LpH of 4.5, reacting for 10min, 20min, 30min, 40min, 50min and 60min respectively at the temperature of 37 ℃, and measuring the absorbance value at 652nm by using a microplate reader respectively, wherein the test result is shown as B in figure 3. As shown in B in FIG. 3, ZIF-67/Cu 0.76 Co 2.24 O 4 The optimum reaction time for peroxidase activity of (2) was 30 min.
10 μ L of 1mg/mL ZIF-67/Cu 0.76 Co 2.24 O 4 The aqueous dispersion was placed in a 96-well plate, and 50. mu.L of an aqueous TMB solution having a concentration of 10 μm. and 50. mu.L of H having a concentration of 10 μm. were added 2 O 2 Adding the aqueous solution into a treated 96-well plate, finally adding morpholine ethanesulfonic acid buffer solution with 150 mu LpH values of 3, 4, 5, 6, 6.8, 7, 8 and 9 respectively, reacting for 30min at 65 ℃, and measuring the absorbance value at 510nm by using a microplate reader respectively, wherein the test result is shown as C in figure 3. As shown by C in FIG. 3, ZIF-67/Cu 0.76 Co 2.24 O 4 The pH of the reaction is 6.8.
10 μ L of 1mg/mL ZIF-67/Cu 0.76 Co 2.24 O 4 The aqueous dispersion was placed in a 96-well plate, and 50. mu.L of an aqueous TMB solution having a concentration of 10 μm. and 50. mu.L of H having a concentration of 10 μm. were added 2 O 2 Adding the water solution into the treated 96-well plate, finally adding morpholine ethanesulfonic acid buffer solution with 150 mu LpH value of 6.8, reacting for 10min, 20min, 30min, 40min, 50min and 60min at 37 ℃, respectively, and measuring the absorbance value at 510nm by using a microplate reader, wherein the test result is shown as D in figure 3. As shown in D in FIG. 3, ZIF-67/Cu 0.76 Co 2.24 O 4 The optimal reaction time for laccase activity of (3) is 30 min.
Example 9
Anti-CD44 was added dropwise to a 96-well plate, and 200. mu.L of MCF-7 cell suspension and MCF-10A (normal mammary epithelial cells) cell suspension were placed in the material-coated wells, respectively, at 37 ℃ with 5% CO 2 Culturing under the condition for 60 min. Followed by 3 washes with PBS to remove free cells. Cells captured in 96-well plates were stained with Hoechst. Cells trapped in dark and bright fields in the ultraviolet excited state were observed using a fluorescence microscope. The test results are shown in fig. 4. FIG. 4 is a contrast image of fluorescent staining of MCF-10A and MCF-7 by microscope, wherein (A) to (C) are MCF-10A, (D) to (F) are MCF-7, (A) and (D) are images in bright field to which only Anti-CD44 antibody was added, (B) are images in bright field to which Anti-CD44 antibody and MCF-10A were added, (C) are images in dark field to which Anti-CD44 antibody and MCF-10A were added, (E) are images in bright field to which Anti-CD44 antibody and MCF-7 were added, and (F) are images in dark field to which Anti-CD44 antibody and MCF-7 were added. As is apparent from FIGS. 4 (E) and (F), cells were captured in the 96-well plate, indicating that the sensor was capable of specifically capturing human breast cancer cell MCF-7, but not normal cell MCF-10A.
Example 10
The application of the cell sensor in the actual sample detection was evaluated by quantifying each MCF-7 cell concentration by a standard addition method. MCF-7 cells are dispersed in human serum to obtain solutions to be detected with the concentrations of 500cells/mL, 5000cells/mL and 10000cells/mL respectively, the detection is carried out according to the method of example 2, the concentration of MCF-7 in the solution to be detected is calculated according to the light absorption value of the solution to be detected at 510nm and Y (0.08702X + 0.34102), the recovery rate and the relative standard deviation are calculated, and the test results are shown in Table 1:
TABLE 1 results of human breast cancer cell assay in serum samples
| Sample (I) | Add value (cells/mL) | Measured value (cells/mL) | Relative standard deviation (%) | Recovery (%) |
| 1 | 500 | 490±25 | 4.5 | 98.0 |
| 2 | 5000 | 5158±140 | 3.8 | 103.2 |
| 3 | 10000 | 10523±275 | 1.3 | 105.2 |
As can be seen from Table 1, the recovery rate of the human breast cancer cells is 98.0-105.2%, and the relative standard deviation is 1.3-4.5%, which indicates that the ELISA kit provided by the invention has potential application value in clinical detection and diagnosis.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Yunnan
<120> metal organic framework nano enzyme biological probe and ELISA kit
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<170> SIPOSequenceListing 1.0
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tttttgcagt tgatcctttg gataccctgg 30
Claims (7)
1. The application of the metal organic framework nano composite material in the preparation of a biological probe for specifically detecting breast cancer cells or the preparation of an ELISA kit for specifically detecting breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 ；
The preparation method of the metal organic framework nano composite material comprises the following steps: mixing cobalt acetate, 2-methylimidazole, triethylamine and water, and reacting to obtain a zeolite imidazole ester framework material; mixing the zeolite imidazole ester framework material, copper nitrate and ethanol, and carrying out a solvothermal reaction to obtain a metal organic framework nano composite material; the mass ratio of the cobalt acetate to the 2-methylimidazole is 1: 1.2-5; the molar ratio of the cobalt acetate to the triethylamine is 1: 0.1 to 1; the mass ratio of the zeolite imidazole ester framework material to the copper nitrate is 1: 0.5 to 5; the ratio of the mass of the zeolite imidazole ester framework material to the volume of ethanol is 1 g: 10-40 mL.
2. A metal organic framework nanoenzyme biological probe comprises a sulfhydryl-containing biological material and a metal organic framework nano composite material, wherein the sulfhydryl-containing biological material can specifically identify breast cancer cells; the chemical composition of the metal organic framework nano composite material is ZIF-67/Cu 0.76 Co 2.24 O 4 ；
The sulfhydryl-containing biomaterial comprises one or more of sulfhydryl-modified aptamer, recognition polypeptide and antibody;
the mass ratio of the metal organic framework nano composite material to the mercapto-containing biological material is 1: 10 -5 ~10 -4 ；
The preparation method of the metal organic framework nano composite material comprises the following steps: mixing cobalt acetate, 2-methylimidazole, triethylamine and water, and reacting to obtain a zeolite imidazole ester framework material; mixing the zeolite imidazole ester framework material, copper nitrate and ethanol, and carrying out a solvothermal reaction to obtain a metal organic framework nano composite material; the mass ratio of the cobalt acetate to the 2-methylimidazole is 1: 1.2-5; the molar ratio of the cobalt acetate to the triethylamine is 1: 0.1 to 1; the mass ratio of the zeolite imidazole ester framework material to the copper nitrate is 1: 0.5 to 5; the ratio of the mass of the zeolite imidazolate framework material to the volume of the ethanol is 1 g: 10-40 mL.
3. The use of the metal organic framework nanoenzyme bioprobe of claim 2 in the preparation of an ELISA kit for specifically detecting breast cancer cells.
4. An ELISA kit for specifically detecting breast cancer cells, comprising the metal organic framework nanoenzyme bioprobe of claim 2, an ELISA plate coated with a breast cancer antibody, non-specific protein, a washing solution and a chromogenic substrate.
5. The ELISA kit of claim 4 wherein the non-specific proteins comprise one or more of bovine serum albumin, casein and ovalbumin.
6. The ELISA kit of claim 4, wherein the chromogenic substrate comprises a peroxidase-active chromogenic substrate and/or a laccase-active chromogenic substrate.
7. The ELISA kit of claim 6 wherein the peroxidase chromogenic substrate comprises 3,3',5,5' -tetramethylbenzidine and H 2 O 2 ；
The laccase active chromogenic substrate comprises 2, 4-dichlorophenol.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105372236A (en) * | 2015-11-18 | 2016-03-02 | 云南大学 | Highly sensitive cancer cell detecting method |
| CN107930586A (en) * | 2017-09-29 | 2018-04-20 | 天津大学 | A kind of method by copper incorporation zeolite imidazole salt frame synthesis Cu ZIF 67 |
| CN109647478A (en) * | 2018-12-20 | 2019-04-19 | 首都师范大学 | The preparation method of the nitrogenous carbon-coating catalyst of copper and cobalt doped |
| CN109748327A (en) * | 2019-02-25 | 2019-05-14 | 武汉理工大学 | One kind preparing CuCoO based on MOFs material at low temperature2The method of nanocrystalline material |
| CN110057877A (en) * | 2018-12-24 | 2019-07-26 | 中山大学 | The biosensor and its preparation method for being used to detect tumour cell of repeatable modification |
| CN110280245A (en) * | 2019-05-28 | 2019-09-27 | 浙江工业大学 | A kind of ZIF-67/CuBTC composite material and its derivative and method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898821A (en) * | 2019-12-14 | 2020-03-24 | 燕山大学 | Ternary metal-containing porous carbon Cu-ZnCo @ C adsorption desulfurizer and preparation method thereof |
| CN112691691A (en) * | 2021-01-17 | 2021-04-23 | 北京工业大学 | Preparation method of modified ZIFs-derived Co-N-C-MT/EA catalyst |
-
2021
- 2021-12-20 CN CN202111562911.XA patent/CN114113582B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105372236A (en) * | 2015-11-18 | 2016-03-02 | 云南大学 | Highly sensitive cancer cell detecting method |
| CN107930586A (en) * | 2017-09-29 | 2018-04-20 | 天津大学 | A kind of method by copper incorporation zeolite imidazole salt frame synthesis Cu ZIF 67 |
| CN109647478A (en) * | 2018-12-20 | 2019-04-19 | 首都师范大学 | The preparation method of the nitrogenous carbon-coating catalyst of copper and cobalt doped |
| CN110057877A (en) * | 2018-12-24 | 2019-07-26 | 中山大学 | The biosensor and its preparation method for being used to detect tumour cell of repeatable modification |
| CN109748327A (en) * | 2019-02-25 | 2019-05-14 | 武汉理工大学 | One kind preparing CuCoO based on MOFs material at low temperature2The method of nanocrystalline material |
| CN110280245A (en) * | 2019-05-28 | 2019-09-27 | 浙江工业大学 | A kind of ZIF-67/CuBTC composite material and its derivative and method and application |
Non-Patent Citations (4)
| Title |
|---|
| Cu掺杂ZIF-67的简单制备和调控;袁尧等;《中国科技论文在线》;20191108;第1-7页 * |
| Doping copper into ZIF-67 for enhancing gas uptake capacity and visible-light-driven photocatalytic degradation of organic dye;Yang, H等;《JOURNAL OF MATERIALS CHEMISTRY》;20120911;第22卷(第41期);第21849-21851页 * |
| 金属有机骨架材料(MOFs)优化方法综述;李天佑;《广州化工》;20200808;第48卷(第15期);第56-60页 * |
| 金属有机骨架材料在催化中的应用;黄刚等;《化学学报》;20160215;第74卷(第02期);第113-129页 * |
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