CN111855625B - CA125 detection kit based on Cu-MOF and application thereof - Google Patents
CA125 detection kit based on Cu-MOF and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and nucleic acid chemistry, and relates to a CA125 detection kit based on Cu-MOF and application thereof. The kit is based on the utilization of the fluorescence quenching effect of a metal organic framework material and the strong adsorption force of metal MOF and a DNA aptamer, when the DNA aptamer and the MOF coexist, the DNA aptamer is combined with the MOF, and the fluorescence is quenched; and when the target protein exists, the DNA aptamer is preferentially combined with the target protein or is displaced from the MOF, and the fluorescence value is recovered. Synthesizing a DNA aptamer specific to CA125, and adding a fluorescent label of Cy5.5, wherein the sequence is as follows: TGCCTTATTACTCTCTCTGTTAAC-Cy5.5. And co-incubating standard proteins with different concentrations, the DNA aptamer and the MOF to obtain a fluorescence recovery standard curve, optimizing a reaction system to obtain an optimal detection range and detection sensitivity, and obtaining different target protein concentrations by using different protein standard curves and target protein recovery conditions with different fluorescence values in the detection system. The detection method is rapid, low in cost, high in sensitivity and free of complex amplification and detection instruments.
Description
Technical Field
The invention belongs to the technical field of molecular biology and nucleic acid chemistry, and particularly relates to a CA125 detection kit based on Cu-MOF and application thereof.
Background
Ovarian Cancer is the highest-mortality gynecologic malignancy, with a 5-year survival rate of only about 40% (Siegel RL, miller KD, jemal a. Cancer statistics,2018.Ca Cancer J Clin 2018 (1): 7-30.) that severely threatens women's life health. Ovarian cancer is occult, early ovarian cancer usually has no obvious symptoms, so about 70 percent of ovarian cancer is discovered at an advanced stage, and extensive metastasis of the abdominal cavity occurs, which is also the main reason for the high mortality rate of ovarian cancer. The survival rate of early ovarian cancer can reach more than 90 percent, so that the improvement of the early diagnosis efficiency of ovarian cancer is a key problem for improving the overall survival rate of ovarian cancer (references: kurman RJ., visvanathan K., roden R., wu TC., shih IeM. Am J Obstet Gynecol,2008,198 (4), 351-6.). The main clinical indicator for early diagnosis of ovarian cancer is CA125.
The detection method of CA125 in clinic is mainly an ELISA method, and has the disadvantages of long time consumption, complex operation, relatively high price of an ELISA kit and the like. Therefore, the invention focuses on finding a detection technology which is simple in operation, stable in system, low in price, good in sensitivity and quick in response. In recent years, detection methods based on metal MOF and DNA aptamers are popular in the current detection field, for example, yu Chao group adopts a trimetal signal amplification strategy based on Ce-MOF @ Au and AuPtRu NPs, so as to sensitively detect TSP1 by using DNA aptamers (refer to the references: xiaooxue Fu, junin He., chengli Zhang., jun Chen, yilin Wen., jia Li., weiran Mao., hangtian Zong., jiahao Wu., xingduo Ji, chaoYu.biosense Bioelctron, 2019,132, 302-309.). The Yinji Chen group developed an electrochemical method for detecting multiple antibiotics based on efficient Y-type DNA probes and nanometal-organic frameworks (NMOF) (references: meng Chen., ningGa., tianhua Li., ye Wang., qing xu., yinji Chen. Animal Chim Acta,2017,968, 30-39.); the group also reported that an ultrasensitive electrochemical probe for simultaneously detecting Oxytetracycline (OTC) and Kanamycin (KAN) using an anti-ssDNA antibody and an aptamer was developed based on cyclic amplification of a target region catalyzed by RecJf exonuclease using metal organic framework Materials (MOFs) as signal tracers. (references: meng Chen., ning Ga., you Zhou., tianhua Li., qing xu., yuting Cao., yinji Chen. Talanta,2016,161, 867-874.); however, to date, there is no detection method for CA125 that can be based on Cu-MOF.
Disclosure of Invention
The problems to be solved by the invention are as follows: the detection method based on the MOF and the DNA aptamer is designed by utilizing the fluorescence quenching effect of the combination of the DNA aptamer carrying fluorescence and the MOF and the high affinity of the DNA aptamer and a target protein thereof, and aims at the defects of the existing CA125 detection, such as long time consumption, complex operation, high relative price of an ELISA kit and the like. Provides a method for rapidly detecting CA125, and assembles a simple and easy-to-operate kit, and can carry out rapid, low-cost and high-sensitivity detection on CA125.
The general technical route of the invention is as follows: collecting a sample, and incubating with the DNA aptamer of CA 125; cu-MOF was added, immediately excited at 650nm and Cy5.5 fluorescence signal was measured at 700 nm. Key reaction conditions, reagent components and concentrations in the experimental process are optimized, and a CA125 aptamer, a Tris-HCl buffer solution and Cu-MOF are developed into a kit for detecting CA125.
The technical scheme of the invention is as follows: (1) Pre-incubation of CA125 DNA aptamer with CA125 protein, hybridization system: 1 mu L of 5 mu M CA125 aptamer, 5 mu L of sample to be detected containing CA125, and supplementing Tris-HCl to 10 mu L; hybridization conditions: room temperature, 20min.
(2) In the precoated ELISA plate, 90 μ L of Cu-MOF diluted with Tris-HCl buffer solution with pH7.4 and with final concentration of 0.5 μ g/mL is added into each well, the mixture is gently mixed and then excited at 650nm, and Cy5.5 fluorescence signals are measured at 700 nm.
The specific content is as follows:
1. study on the binding of CA125 DNA aptamer to Cu-MOF and the optimal concentration of Cu-MOF
Preparing 10mg/mL Cu-MOF by using DMF; the synthesized CA125 DNA aptamer (Cy5.5-labeled CA125 aptamer sequence: cy5.5-TGCCTATATTACTCTCTCTCCTGTTAAC) was dissolved with an appropriate amount of TM buffer to a final concentration of 5. Mu.M. Taking Cu-MOF with different concentrations (final concentration: 5 mu g/mL,1 mu g/mL,0.5 mu g/mL,0.1 mu g/mL and 0.02 mu g/mL), adding DNA aptamer with final concentration of 0.05 mu M CA125, incubating for 0.5h at room temperature, setting different wavelengths of activated light and emitted light, and detecting quenching of fluorescence value by using a microplate reader. As a result, it was found that the fluorescence quenching effect reached a plateau substantially when the final concentration of Cu-MOF was 0.5. Mu.g/mL, and the concentration was selected as the optimum concentration.
2. Research on influence of different conditions on binding force of CA125 aptamer and Cu-MOF
Preparing 100mM NaCl,300mM NaCl,1 mu g/mu L BSA, pH3.0, pH5.0, pH7.0 and pH9.0, preparing 25 ℃,37 ℃,60 ℃ and 80 ℃ water baths, incubating DNA aptamers of Cu-MOF and CA125 for 0.5h under the conditions, setting excitation light and emission light wavelength, detecting quenching condition of fluorescence value by using a microplate reader, analyzing different salt ion concentrations, and analyzing the influence of different pH and different concentrations on DNA suitability and Cu-MOF binding force, and checking whether the DNA aptamer of CA125 and BSA protein have non-specific binding. The result shows that the fluorescence quenching effect is good under the conditions of normal temperature and 25 ℃ and 37 ℃, the fluorescence quenching effect is basically equivalent when the pH is 5-9, the fluorescence quenching effect is not greatly influenced by 100mM NaCl, and no obvious nonspecific binding effect exists between the CA125 aptamer and BSA.
3. The linear relation between the concentration of the CA125 standard protein and the fluorescence intensity is researched to obtain the optimal concentration range of the aptamer detection protein
CA125 standard protein (ab 164957, abcam, final concentration of 100. Mu.L reaction system 1500ng/mL,1200ng/mL,800ng/mL,400ng/mL,200ng/mL,100ng/mL,50ng/mL,20ng/mL,10ng/mL,1ng/mL,0.1 ng/mL) was prepared in different concentrations and incubated with CA125 DNA aptamer for 20min at room temperature. Then Cu-MOF was added to a final concentration of 0.5. Mu.g/mL and immediately subjected to fluorescence detection. Fluorescence measurements were performed by an F2500 fluorescence spectrometer (hitachi). Cy5.5 was excited at 650nm and the Cy5.5 fluorescence signal was measured at 700 nm. All excitation and emission slit widths were set at 5nm and pmt voltage at 800V. All these measurements were recorded at room temperature.
The results found that the detection range of CA125 based on Cu-MOF is: the linear detection range is shown in the specification, and the CA125 detection kit based on the Cu-MOF has a very good detection range and sensitivity.
The kit is used for the in vitro CA125 detection method:
(1) Pre-incubation of CA125 DNA aptamer with CA125 protein, hybridization system: mu.L of 5 mu M CA125 aptamer and 5 mu.L of a sample to be detected containing CA125 are supplemented with Tris-HCl to 10 mu.L; hybridization conditions: room temperature, 20min.
(2) In the pre-coated ELISA plate, 90 μ L of Cu-MOF diluted with Tris-HCl buffer solution with pH7.4 and with final concentration of 0.5 μ g/mL is added into each hole, after being mixed gently, the mixture is excited at 650nm immediately, and Cy5.5 fluorescence signal is measured at 700 nm.
Compared with the prior art, the detection method for detecting the CA125 by using the DNA aptamer of the Cu-MOF and the CA125 has the beneficial effects that an antibody is not needed, but the DNA aptamer of the fluorescent marker is hybridized with the CA125 protein to form a DNA protein hybrid, so that the DNA probe of the fluorescent marker is protected, and the fluorescent quenching caused by the combination of the DNA probe and the MOF is avoided. Aiming at the defects of the existing CA125 detection, such as long time consumption, complex operation, relatively high price of an ELISA kit and the like, the invention provides a method for quickly detecting CA125, and the kit which is simple and easy to operate is assembled, so that the rapid, low-cost and high-sensitivity detection can be carried out on CA125.
Drawings
FIG. 1: method schematic of the invention
FIG. 2: binding of CA125 DNA aptamer to Cu-MOF
FIG. 3: influence of different conditions on binding force of CA125 aptamer and Cu-MOF
Influence of different temperatures of A on binding force of CA125 aptamer and Cu-MOF
Influence of different pH values on binding force of CA125 aptamer and Cu-MOF
Influence of C salt ions on binding force of CA125 aptamer and Cu-MOF
Influence of the Presence of D-nonspecific protein on the binding force of CA125 aptamer to Cu-MOF
FIG. 4: CA125 standard protein concentration and fluorescence intensity standard curve and optimal linear range analysis for CA125 protein detection by CA125 DNA aptamer
A. Specific analysis result chart applied to detection of CA125 in the invention
B. Results plot of the present invention applied to the detection of the linear concentration range of CA125
Detailed Description
The following specific examples are provided to further illustrate the technical solutions of the present invention, but the application of the technology of the present invention is not limited to the examples.
Example 1 study of the binding of CA125 DNA aptamer to Cu-MOF and the optimal concentration of Cu-MOF
Preparing 10mg/mL Cu-MOF by using DMF; the synthesized CA125 DNA aptamer (Cy5.5-labeled CA125 aptamer sequence: cy5.5-TGCCTTATTACTTACTCTCCTGTTAAC) was dissolved in an appropriate amount of TM buffer to a final concentration of 5. Mu.M. Taking Cu-MOF with different concentrations (final concentration: 5 mu g/mL,1 mu g/mL,0.5 mu g/mL,0.1 mu g/mL,0.02 mu g/mL), adding DNA aptamer with a final concentration of 0.05 mu M CA125, incubating for 0.5h at room temperature, setting different activated light and emitted light wavelengths, and detecting quenching of a fluorescence value by using a microplate reader. As a result, it was found that the fluorescence quenching effect reached a plateau substantially when the final concentration of Cu-MOF was 0.5. Mu.g/mL, and the concentration was selected as the optimum concentration.
Example 2 study of the Effect of different conditions on the binding force of CA125 aptamer and Cu-MOF
Preparing 100mM NaCl,300mM NaCl,1 mu g/mu L BSA, pH3.0, pH5.0, pH7.0 and pH9.0, preparing 25 ℃,37 ℃,60 ℃ and 80 ℃ water baths, incubating the DNA aptamers of Cu-MOF and CA125 for 0.5h under the conditions, setting excitation light and emission light wavelength, detecting the quenching condition of fluorescence value by using a microplate reader, analyzing different salt ion concentrations, and analyzing the influence of different pH and different concentrations on DNA suitability and Cu-MOF binding force, and detecting whether the DNA aptamer of CA125 and BSA protein have non-specific binding. The result shows that the fluorescence quenching effect is better under the conditions of normal temperature and 25 ℃ and 37 ℃, the fluorescence quenching effect is basically equivalent when the pH is 5-9, the influence of 100mM NaCl on the fluorescence quenching effect is small, and no obvious non-specific binding effect exists between the CA125 aptamer and BSA.
Example 3 study of the Linear relationship between CA125 Standard protein concentration and fluorescence intensity to obtain the optimal concentration Range of aptamer-detected protein
Different concentrations of CA125 standard protein (ab 164957, abcam, final concentrations of 1500ng/mL,1200ng/mL,800ng/mL,400ng/mL,200ng/mL,100ng/mL,50ng/mL,20ng/mL,10ng/mL,1ng/mL,0.1ng/mL, respectively) were prepared and incubated with CA125 DNA aptamer for 20min at room temperature. Then Cu-MOF was added to a final concentration of 0.5. Mu.g/mL and immediately subjected to fluorescence detection. Fluorescence measurements were performed by an F2500 fluorescence spectrometer (hitachi). Cy5.5 was excited at 650nm and the Cy5.5 fluorescence signal was measured at 700 nm. All excitation and emission slit widths were set at 5nm and pmt voltage at 800V. All these measurements were recorded at room temperature.
As a result, the detection range of CA125 based on Cu-MOF is found as follows: the linear detection range is shown in the specification, and the CA125 detection kit based on the Cu-MOF has very good detection range and sensitivity.
Method for detecting CA125 in vitro by using kit
(1) Pre-incubation of CA125 DNA aptamer with CA125 protein, hybridization system: mu.L of 5 mu M CA125 aptamer and 5 mu.L of a sample to be detected containing CA125 are supplemented with Tris-HCl to 10 mu.L; hybridization conditions: room temperature, 20min;
(2) In the pre-coated ELISA plate, 90 μ L of Cu-MOF diluted with Tris-HCl buffer solution with pH7.4 and with final concentration of 0.5 μ g/mL is added into each hole, after being mixed gently, the mixture is excited at 650nm immediately, and Cy5.5 fluorescence signal is measured at 700 nm.
Sequence listing
<110> Nanjing City health care hospital for women and children
<120> CA125 detection kit based on Cu-MOF and application thereof
<160>1
<210>1
<211>25
<212>DNA
<213> Artificial sequence
<400>1
tgccttattactctctcctgttaac
<110> Nanjing city women and children health care hospital
<120> CA125 detection kit based on Cu-MOF and application thereof
<160>1
<210>1
<211>25
<212>DNA
<213> Artificial sequence
<400>1
tgccttattactctctcctgttaac
Claims (2)
1. A CA125 detection kit based on Cu-MOF is characterized by comprising the following components:
(1) A common 96-well plate;
(2) Probe mixture 1: DNA aptamer to fluorescently labeled CA125 at a concentration of 5. Mu.M;
fluorescently labeled CA125 DNA aptamer: SEQ ID NO1, sequence (5 '-3'): cy5.5-TGCCTTATTACTTACTCTCCTGTTAAC;
(3) Cu-MOF: preparing 10mg/mL of DMF (dimethyl formamide) and Cu-MOF storage solution;
(4) Buffer solution: 10mM Tris-HCl pH 7.4.
2. Use of a CA125 test kit based on Cu-MOF according to claim 1 for in vitro CA125 test, characterized in that it consists of the following steps:
(1) Pre-incubation of CA125 DNA aptamer with CA125 protein, hybridization system: fluorescence labeled DNA aptamer of CA125, 5 mu L of sample to be detected containing CA125, and adding Tris-HCl to 10 mu L; hybridization conditions: room temperature, 20min;
(2) And (2) adding 90 mu L of Cu-MOF diluted by Tris-HCl buffer solution with the pH value of 7.4 and having the final concentration of 0.5 mu g/mL into each hole of the enzyme label plate pre-coated by the hybridization system in the step (1), gently mixing the mixture uniformly, immediately exciting the mixture at 650nm, and measuring a Cy5.5 fluorescence signal at 700 nm.
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