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CA125 detection kit based on Cu-MOF and application of CA125 detection kit

A CA125, detection kit technology, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of complicated operation, long time and high price of ELISA kits

Active Publication Date: 2020-10-30
NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The problem to be solved in the present invention: the detection method based on MOF and DNA aptamer is a detection method designed by using the fluorescence quenching effect of the combination of DNA aptamer carrying fluorescence and MOF and the high affinity of DNA aptamer and its target protein, For the shortcomings of existing CA125 detection, such as long time-consuming, complicated operation, relatively high price of ELISA kit, etc.

Method used

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  • CA125 detection kit based on Cu-MOF and application of CA125 detection kit
  • CA125 detection kit based on Cu-MOF and application of CA125 detection kit
  • CA125 detection kit based on Cu-MOF and application of CA125 detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Example 1 Study on the combination of CA125 DNA aptamer and Cu-MOF and the optimal concentration of Cu-MOF

[0032] Prepare 10 mg / mL Cu-MOF with DMF; add an appropriate amount of TM buffer to dissolve the synthetic CA125 DNA aptamer (Cy5.5-labeled CA125 aptamer sequence: Cy5.5-TGCCTTATTACTCTCTCCTGTTAAC) to a final concentration of 5 μM. Take different concentrations of Cu-MOF (final concentration: 5 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 0.02 μg / mL), add DNA aptamer with a final concentration of 0.05 μM CA125, and incubate at room temperature for 0.5 h, set different activation light and emission light wavelengths, and use a microplate reader to detect the quenching of the fluorescence value. It was found that when the final concentration of Cu-MOF was 0.5 μg / mL, the fluorescence quenching effect basically reached the plateau, which was selected as the optimal concentration.

Embodiment 2

[0033] Example 2 Study on the Effect of Different Conditions on the Binding Force of CA125 Aptamer and Cu-MOF

[0034] Prepare 100mM NaCl, 300mM NaCl, 1μg / μL BSA, pH3.0, pH5.0, pH7.0, pH9.0 buffer, prepare 25℃, 37℃, 60℃ and 80℃ water baths, put Cu-MOF After incubating with the DNA aptamer of CA125 for 0.5h under the above conditions, set the excitation light and emission light wavelengths, use a microplate reader to detect the quenching of the fluorescence value, and analyze the suitability and The influence of Cu-MOF binding force, to test whether there is non-specific binding between CA125 DNA aptamer and BSA protein. The results showed that the fluorescence quenching effect was better at room temperature and 25°C and 37°C, the fluorescence quenching effect was basically the same between pH 5-9, 100mM NaCl had little effect on the fluorescence quenching effect, and the difference between CA125 aptamer and BSA There was no obvious non-specific binding between them.

Embodiment 3

[0035] Example 3 Study the linear relationship between CA125 standard protein concentration and fluorescence intensity, and obtain the optimal concentration range of aptamer detection protein

[0036] Prepare different concentrations of CA125 standard protein (ab164957, abcam, the final concentration of 100μL reaction system is 1500ng / mL, 1200ng / mL, 800ng / mL, 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 20ng / mL mL, 10ng / mL, 1ng / mL, 0.1ng / mL), and incubated with CA125 DNA aptamer at room temperature for 20min. Then Cu-MOF was added at a final concentration of 0.5 μg / mL, and fluorescence detection was performed immediately. Fluorescence measurements were performed by a F2500 fluorescence spectrometer (Hitachi). Cy5.5 was excited at 650nm and the Cy5.5 fluorescence signal was measured at 700nm. All excitation and emission slit widths were set to 5 nm, and the PMT voltage was 800 V. All these assays were recorded at room temperature.

[0037] The results showed that the detection ra...

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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 of the CA125 detection kit. The kit is based on the utilization of the fluorescence quenching effect of a metal organic framework material and the strong adsorption capacity of metal MOF and a DNA aptamer, and when the DNA aptamer and the MOF coexist, the DNA aptamer and the MOF are combined, and fluorescence quenching is carried out; and in the presence of the target protein, the DNA aptamer is preferentially combined with the target protein or is replaced from the MOF, and the fluorescence value is recovered. The method comprises the following steps: synthesizing the DNA aptamer specifically aiming at CA125, adding a fluorescence label of Cy5.5, and the sequence of the DNA aptamer being TGCCTTATTACTCTCTCCTGTTAAC-Cy5.5; co-incubating standard proteins with different concentrations, the DNA aptamer and the MOF to obtain a fluorescence recovery standard curve, optimizing a reaction system, obtaining an optimal detection range and detection sensitivity, and obtaining different target protein concentrations according to different protein standard curves and recovery conditions of target proteins with different fluorescence values in the detection system. The detection method is rapid, low in cost and high in sensitivity,and does not need complex amplification and detection instruments.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and nucleic acid chemistry, and in particular relates to a Cu-MOF-based CA125 detection kit and its application. Background technique [0002] Ovarian cancer is the gynecological malignancy with the highest mortality rate, and the 5-year survival rate is only about 40% (Siegel RL, MillerKD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018; 68(1): 7-30.) Severe Threat to women's life and health. The onset of ovarian cancer is occult, and early ovarian cancer usually has no obvious symptoms. Therefore, about 70% of ovarian cancers are found in the advanced stage and have extensive metastasis in the abdominal cavity. This is also the main reason for the high mortality rate of ovarian cancer. It has been reported in the literature that the survival rate of early ovarian cancer can reach more than 90%, so improving the early diagnosis efficiency of ovarian cancer is a key issue to im...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 徐娟马洁桦贾雪梅沈嵘季晨博钱冰
Owner NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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