High-sensitivity microRNA fluorescence in-situ hybridization quantitative labeling probe based on semiconductor polymer points and preparation method of high-sensitivity microRNA fluorescence in-situ hybridization quantitative labeling probe
A fluorescence in situ hybridization, polymer dot technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome operation, unsatisfactory physical and chemical stability, complicated preparation process, etc. Stable and efficient target RNA labeling ability, non-toxic biocompatibility effect
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Embodiment 1
[0040] (1) Prepare 3mL THF solutions of PFO, PFBT, PFDTBT and CN-PPV semiconducting polymer and functional polymer (polystyrene maleic anhydride (PSMA)) respectively, the concentrations of semiconducting polymer and PSMA are 40 μg / mL and 10 μg / mL. The near-infrared dye NIR 775 is also doped in the tetrahydrofuran solution of CN-PPV, and its concentration is 0.5μg / mL; then it is filtered with a 220nm organic filter head, and then the semiconductor obtained after filtration is ultrasonically The mixed solution of polymer and PSMA was quickly injected into 4 parts of 10mL DEPC water respectively and continued to sonicate for 2 minutes;
[0041] (2) Under the protection of nitrogen, the solution obtained in step (1) was heated on a heating platform (85°C) for 4 hours to remove the tetrahydrofuran solvent, and after cooling to room temperature, it was filtered through a 220nm water filter head to remove large particles, and the obtained particle Semiconducting polymer dots with a ...
Embodiment 2
[0051] Refer to the preparation method of Example 1 to obtain 4 kinds (PFO Pdos standard hsa-microRNA-1292-5p, PFBTPdos standard hsa-microRNA-1301-3p, PFDTBT Pdos standard hsa-microRNA-3614-5p and NIR Pdos standard hsa-microRNA- 5589-3p) Semiconducting polymer dot-based microRNA fluorescence in situ hybridization quantitative labeling probe. When multi-target microRNA labeling, the particle size distribution and surface potential of different types of semiconducting polymer dots should be at an approximate level as far as possible. When we prepare the four kinds of semiconducting polymer dots, we must keep the preparation conditions consistent, including the concentration and volume of the THF solution of the semiconducting polymer and the functional polymer, the volume of DEPC water injected, the ultrasonic time, the temperature and time when heating to remove THF, etc. . The particle size distribution and surface potential of the semiconducting polymer dots thus obtained ca...
Embodiment 3
[0058] Firstly, tetrahydrofuran solutions of semiconducting polymers PFO and PSMA were prepared with concentrations of 100 μg / mL and 1 μg / mL, respectively. Then go through the same process as in Example 1 to modify the target microRNA-specific antisense complementary sequence to obtain a fluorescence in situ hybridization quantitative labeling probe for the target microRNA (hsa-microRNA-1292-5p).
[0059] The sequence of Hsa-microRNA-1292-5p is as follows:
[0060] Hsa-miR-1292-5p
[0061]
[0062] In order to further verify the sensitivity and specificity of the microRNA fluorescent in situ hybridization quantitative labeling probe designed in the present invention for the target microRNA, we introduced a resonance based on the semiconductor polymer dots (PFO Pdots) and the base side chain labeling dye Cy3dt labeling label energy transfer( (or fluorescence) resonance energy transfer, FRET) model. First, based on the preparation process in Example 1, we prepared labeled ...
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