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Fluorescent probe for monitoring apoptosis as well as preparation method and application of fluorescent probe

A fluorescent probe and apoptosis technology, which are applied in the field of fluorescent probes for monitoring apoptosis and their preparation, and can solve problems such as difficulty in detecting apoptosis

Active Publication Date: 2020-10-20
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current method can only be used for the detection of a single marker in cytochrome c or telomerase, and there is no report on the simultaneous detection of probes for both, so it is difficult to detect the whole process of apoptosis

Method used

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  • Fluorescent probe for monitoring apoptosis as well as preparation method and application of fluorescent probe
  • Fluorescent probe for monitoring apoptosis as well as preparation method and application of fluorescent probe
  • Fluorescent probe for monitoring apoptosis as well as preparation method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] The preparation process of a fluorescent probe for monitoring the whole process of apoptosis of DNA tetrahedral nucleic acid framework is as follows:

[0095] S1. Preparation of DNA tetrahedral nucleic acid framework:

[0096] Mix equal amounts of ssDNA A, ssDNA B, ssDNA C, ssDNA D, ssDNA E, and ssDNAF in 2×PBS buffer, anneal at 95°C for 10 minutes, and then cool at 37°C for 1 hour to obtain the DNA tetrahedral nucleic acid framework structure.

[0097] S2. Preparation of double-stranded probes:

[0098] 1) Cytochrome c probe: Mix equal amounts of cytochrome c capture chain and cytochrome c aptamer in 2×PBS buffer, anneal at 95°C for 10 minutes, and finally cool at 37°C for 1 hour.

[0099] 2) Telomerase probe: Mix equal amounts of telomerase capture strand, telomerase template strand and telomerase dye strand in 2×PBS buffer, anneal at 95°C for 10 minutes, and finally cool at 37°C for 1 hour.

[0100] S3. Cell apoptosis probe assembly:

[0101] Mix the same amount o...

Embodiment 2

[0102] Example 2 DNA Tetrahedral Nucleic Acid Framework Assembly Process and Reaction Mechanism Characterization

[0103] How the DNA tetrahedron works figure 1 As shown, in order to verify the correctness of the mechanism, use PAGE gel for gel electrophoresis experiment, take 5 μL of DNA sample and mix with 1 μL of 6×DNA loading buffer, run at 80V for 85 minutes and image. PAGE gel electrophoresis to characterize DNA tetrahedron nucleic acid framework-type apoptosis monitoring fluorescent probe construction and its working mechanism.

[0104] In addition to the aforementioned DNA strands, two extended strands of the DNA tetrahedral nucleic acid framework were used for simulating probe binding, the sequences of which are as follows:

[0105] ssDNA C extension:

[0106] ssDNA E extension:

[0107] The formation of the DNA tetrahedral nucleic acid framework was first characterized using 5% PAGE. Such as figure 2 As shown in A, the different bands in lanes 1-6 correspon...

Embodiment 3

[0110] Example 3 Cell Apoptosis Probe Detection of Cell Apoptosis Markers

[0111] To the apoptosis probe constructed in Example 1, add cytochrome c or 0, 0.625, 2.5, 5, 12.5, 25, 37.5, 50, 62.5, 70cells / μL of telomerase, the final concentration of the probe is 50nM, and after incubation at 37°C for two hours, the fluorescence intensity is measured using a fluorescence spectrometer, wherein the FAM excitation light is 495nm, and the Cy5 excitation light is 640nm. image 3 Take B and D respectively image 3 Plotting the fluorescence intensity at 660nm and 520nm in A and C.

[0112] image 3 In A, the cytochrome c concentration-dependent Cy5 fluorescence emission line increases with increasing cytochrome c concentration. image 3 B is the linear function of the fluorescence intensity (660nm) of Cy5 as the concentration of cytochrome c changes, that is, I Cyt c =0.07C Cyt c +52.61, R 2 =0.998, LOD=85.93nm, the linear range is 20nm~2000nm. image 3 In C and 3D, the apoptosi...

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Abstract

The invention discloses a fluorescent probe for monitoring apoptosis as well as a preparation method and application of the fluorescent probe. The fluorescent probe comprises a DNA tetrahedral nucleicacid framework nanostructure with three probe binding arms, a Cy3-modified DNA single chain as a positioning probe, a cytochrome c probe and a telomerase probe, the positioning probe, the cytochromec probe and the telomerase probe are combined with three probe combination arms of the DNA tetrahedron nucleic acid framework nano structure through nucleic acid complementary hybridization, the cytochrome c probe comprises a cytochrome c aptamer chain and a hybrid double chain of a cytochrome c capture chain, and the telomerase probe comprises a telomerase capture chain, a telomerase template chain and a hybrid double chain of a telomerase dye chain. The DNA tetrahedron nucleic acid framework type cell apoptosis whole-process monitoring fluorescence probe has the characteristics of high sensitivity, good stability, strong selectivity and low toxicity, and shows good application performance in the aspects of cell apoptosis monitoring, drug evaluation and screening and the like.

Description

technical field [0001] The invention belongs to the field of functional nanomaterials and biological detection, and in particular relates to a fluorescent probe for cell apoptosis monitoring and its preparation method and application. Background technique [0002] Cytochrome c, which contains metalloproteins, resides in the mitochondrial intermembrane space and is released into the blood under pathological conditions. The release of cytochrome c from mitochondria is a key active step in the activation of cell death pathways. It is generally believed that the leakage of cytochrome c is an early event of cell apoptosis and at the same time indicates the beginning of apoptosis. Therefore, the detection of cytochrome c is very important for the monitoring of early cell apoptosis. is of great significance. Telomerase is a ribonucleoprotein reverse transcriptase responsible for maintaining cellular telomere length. In the late stage of apoptosis, as the nuclear membrane breaks d...

Claims

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Application Information

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IPC IPC(8): C12Q1/6818C12N15/11C12N15/10C12Q1/02
CPCC12Q1/6818G01N33/5011C12N2503/02G01N2500/10C12Q2563/107C12Q2525/205C12Q2521/113C12Q2525/30
Inventor 宋春元董晨张晶晶汪联辉
Owner NANJING UNIV OF POSTS & TELECOMM
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