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Method for detecting tumor cell marker miRNA-21 and tumor cells

A technology of miRNA-21 and tumor cells, which is applied in the detection field of tumor cell marker miRNA-21 and tumor cells, can solve the problems of low signal, high accuracy, and limited sensitivity of detection results, and achieve the detection of fewer samples required , Large linear range and high sensitivity

Active Publication Date: 2019-11-15
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] (1) The existing technology is to carry out the recognition trigger reaction on the surface of the cell membrane, and then enter the cell for Raman detection, the background signal is high and the sensitivity is limited
[0007] (2) The probes of the prior art do not combine the signal amplification of the WalkerDNA walker with the ratiometric detection method, and the detection result signal is low, the sensitivity is high, and the accuracy is poor
[0008] (3) The probes of the prior art are only monitored by fluorescence imaging and Raman imaging, and no detection and analysis of fluorescence and Raman are performed
[0013] Due to the low detection accuracy and poor practicability of the probes in the prior art for tumor cells and their markers; without combining Walker signal amplification with ratiometric detection, the generated signal is weak and the sensitivity of the detection target is not high ; No DNA tetrahedron is used to bring signal molecules into cells, the recognition sensitivity of the target is not high, and the background signal is high; the detection method is single, and the reliability of the detection results is not high. high

Method used

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  • Method for detecting tumor cell marker miRNA-21 and tumor cells
  • Method for detecting tumor cell marker miRNA-21 and tumor cells
  • Method for detecting tumor cell marker miRNA-21 and tumor cells

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Experimental program
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Effect test

Embodiment 1

[0078] Raman assay was performed on different concentrations of miRNA-21, image 3 -A is the Raman signal of different concentrations of miRNA-21, from curve a to curve h are the corresponding Raman signal curves of 0nM, 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM, 0.01pM concentrations of miRNA-21 picture. Choose 1600cm -1 It is the Raman characteristic absorption peak of Cy5, 1640cm -1 is the Raman characteristic absorption peak of Rox, the peak intensity of its peak position is respectively subtracted from the blank processing, and using I 1640 / I 1600 Performing a linear analysis for the final linear analysis object yields image 3 -B.

Embodiment 2

[0080] For Raman imaging detection of tumor cells such as image 3 As shown in -C, 200 μL of trypsinized Hela cells were seeded on a gold glass slide overnight, and 20 μL of biological barcode probe and 20 μL of 1 μM hairpin DNA H were added. 2 -DNA tetrahedral complexes, incubated for different times. Fix the cell culture dish on the microscope stage, and perform SERS imaging in the state of cell culture. Use the 633nm laser of the Raman spectrometer and perform SERS cell imaging with a 50x objective lens. Depend on image 3 In -C, it can be seen that the Raman signal intensity of Cy5 was significantly weakened after the reaction, and the Raman signal intensity of Rox was significantly enhanced, indicating the presence of miRNA-21 in Hela cells.

Embodiment 3

[0082] Fluorescent assays were performed on different concentrations of miRNA-21, Figure 4 -A is the fluorescence signal of Cy5 of different concentrations of miRNA-21, Figure 4 -B is the fluorescence signal of Rox with different concentrations of miRNA-21, from curve a to curve h are the fluorescence signal curves corresponding to the concentrations of miRNA-21 of 0nM, 100nM, 50nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM respectively picture. Fluorescence spectra were determined for Cy5 using an excitation wavelength of 648 nm with a maximum emission wavelength of 688 nm. Fluorescence spectra were determined for Rox using an excitation wavelength of 550 nm with a maximum emission wavelength of 610 nm. The peak intensity was deducted from the blank respectively, and the F 610 / F 688 Linear analysis is performed for the final linear analysis object to get Figure 4 -C.

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Abstract

The invention belongs to the technical field of tumor detection and discloses a method for detecting a tumor cell marker miRNA-21 and tumor cells. A composite nano-probe comprises a gold nanoparticlecore, hairpin DNA H1 and swing arm Walker-Blocker; the apex of DNA tetrahedron complex is connected with hairpin DNA H2; miRNA-21 is a 3D DNA Walker activator; by means of DNA walker cycle signal amplification of Walker DNA, H1 and H2, miRNA-21 ratiometricand fluorescence Raman dual-mode detection is achieved; gold nanoparticles and DNA tetrahedral structure enter tumor cells for ratiometric and fluorescence Raman dual-mode imaging detection of tumor cells. 3D DNA Walker nano-machine amplification and the DNA tetrahedral nano-probe are combined for ratiometric and dual-mode detection of the tumor cell marker miRNA-21 and the tumor cells.

Description

technical field [0001] The invention belongs to the technical field of tumor detection, and in particular relates to a tumor cell marker miRNA-21 and a detection method for tumor cells. Background technique [0002] Currently, the closest prior art: [0003] Prior art one: (YangL.; MengL.; SongJ.; XiaoY.; WangR.; KangH.; HanD..DynamicColloidalNanoparticleAssemblyTriggeredbyAptamer–receptor InteractionsonlivecellMembranes.Chem.Sci., 2019, 10, 7466-7471.) Proposed: design An aptamer and DNA displacement reaction-based dynamic system, which consists of core nanoparticles and small satellite nanoparticles, is partially hybridized with different DNA-modified hairpin strands and swing arm strands to form an aptamer , which specifically recognize EpCAM, these two separated particles can dynamically interact through the aptamer-receptor on the cell membrane to assemble into nucleated satellite assemblies on the surface. Structural changes of the system from isolated particles to c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/65
CPCG01N21/6428G01N21/6486G01N21/658
Inventor 何鹏毕成韩文豪宋维玲牛淑妍
Owner QINGDAO UNIV OF SCI & TECH
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