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Surface-enhanced Raman technology based on signal-off and used for detecting intracellular telomerase activity

A telomerase, cell technology, applied in the field of detecting intracellular telomerase activity

Inactive Publication Date: 2015-05-13
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of the currently used SERS detection methods are based on the signal-on mode, and there are few reports in the literature on the method of detecting tumor markers by SERS in the signal-off mode.

Method used

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  • Surface-enhanced Raman technology based on signal-off and used for detecting intracellular telomerase activity
  • Surface-enhanced Raman technology based on signal-off and used for detecting intracellular telomerase activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment one combine figure 1 , to synthesize a nanoprobe that can detect intracellular telomerase.

[0018] After mixing 20 μL hairpin DNA (150 μM) and 20 μL telomerase primer (150 μM) with 2 mL carrier-loaded nanoparticles, stir at room temperature for 20 h. Then 0.2 mL of phosphate buffered saline (PBS, pH 7.8) containing 1M NaCl was added dropwise to the above mixture to stabilize the nanoprobes. After the above solution was centrifuged and washed with PBS, it was redispersed in 1 mL of PBS to obtain nanoprobes, which were stored at 4°C for future use.

Embodiment 2

[0019] Embodiment two combine figure 2 , Activity detection of intracellular telomerase activity using nanoprobes.

[0020] HeLa cells were co-incubated with the probe for 2 h, and observed with a laser confocal Raman spectrometer. In telomerase-positive HeLa cells, the probe of the hairpin structure is opened, the Raman signal is weakened, and normal cells can be distinguished from tumor cells. Incubate the telomerase inhibitor drug catechin with HeLa cells for 48 hours, then add 20 μL of probe and incubate for 2 hours, observe with a Raman spectrometer, it can be seen that the Raman signal in the cell is much higher than that of the tumor cell, indicating that the cell Internal telomerase activity is inhibited by drugs.

[0021] Probe sequence: 5'-R6G-ACG TAG TCC GCT TTA AAC TCT GCT CG A CGG TAA AGC GGA CTA CGT - M -3'

[0022] M is mercapto, amino or carboxyl, etc.

[0023] Telomerase Primer: 5’- AAT CCG TCG AGC AGA GTT-3’

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Abstract

The invention relates to a surface-enhanced Raman technology based on signal-off and used for detecting intracellular telomerase activity. According to the technology, graphene, carbon nitride, MoS2, SiO2 and the like are taken as carriers, nano-gole or nano-silver with controllable dimension and morphology or a composite magnetic nano-particle with a core-shell structure is supported by the carriers, and then a hairpin probe containing a telomerase primer and a Raman molecule beacon is fixedly disposed on the nano=particle surface, so that the high-sensitivity high-selectivity SERS detection method based on signal-off is established. When a target exists, the primer extends and generates a DNA chain with a repeat sequence under the effect of telomerase, and the DNA chain is hybridized with the probe molecule, then the DNA hairpin structure is opened, a Raman signal molecule with the single-chain labeled end is far away from a Raman substrate, the Raman signal is reduced, and high-sensitivity detection on telomerase is realized. The probe prepared by taking Hela cervical carcinoma cell as a model realizes intracellular telomerase activity detection and dynamic detection on intracellular telomerase activity variation under effect of a telomerase inhibiting medicament. By using multiple cells for imaging, the probe is confirmed to be capable of distinguishing tumor cells and normal cells.

Description

technical field [0001] The invention relates to a signal-off-based surface-enhanced Raman technique for detecting intracellular telomerase activity. Background technique [0002] Cancer is currently one of the main killers threatening human health. The best way to treat cancer is early detection, early diagnosis, and early treatment. Therefore, it is of great significance to develop novel, sensitive and specific early diagnostic probes. [0003] At present, the clinical diagnosis methods of cancer are mainly based on imaging examination of tissue and cell morphology, which has low resolution and lack of clear and unified marker molecules, making it difficult to achieve early diagnosis of cancer. How to find a method that can really detect tumors early and eliminate them in the bud is particularly urgent. Now clinically, tumor markers are mainly used for early detection and diagnosis of cancer. Among them, telomerase is considered to be a promising tumor marker. [0004] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 孙召梅修宝郭英姝张书圣
Owner LINYI UNIVERSITY
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