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Molecular beacon transmission system for directly detecting circulating tumor cells in blood as well as preparation method and application of molecular beacon transmission system

A molecular beacon and tumor cell technology, applied in the field of molecular beacon delivery system, can solve the problems of cumbersome process, invasive injury to patients, and damage to circulating tumor cells, and achieve simple preparation process, good stability and biocompatibility. , has the effect of stability and biocompatibility

Active Publication Date: 2020-12-01
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional clinical diagnosis methods mainly include tissue biopsy and imaging. Tissue biopsy is to obtain the tissue of the lesion through invasive puncture or surgery to interpret the tumor type. This method will not only bring invasive harm to the patient, but also due to the Hysteresis causes false positive or false negative results; the tumor size detectable by imaging is only more than 100 million cells (about 1 cubic centimeter), and the tumor is already mature when the volume is less than 1 million cells (about 0.01 cubic centimeter) start transfer
The main defects of this detection method of enrichment first and then characterization are as follows: 1. Circulating tumor cells are easy to cause damage during the enrichment process; 2. Real-time detection of living cells cannot be realized; 3. The interpretation of enriched cells is overly dependent on Due to various staining methods, the process is cumbersome, and at the same time, the results of segment judgment are prone to false negatives due to the lack of surface antigens
Therefore, it is particularly important to develop a nanomaterial that can detect circulating tumor cells in the blood and then enrich them. Most of the reported similar nanomaterials are composed of metal or inorganic non-metallic materials, which have poor biocompatibility and are only available. The protein molecules on the surface of CTCs can be detected by antibodies or aptamers, but the nucleic acid molecules inside the cells cannot be detected. Therefore, it is imminent to develop a nanomaterial with good biocompatibility and real-time reflection of the expression of nucleic acids inside the cells.

Method used

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  • Molecular beacon transmission system for directly detecting circulating tumor cells in blood as well as preparation method and application of molecular beacon transmission system
  • Molecular beacon transmission system for directly detecting circulating tumor cells in blood as well as preparation method and application of molecular beacon transmission system
  • Molecular beacon transmission system for directly detecting circulating tumor cells in blood as well as preparation method and application of molecular beacon transmission system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment one: molecular beacon delivery system Synthetic applications of nanoparticles.

[0047] 1. Synthesis of Molecular Beacon Delivery System Nanoparticles

[0048] The specific process is to dissolve sodium hyaluronate or sodium alginate or sodium heparin (150 μg) in PBS buffer solution (pH=6.0, 1 mL) with catalyst EDC / HoBt (-COOH:EDC:HoBt=1:1.2:1.2) Molar ratio) was activated at room temperature for 1 hour, and then an aminated aptamer or polypeptide (150 μg) was added and reacted at room temperature for 24 hours. Alternatively, the carboxylated aptamer or polypeptide (150 μg) was activated with catalyst EDC / HoBt at room temperature for 1 hour, and then carboxymethyl chitosan (150 μg) was added and reacted at room temperature for 24 hours. The product obtained after the reaction of the four polymer materials was placed in a dialysis bag (MWCO 10000), dialyzed with ultrapure water for 3 days, and finally obtained a functional polymer material after freeze-dr...

Embodiment 2

[0055] Example 2: Characterization of SHA / HA / PS / CHA nanoparticles.

[0056] 1. SHA / HA / PS / CHA Determination of particle size, potential and encapsulation efficiency of nanoparticles.

[0057] Specific implementation method: the SHA / HA / PS / CHA nanoparticle solution prepared in Example 1 was diluted with deionized water to a total volume of 1 mL. The size and potential of SHA / HA / PS / CHA nanoparticles in deionized water were measured by Zetasizer (Nano ZS, Malvern Instruments). Data are expressed as mean ± standard deviation (SD) based on three independent assays. To determine the encapsulation efficiency of molecular beacons, the solution containing SHA / HA / PS / CHA nanoparticles was centrifuged (10,000 rpm) at a specific speed for 1 hour at 4°C, and then the remaining unprecipitated free molecular signals in the supernatant were measured. The amount of the target, the encapsulation rate of the molecular beacon is calculated as the ratio of the amount of the precipitated beacon ...

Embodiment 3

[0069] Embodiment 3: SHA / HA / PS / CHA nanoparticles are used to detect target nucleic acid molecules in a sample.

[0070] 1. Detection of SHA / HA / PS / CHA nanoparticles at cell line level.

[0071] Specific implementation methods: the cellular uptake and intracellular distribution of SHA / HA / PS / CHA nanoparticles are mainly measured by confocal laser light (CLSM). First, tumor cells (MCF-7, HeLa) and normal cells (MCF-10A, 293T) were inoculated into a 35 mm confocal dish for 24 hours, and then 1 ml of SHA / HA / PS / CHA nanoparticle solution and Cells were incubated for four hours. Nuclei were then stained with Hoechest 33342, and cells were finally visualized by CLSM (PerkinElmer MltraVIEW VoX). Experimental results such as Figure 8 As shown, it can be seen from the figure that the light emitted by the two cancer cells (MCF-7, HeLa) is much stronger than that of the two normal cells (MCF-10A, 293T), which is due to the microRNA- 21 content is much higher than normal cells. This r...

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Abstract

The invention relates to the technical field of biomedicine, and in particular relates to a molecular beacon transmission system for directly detecting circulating tumor cells in blood as well as a preparation method and application of the molecular beacon transmission system, and the molecular beacon transmission system for directly detecting the circulating tumor cells in the blood is nanoparticles self-assembled by a high polymer material, protamine and a molecular beacon. All materials of the molecular beacon transmission system are biocompatible materials, and direct incubation in blood does not cause toxic and side effects of blood cells and the circulating tumor cells. The particle size, potential and surface topography of the nanoparticles of the molecular beacon transmission system can meet the requirements of cell entry, and meanwhile, the nanoparticles have good stability and biocompatibility.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a molecular beacon delivery system for directly detecting circulating tumor cells in blood, its preparation method and application. Background technique [0002] Today, with the influence of many factors such as people's lifestyle, living environment and population aging, the prevention and treatment of cancer has become a major issue of public health in our country. According to the latest World Health Organization data, about 1 / 6 of the world's people die of cancer. Although malignant tumors are a great threat to people's lives and health, early diagnosis and optimal treatment can greatly improve the survival rate of cancer patients. The traditional clinical diagnosis methods mainly include tissue biopsy and imaging. Tissue biopsy is to obtain the tissue of the lesion through invasive puncture or surgery to interpret the tumor type. This method will not only bring invasive...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/569C12Q1/6886G01N21/64
CPCG01N33/574G01N33/56966C12Q1/6886G01N21/6458G01N21/6428C12Q2600/158C12Q2600/178C12Q2600/118
Inventor 程巳雪徐唱
Owner WUHAN UNIV
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