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Application of a nanoparticle-coupled probe system in the highly sensitive detection of ctdna

A technology of nanoparticles and coupled probes, which is applied in the field of medical bio-nanomaterials, can solve the problems of cumbersome pretreatment, high false positive probability, and high cost, and achieve the effect of high specificity, low false positive probability, and high sensitivity detection

Active Publication Date: 2020-08-14
SHANGHAI INST OF CERAMIC CHEM & TECH CHINESE ACAD OF SCI
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Problems solved by technology

[0004] At present, the international research on ctDNA detection of early cancer is in its infancy. The problem with the detection method based on PCR amplification is that the test results are largely dependent on gene amplification, the probability of false positive is high, the pretreatment is cumbersome, and it depends on biopsy. Provided gene mutation information, detection equipment and reagents are completely dependent on imports
Although the detection method based on gene sequencing has a low error rate, the cost of R&D and testing is very high, and the detection cycle is as long as more than one week, so it is difficult to be widely used in clinical detection
In general, the existing detection methods have not been widely promoted in clinical medicine due to the limitations of sensitivity, detection limit, detection price and detection cycle.

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  • Application of a nanoparticle-coupled probe system in the highly sensitive detection of ctdna
  • Application of a nanoparticle-coupled probe system in the highly sensitive detection of ctdna
  • Application of a nanoparticle-coupled probe system in the highly sensitive detection of ctdna

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preparation example Construction

[0055] Regarding the preparation of the magnetic masking agent, the complementary nucleic acid strand DNA of the wild-type gene can be 3 Modification on the surface of magnetic nanoparticles to prepare magnetic masking agent MNP@X-DNA 3 . In one example, masked DNA is prepared by modifying carboxyl groups at the end of the complementary sequence of wild-type ctDNA, for example 3 , through MNP@X surface amino groups and DNA 3 DNA 3 Grafting onto MNP@X surface to prepare magnetic masking agent MNP@X-DNA 3 .

[0056] Next, after the mutant ctDNA is hybridized with the magnetic nanoprobe and the reporter probe added to the detection system, magnetic separation is performed. In the present invention, when the target ctDNA exists, the magnetic nanoprobe and the reporter probe are coupled through ctDNA, and magnetically separated to obtain nanoparticles with reporter elements on the surface of the magnetic nanoparticles; Nanoparticles with no reported elements. Combined with L...

Embodiment 1

[0066] 1. AFe@SiO 2 -DNA 1 , Au-DNA 2 Probe preparation.

[0067] 1. Preparation of amorphous iron (AFe) nanoparticles: Weigh 0.36mmol (175mg) ferric ammonium citrate, 9mmol PVP (Average Mw=55000) and F127 (1.0g) respectively and dissolve them in 40mL anaerobic water, under argon atmosphere Raise the temperature to 70°C, mechanically stir (600rpm) for 60min, and remove the oxygen in the reaction system. 7.5mmol (283g) NaBH 4 Dissolve in 6mL of water, slowly (0.1mL / min) dropwise added to the reaction system to generate a lot of bubbles. After 4 hours, 5 mL of ethanol was added to remove air bubbles, magnetically separated, and the collected amorphous iron nanoparticles were ultrasonically washed three times with ethanol, and dispersed in ethanol for storage.

[0068] figure 1 The TEM spectrum of the amorphous iron (AFe) nanoparticle that this embodiment makes is dispersed in water, by figure 1 It can be seen that the prepared nanoparticles are spherical, have good dispersi...

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Abstract

The invention relates to application of a nano-particle coupling probe system in high-sensitivity ctDNA detection. The nano-particle coupling probe system comprises a first probe and a second probe, wherein the expression of the first probe is MNP@X-DNA1, MNP is a magnetic-nano-particle core, X is a hydrophilic shell wrapping the magnetic-nano-particle core, and DNA1 is a first complementary nucleic acid chain which is modified on the hydrophilic shell and can be complementary to the first part of target circulating tumor DNA; the expression of the second probe is Rep-DNA2, Rep is a reporting-element core formed by reporting elements which can be detected by using ICP-MS, and DNA2 is a second complementary nucleic acid chain which is modified on the reporting-element core and can be complementary to the second part of the target circulating tumor DNA. The double nano probes MNP@X-DNA1 and Rep-DNA2 are simple to prepare, controllable and easy in repetition.

Description

technical field [0001] The invention belongs to the technical field of medical biological nanometer materials, and in particular relates to a nanoparticle-coupled probe material for ctDNA detection, a preparation method and application thereof. Background technique [0002] The first step of tumor generation is gene mutation, and cancer is ultimately a genetic disease. With the deepening of cancer research, people have detected in the peripheral blood of cancer patients a mutated gene consistent with tumor cells, that is, circulating tumor DNA (circulating tumor DNA, ctDNA). ctDNA is a gene fragment released by tumor cells into the blood circulation system, which is the "information code" scattered in the blood by cancer. The stage of cancer is directly related to the concentration of ctDNA in the blood, and the concentration of ctDNA in middle-advanced patients is significantly higher than that in early or ultra-early stage patients. The concentration of ctDNA increases w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6818
CPCG01N1/28G01N27/64
Inventor 胡萍施剑林
Owner SHANGHAI INST OF CERAMIC CHEM & TECH CHINESE ACAD OF SCI
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