Hydrogen peroxide responsive ratio meter nanoprobe and its use

A technology of hydrogen peroxide and nano-probes, applied in chemical instruments and methods, material analysis through optical means, instruments, etc., can solve the problems of insufficient sensitivity or specificity, and achieve easy cytotoxicity, excellent fluorescence performance, good The effect of water solubility

Inactive Publication Date: 2018-01-05
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the existing detection methods are insufficient in sensitivity or specificity to a certain extent

Method used

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  • Hydrogen peroxide responsive ratio meter nanoprobe and its use
  • Hydrogen peroxide responsive ratio meter nanoprobe and its use
  • Hydrogen peroxide responsive ratio meter nanoprobe and its use

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0027] Example 1. Synthesis, assembly and characterization of ratiometric nanoprobes

[0028] Dissolve succinimide-activated-coumarin-phenylboronic acid pinacol ester (62.6mg, 0.125mmol) and isothiocyanate-activated rhodamine B (6.65mg, 0.0125mmol) in dimethylmethylene Sulfone (DMSO), was added dropwise to an aqueous solution of ethylene glycol chitosan (160 mg, 0.75 mmol), and finally 20 μL of triethylamine was added, and reacted at room temperature for 24 h. After the reaction, first in DMSO / H 2 Dialyzed in O(1 / 4, V / V) for 48h, and then dialyzed in ultrapure water for 48h, assembled to obtain the ratiometric nanoprobe (GC-Cou-Bpin-RhBnanoprobe), figure 1 .

[0029] The chemical structure of the ratiometric nanoprobe was characterized by NMR spectroscopy, as figure 2 (a) 1 As shown in the H NMR spectrum, the characteristic peaks of two fluorescent molecule benzene rings appeared at 6.5-8.0ppm, and the characteristic peaks of the methyl group on phenylboronic acid pinacol...

Embodiment 2

[0031] Embodiment 2. Experiment of colon cancer cell line

[0032] We selected two normal cells (HEK 293 and NIH-3T3) and three colon cancer tumor cells (HCT 116, HT 29 and SW620) and characterized GC-Cou-Bpin-RhB by flow cytometry and confocal laser Intracellular Capability of Ratiometric Nanoprobes and Detection of H in Cells 2 o 2 content. Analyze flow cytometry results from different cell lines such as image 3 As shown, according to the fluorescence intensity of the internal standard RhB, we found that both normal cells and tumor cells can enter the ratiometric nanoprobes to varying degrees. The cell entry ability of colon cancer cell HCT 116 was similar to that of normal cell HEK 293, but the H in HCT116 cells 2 o 2 The concentration is much higher than that of HEK 293. As for the colon cancer cell HT29, its ability to enter cells is stronger than that of the other two colon cancer cells HCT 116 and SW 620, but its H 2 o 2 The concentration is much lower than the...

Embodiment 3

[0035] Example 3. Ratiometric nanoprobes assist in the detection of CTC in patients with gastric cancer

[0036] We enriched CTCs in the peripheral blood of patients with gastric cancer by negative screening method, and used GC-Cou-RhB ratiometer nanoprobes and tumor cell surface-specific antibody cytokeratin (CK19- FITC) to stain the enriched cells together and observe with laser confocal, where the blue is H 2 o 2 The probe coumarin-phenylboronic acid pinacol ester and H 2 o 2 The fluorescence emitted after the reaction, the red fluorescence is the internal standard probe Rhodamine B, and the green fluorescence is CK19-FITC, such as Figure 7 shown. Figure 7 (27), (28), and (29) showed that although the amount of the probe entering the cell was not much (internal standard fluorescence was weak), the concentration of hydrogen peroxide in the cell was high (blue fluorescence was strong), among which Figure 7 (29) had relatively strong expression of CK-19 on the surface ...

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Abstract

The invention discloses a hydrogen peroxide responsive ratio meter nanoprobe and its use. The nanoprobe comprises a hydrogen peroxide-responsive fluorescent molecule, a hydrogen peroxide-inert fluorescent molecule and a polymer with good biocompatibility. The invention also discloses a use of the hydrogen peroxide responsive ratio meter nanoprobe in preparation of a detection agent of circulatingtumor cells in peripheral blood of malignant solid tumors. The ratio meter nanoprobe has good water solubility, can be easily taken, has low cytotoxicity and can be used for quantitative imaging of hydrogen peroxide in circulating tumor cells. The ratio meter nanoprobe has excellent fluorescence properties, has good sensitivity and specificity to hydrogen peroxide and can detect difference of levels of hydrogen peroxide in malignant solid tumor cells and difference of intracellular intake amounts.

Description

technical field [0001] The invention belongs to the biological field, and more specifically relates to a hydrogen peroxide-responsive ratiometric nanometer probe and its application in detecting peripheral blood circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) are tumor cells with special properties that shed from the primary tumor and enter the peripheral blood circulation. In the clinical diagnosis and treatment of cancer, CTC is considered as a biomarker with tumor diagnostic properties. Therefore, the capture and analysis of CTCs has a great application prospect in the biological classification of cancer patients, the adoption of specific treatment methods, and the monitoring of curative effects. However, due to the extremely small number of CTCs in peripheral blood, there are great challenges in the enumeration and identification of CTCs. [0003] At present, there are many methods for detecting "circulating tumor cells" in human p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCC08B37/0072C09K11/025C09K11/06C09K2211/1088C09K2211/1491G01N21/6428
Inventor 李培勇李纯婷朱新远
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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