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Ratio-dependent fluorescent probe and preparation method and application thereof

A fluorescent probe, ratiometric technology, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of long analysis time, low detection sensitivity, complex instruments and equipment, etc., to overcome low sensitivity, biological Good compatibility and high sensitivity detection effect

Pending Publication Date: 2021-11-12
QINGDAO UNIV OF SCI & TECH
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] In order to overcome the problems of low detection sensitivity, long analysis time, high cost and complex equipment of traditional technology, the present invention proposes a metal-organic framework (MOFs) ratiometric fluorescent probe based on loaded gold nanoclusters (AuNCs)

Method used

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  • Ratio-dependent fluorescent probe and preparation method and application thereof
  • Ratio-dependent fluorescent probe and preparation method and application thereof
  • Ratio-dependent fluorescent probe and preparation method and application thereof

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Embodiment 1

[0024] A method for preparing a novel metal-organic framework ratiometric fluorescent probe based on loaded gold nanoclusters proposed by the present invention, comprising the steps of:

[0025] (1) Design and synthesize BHQ-DNA1 chain and FAM-DNA2 chain, the sequences from 5' to 3' are: COOH-TCAGACTGATGTTGAA-BHQ1 and 6-FAM-ATCAACATCAGTCTGATAAGCTA, in which, DNA1 chain and DNA2 chain are separated by 3' The 15 bases starting from the 8th base at the end are complementary, and the DNA2 strand is the complementary strand of the target miRNA-21 + A;

[0026] (2) Mix 5.0 μL of BHQ-DNA1 (2.0 μM) and the same amount of FAM-DNA2 (2.0 μM) and then perform annealing treatment, that is, first place the above mixture in a water bath at 90 ° C, gradually cool down to 37 ° C and keep it for 30 min , forming the required double-stranded probe at this time;

[0027] (3) Add 10 mL of deionized water to the mixture of 0.1917 g EDC and 0.1151 g NHS, take 10 μL of this solution and mix with the...

Embodiment 2

[0035] A method for detecting miRNA-21 using the metal-organic framework ratiometric fluorescent probe proposed by the present invention, comprising the steps of:

[0036] (1) Add different concentrations of miRNA-21 (0, 1.0, 2.0, 5.0, 10, 20, 40, 60, 80, 100, 120, 140nM) to 100μL ratiometric fluorescent probe (0.2mg / mL) in solution;

[0037] (2) After incubating at 37°C for 20 minutes, the F-4600 fluorescence spectrometer was used for detection. The specific detection conditions were 488nm as the single excitation wavelength, and the fluorescence intensity of FAM at 521nm and the fluorescence intensity of AuNCs at 655nm were detected respectively, and the slit width was 5.0nm. ;

[0038] (3) Use (F-F 0 ) / F AuNCs As the output signal, F is the fluorescence intensity of the probe at 521nm, F 0 is the blank value at 521nm, F AuNCs is the fluorescence intensity of the probe at 655 nm.

[0039] The experimental results showed that as the concentration of miRNA-21 increased, ...

Embodiment 3

[0041] (1) Human breast cancer MCF-7 cells with high expression of miRNA-21 were selected as the cell model for in situ imaging, and the MCF-7 cells were divided into two groups, one group was treated with 100 μL ratiometric fluorescent probe (0.2 mg / mL) 37°C, 5% CO 2 co-incubated for 2 hours, and the other group was not treated as a blank control;

[0042] (2) Then wash with PBS buffer, and use laser confocal microscope to perform fluorescence imaging on the two groups of cells respectively, and select the excitation wavelength: 488nm;

[0043] The experimental results show that when the ratiometric fluorescent probe enters MCF-7 cells, the intracellular miRNA-21 reacts with the probe, which restores the fluorescent signal of the FAM dye, while the fluorescent signal of AuNCs remains basically unchanged; untreated There is no generation of fluorescent signals in MCF-7 cells. On the one hand, it shows that MCF-7 cells do not generate spontaneous fluorescent signals. On the ot...

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Abstract

As an important member of the miRNA family, miRNA-21 has already been widely concerned as a tumor biomarker for breast cancer. As the sequence of the miRNA-21 is short, the expression level of the miRNA-21 in cells is low, and the similarity between the miRNA-21 and the sequence of other family members is high, certain difficulty and challenge still exist in high-sensitivity detection of the miRNA-21 clinically. The invention provides a novel ratio-type fluorescent probe based on a metal organic framework loaded with gold nanoclusters, a metal organic framework material with large specific surface area and high porosity is used as a carrier, the gold nanoclusters are generated through an in-situ synthesis method, and the designed and synthesized double-chain probe is assembled on the surface of the carrier. When the probe constructed by the invention is used for detecting miRNA-21, the defects of low detection sensitivity, high background signal and the like of the traditional method are overcome, the problems of complex living body and cell imaging technology, high price and the like are solved, and a new strategy and method are provided for living body and cell detection and in-situ imaging.

Description

technical field [0001] The invention relates to the detection of tumor biomarkers, in particular to a ratiometric fluorescent probe for detecting MicroRNA (miRNA) in breast cancer cells and its preparation method and application, belonging to the fields of photoelectrochemical biosensing analysis and biomedical clinical diagnosis. Background technique [0002] MicroRNA (MiRNA) is a kind of endogenous non-coding single-stranded RNA molecule with a size of 18-39bp existing in eukaryotic organisms. play an important regulatory role in the process. Studies have shown that the abnormal expression of miRNA in the body is highly related to the occurrence and development of tumors. Among them, miRNA-21, as an important member of the miRNA family, has already received extensive attention as a tumor biomarker for breast cancer. Breast cancer is the number one killer of cancer in women worldwide, and its mortality rate remains high. Due to the short sequence of miRNA-21, low express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/02C09K11/06C09K11/58G01N21/64B82Y30/00
CPCC09K11/02C09K11/06C09K11/58B82Y30/00G01N21/6428G01N21/6458C09K2211/1007C09K2211/183
Inventor 王卫唐凯秦力琳宋志灵罗细亮
Owner QINGDAO UNIV OF SCI & TECH
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