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A nanosensor for detecting DNA hydroxylase tet1 and its detection method and application

A nano-sensor and detection method technology, applied in the field of analysis and testing, can solve the problems of low sensitivity, time-consuming separation operation, large TET1-specific antibody, etc., and achieve the effect of sensitive detection

Active Publication Date: 2022-08-02
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the inventors have found that these methods have the disadvantages of requiring a large number of TET1-specific antibodies, high sample input, time-consuming separation operations, and low sensitivity.

Method used

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  • A nanosensor for detecting DNA hydroxylase tet1 and its detection method and application
  • A nanosensor for detecting DNA hydroxylase tet1 and its detection method and application
  • A nanosensor for detecting DNA hydroxylase tet1 and its detection method and application

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Embodiment approach

[0032] A typical embodiment of the present invention provides a nanosensor for detecting DNA hydroxylase TET1, including a hairpin probe, T4 phage β-glucosyltransferase, endonuclease, and quantum dots;

[0033] The hairpin probe is a single-stranded DNA capable of forming a stem-loop structure, one end of the hairpin probe is connected to a fluorescent agent, and the other end can be connected with a quantum dot, and the stem region forming the stem-loop structure contains an endonuclease. A recognition and cleavage site containing 5-methylcytosine;

[0034] Quantum dots and fluorescent agents can cooperate to generate fluorescence resonance energy transfer.

[0035] In some embodiments, the fluorescent agent is Cy5 and the quantum dots are 605QD.

[0036] In some embodiments, the hairpin probes are linked to the quantum dots via biotin-streptavidin. Further, the hairpin probe was linked to biotin, and the surface of the quantum dots was coated with streptavidin.

[0037] I...

Embodiment

[0063] Determination of TET1: First, the detection probe was mixed with buffer A (5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.0), incubated at 95 °C for 5 min, and slowly cooled to room temperature. Then, in a solution containing 50 mM HEPES, 50 mM NaCl, 75 μM Fe(NH 4 ) 2 (SO 4 ) 2 , 2mM ascorbic acid, 2.5mM DTT and 1mM α-KG were added to the reaction solution of 144nM detection probe and different concentrations of TET1, and reacted at 37°C for 30min. Third, the reaction product was mixed with 40 μM uridine diphosphate glucose (UDP-glucose) and 3U T4 bacteriophage β-glucosyltransferase (T4-βGT) and buffer B (50 mM potassium acetate, 20 mM Tris-Ac, 10 mM magnesium acetate, 1mM DTT, pH 7.9) to form a reaction solution, and incubated at 37°C for 2h. Subsequently, the glycosylation product was combined with 30 U MspI and buffer C (10 mM magnesium acetate, 20 mM Tris-Ac, 50 mM potassium acetate, 0.1 mg / mL BSA, pH 7.9) to form a reaction solution, and digested at 37 °C for 2 h. Finall...

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Abstract

The invention discloses a nanometer sensor for detecting DNA hydroxylase TET1, a detection method and application thereof. The nanometer sensor includes a hairpin probe, a T4 phage β-glucosyltransferase, an endonuclease and a quantum dot; the hairpin The probe is a single-stranded DNA that can form a stem-loop structure. One end of the hairpin probe is connected to a fluorescent agent, and the other end can be connected to a quantum dot. The stem region that forms the stem-loop structure contains an endonuclease that is recognized and cleaved. The site contains 5-methylcytosine; the quantum dot and the fluorescent agent can cooperate to generate fluorescence resonance energy transfer. The nanosensor provided by the present invention does not involve any specific antibody, and can simply, rapidly and quantitatively detect TET1.

Description

technical field [0001] The invention belongs to the technical field of analysis and testing, and relates to a nano-sensor for detecting DNA hydroxylase TET1, a detection method and application thereof. Background technique [0002] The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. [0003] The DNA hydroxylase TET1 is an Fe(II) and 2-oxoglutarate-dependent dioxygenase that is responsible for DNA 5-methylcytosine (5mC) hydroxymethylation in mammals. Dysregulated TET1 expression is closely associated with a variety of genetic diseases and cancers. Therefore, accurate and sensitive quantification of TET1 activity is of great significance. [0004] According to the inventor's research and understanding, the curren...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6818C12Q1/6825C12Q1/48C12Q1/26
CPCC12Q1/6818C12Q1/6825C12Q1/48C12Q1/26C12Q2525/301C12Q2563/107C12Q2565/101C12Q2565/607
Inventor 张春阳胡娟姚洁潘丽媛
Owner SHANDONG NORMAL UNIV
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