A self-assembled nucleic acid aptamer/protein composite nanoprobe, preparation method, kit and application thereof

A nucleic acid aptamer and protein complex technology, applied in the field of molecular biology, achieves the effects of low detection limit, improved capture efficiency, high precision and accuracy

Active Publication Date: 2021-04-06
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, rare tumor cell detection strategies based on Aptamer binding to proteins

Method used

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  • A self-assembled nucleic acid aptamer/protein composite nanoprobe, preparation method, kit and application thereof
  • A self-assembled nucleic acid aptamer/protein composite nanoprobe, preparation method, kit and application thereof
  • A self-assembled nucleic acid aptamer/protein composite nanoprobe, preparation method, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Preparation of self-assembled nucleic acid aptamer / protein composite nanoprobe: mix 75nmol / L hybridization probe 1, 150nmol / L hybridization probe 2 and 150nmol / L hybridization probe 3 and add to the vortex mixer, vortex at room temperature Spin hybridization for 1 h to obtain the self-assembled nucleic acid aptamer / protein composite nanoprobe. The preparation process is as follows: figure 1 shown.

[0058] Characterization of hybridization results: Dilute ApDNA, H1 DNA, and H2 DNA to a concentration of 2 μmol / L, and then take 10 μL of the above solution, 10 μL of a mixed solution of 1 μmol / L ApDNA, 2 μmol / L H1, and 2 μmol / L H2, and perform 2% Agarose gel electrophoresis, the electrophoresis conditions are voltage 100V, time 1h, and finally gel imaging, such as figure 2 shown, where figure 2 Middle lane 1: primer; lane 2: binding buffer; lane 3: 2 μmol / L ApDNA; lane 4: 2 μmol / L H1 DNA; lane 5: 2 μmol / L H2; lane 6: mixed solution of ApDNA, H1 and H2, The concentratio...

Embodiment 2

[0060] The preparation method of Sgc8c-MNPs comprises the following steps:

[0061] A: Wash magnetic beads: Take 300 μL 2mg / mL magnetic beads (MNPs) in a 1.5 mL low adsorption centrifuge tube, magnetically separate, and discard the supernatant. Then add 300 μL of PBS buffer solution, mix thoroughly, magnetically separate, discard the supernatant, and wash 3 times in total.

[0062] B: Combination of MNPs and Sgc8c: Add 500 μL of 400 nmol / L Sgc8c (another portion of Sgc8c with the same concentration, marked as pre) to the above-mentioned low-adsorption centrifuge tube, and mix thoroughly. Vortex reaction at room temperature for 30 minutes, magnetically separate, save the supernatant, marked as post, wash the magnetic beads twice with PBS, 500 μL each time, save the supernatant separately, marked as wash 1 and wash 2;

[0063] C: The MNPs modified with Sgc8c (Sgc8c-MNPs) were resuspended in 600 μL of immunomagnetic bead preservation solution, and stored at 4°C for use.

[0064...

Embodiment 3

[0071] Capture of target cells by Sgc8c-MNPs in a single CCRF-CEM suspension:

[0072] (1) Wash cells: wash CCRF-CEM 3 times with binding buffer, then resuspend for 10 6 cells / mL cell suspension;

[0073] (2) Cell staining: 5 μL of 2 mmol / L DiD dye was added to each ml of CCRF-CEM cell suspension, and incubated at 37° C. for 5 min. Wash cells 3 times with binding buffer;

[0074] (3) Sgc8c-MNPs capture CCRF-CEM: Dilute the stained cells into five different concentrations: 500, 1000, 2500, 5000, 10000 cells / mL. Take 1 mL of different concentrations of CCRF-CEM and mix them with Sgc8c-MNPs (the amount of Sgc8c-MNPs is 20 μL of stock solution), incubate at 4°C for 20 min, magnetically separate, and collect the supernatant. Centrifuge at 1000r / min for 5min, and disperse the cell pellet in 100μL binding buffer;

[0075] (4) Cell counting: Under the laser confocal microscope, the CCRF-CEM in the supernatant was counted for fluorescence, and the capture efficiency (Capture effici...

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Abstract

The invention discloses a self-assembled nucleic acid aptamer / protein composite nanoprobe, a preparation method, a kit and an application thereof. The invention uses three kinds of DNA probes, one of which is a DNA containing a nucleic acid aptamer sequence. The probe, a kind of labeled horseradish peroxidase, is hybridized to prepare a self-assembled nucleic acid aptamer / protein composite nanoprobe, and the magnetic nanoparticle functionalized by the nucleic acid aptamer is used as a magnetic separation solid phase carrier. Specific capture of human acute lymphoblastic leukemia T lymphocytes (CCRF‑CEM) in the blood to improve the capture efficiency of the target, used in conjunction with the above self-assembled nucleic acid aptamer / protein composite nanoprobe to detect CCRF‑CEM With wide linear range, low detection limit, high precision and accuracy, it can achieve high sensitivity, high specificity and rapid detection of CCRF‑CEM in peripheral blood samples, which is of great significance for auxiliary diagnosis, curative effect evaluation and prognosis judgment of leukemia significance.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a self-assembled nucleic acid aptamer / protein composite nanoprobe, a preparation method, a kit and an application thereof. Background technique [0002] Currently, self-assembly, as a powerful technique to spontaneously organize or aggregate functional building blocks into multifunctional materials in a controllable manner, has been successfully used in sensing and drug loading in biomedicine. In particular, self-assembly based on nucleic acid hybridization regulation, due to the diversity of nucleic acid structure and function, and the advantages of programmable design of its sequence, makes it useful in the orderly construction of nanomaterials, nucleic acid, enzyme activity, heavy metal ion detection, etc. It has broad application prospects. In recent years, the construction and application of nucleic acid assemblies using nucleic acid aptamers (Aptamers) as affinit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 何磊良邓猛聪赵梦瑶沙吉旦·布拉了高宁振刘焕岳帅汪俊朱星晨
Owner ZHENGZHOU UNIV
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