A deoxyribozyme-based extracellular ATP rapid sensing method and application kit

A deoxyribozyme and sensing method technology, applied in the field of deoxyribozyme-based extracellular ATP rapid sensing method and application kit, can solve the problems of inability to guarantee the specificity of ATP detection, inability to distinguish ADP and AMP, and achieve Good specificity and accurate sensing results

Active Publication Date: 2022-06-21
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although aptamer-based sensors have been developed for the analysis of intracellular ATP, this method cannot guarantee the specificity of ATP detection in the case of complex signal transmission between cells, and cannot even distinguish between extracellular ADP and AMP, and thus monitoring of extracellular ATP remains a major challenge and technical difficulty

Method used

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  • A deoxyribozyme-based extracellular ATP rapid sensing method and application kit
  • A deoxyribozyme-based extracellular ATP rapid sensing method and application kit
  • A deoxyribozyme-based extracellular ATP rapid sensing method and application kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 utilizes F DK1 molecules image MCF-7 cells

[0042] use F DK1 molecules image MCF-7 cells, the principle is as follows figure 1 As shown, the specific operation is as follows:

[0043] a) MCF-7 cell culture. Add 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin to the modified DMEM medium, place at 37°C, 5% CO 2 and 95% air in a humid atmosphere. Before harvesting the cells, wash the cells with 1 mL of PBS, then add 1 mL of 0.25% (v / v) trypsin-EDTA solution (purchased from Invitrogen), and incubate at 37°C for 5 minutes to separate the cells from the culture dish to obtain Cell suspension, after the cell suspension was centrifuged at 3000g (5 minutes), the cells were resuspended in 1mL of DMEM, and approximately 1×10 5 The cells were transferred to glass-based culture dishes with a diameter of 35 mm and incubated at 37 °C for 24 h.

[0044] b) will F DK1 molecules are fixed on the surface of MCF-7 cell membrane. Tak...

Embodiment 2

[0046] Embodiment 2 utilizes F Confocal fluorescence imaging analysis of DK1 molecule on mouse brain astrocytes stimulated by glutamate

[0047] use F Confocal fluorescence imaging analysis of DK1 molecules on mouse brain astrocytes under glutamate stimulation, the steps are as follows:

[0048] a) Mouse brain astrocytes were obtained from pups of mice in the P1-P4 stage. Take the head of the mouse, peel the cortex of the mouse from the brain with flat-nose forceps under a microscope, carefully separate the cerebral hemisphere cortex from the cortex using fine forceps, mince the obtained cerebral hemisphere cortex, and place in a container containing 0.25 % (v / v) trypsin-EDTA culture medium for 10 minutes (37 ° C) to obtain mouse brain astrocytes. Seed the obtained cells in 25cm 2 Poly-L-lysine-coated culture flasks were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin , at 37°C, 5% CO 2 Cultured in a h...

Embodiment 3

[0054] Embodiment 3 utilizes F Confocal fluorescence imaging analysis of DK1 molecule on rat brain astrocytes under mechanical and electrical stimulation

[0055] useF Confocal fluorescence imaging analysis of DK1 molecules on rat brain astrocytes under mechanical and electrical stimulation, the steps are as follows:

[0056] a) Mouse brain astrocytes were obtained from pups of mice in the P1-P4 stage. Take the head of the mouse and peel the cortex of the mouse from the brain under a microscope using flat-nosed forceps, using fine forceps to carefully separate the hemispheric cortex from the cortex. The obtained cerebral hemisphere cortex was minced, placed in a medium containing 0.25% (v / v) trypsin-EDTA and incubated for 10 minutes (37° C.) to obtain mouse brain astrocytes. Seed the obtained cells in 25cm 2 Polylysine-coated culture flasks were cultured using DMEM medium, which contained 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin, A...

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PUM

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Abstract

The invention discloses a deoxyribozyme-based extracellular ATP rapid sensing method and an application kit, belonging to the field of analysis and detection. A kind of deoxyribozyme, described deoxyribozyme comprises the deoxyribonucleotide chain shown in SEQ ID NO.2 of nucleotide sequence, connects at the 5' end of the deoxyribonucleotide chain shown in SEQ ID NO.2 There are hydroxyl groups, and cholesterol molecules are attached to the 3' end. A deoxyribozyme-based rapid sensing method for extracellular ATP has simple steps and fast response, which can realize the rapid sensing and determination of extracellular secreted ATP molecules, and can effectively distinguish other extracellular nucleotides including ADP, AMP and adenocarcinamide It can be widely used in various physiological and pathological research processes such as cell growth, tissue injury, inflammation, and transplantation immunity. It can better understand the signaling mechanism of ATP and its role in basic pathological and physiological processes. function.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and specifically relates to a deoxyribozyme-based extracellular ATP rapid sensing method and an application kit. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Since the first catalytically active DNA (also known as deoxyribozyme or DNAzyme) was obtained by in vitro screening technology in 1994, many DNAzymes (RCDz for short) that can catalyze the cleavage of RNA have been discovered so far. These RCDz are widely used in the fields of clinical diagnosis, drug development, environmental monitoring, and biosensing. For example, RCDz can be used as sensors and imaging agents for intracellu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/34G01N21/64
CPCC12N15/113C12Q1/34G01N21/6428G01N21/6458C12N2310/127G01N2021/6432
Inventor 刘猛石蒙赵丹
Owner DALIAN UNIV OF TECH
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