Method for detecting tumor marker

A tumor marker and nucleic acid aptamer technology, applied in the field of new nano-probes, can solve the problem of no significant decrease in fluorescence and achieve rapid detection results

Inactive Publication Date: 2017-08-04
浙江汉耀科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the double-stranded probe itself and other rigid DNA structures cannot be adsorb

Method used

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  • Method for detecting tumor marker
  • Method for detecting tumor marker

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0033] Example 1 Detection of Prostate Specific Antigen (PSA) by Nano-Gold-DNA Interaction Principle

[0034] Steps: Take 2μL of 100μM PSA-aptamer solution and 2μL of 100μM cDNA solution respectively, add 18μL of buffer solution (25mM Tris, pH8.2, 0.3M NaCl) and then fully hybridize to form a double-stranded probe (room temperature Reaction for 30 minutes). Then, 2 μL of 1 mM PSA aqueous solution was added to the hybridization solution, and the reaction was carried out fully at room temperature. At the same time, the group without PSA and 2μL of water was used as blank, and the two groups with 2μL of 1mM AFP (N1) and CEA (N2) were used as control group 1 and control group 2, respectively. Then respectively take 2 μL of the above reaction solution and add 100 μL of 3.5 nM nano-gold solution, use buffer solution (25 mM Tris, pH 8.2, 0.3 M NaCl) to make up the total volume of 0.2 mL, and record the fluorescence spectra of the experimental group and the control group.

[0035] Result...

Example Embodiment

[0036] Example 2 Detection of Carcinoembryonic Antigen (CEA) by Nano-Gold-DNA Interaction Principle

[0037] Steps: Take 2μL of 100μM CEA-aptamer solution and 2μL of 100μM cDNA solution respectively, add 18μL of buffer solution (25mM Tris, pH8.2, 0.3M NaCl) and then fully hybridize to form a double-stranded probe (room temperature Reaction for 30 minutes). Then, 2 μL of 0.1 mM CEA aqueous solution was added to the hybridization solution, and the reaction was carried out fully at room temperature. At the same time, the group without CEA and 2μL of water was used as a blank, and the two groups with 2μL of 1mM PSA and AFP were used as controls. Then respectively take 2 μL of the above reaction solution and add 10 μL of 3.5 nM nano-gold solution. After the buffer solution is used to make up the total volume of 0.2 mL, the fluorescence spectra of the experimental group and the control group are recorded.

[0038] Results: In a set of solutions containing CEA, CEA combined with the CEA...

Example Embodiment

[0039] Example 3 Detection of alpha-fetoprotein (AFP) by nano-gold-DNA interaction principle

[0040] Steps: Take 2μL of 100μM AFP-aptamer solution and 2μL of 100μM cDNA solution, add 18μL of buffer solution (20mM Tris-acetic acid, pH7.4, 140mM NaCl, 1mM CaCl 2 , 1mM MgCl 2 ) And then fully hybridized to form a double-stranded probe (reaction for 30 minutes at room temperature). Then, 2 μL of 0.05 mM AFP aqueous solution was added to the hybridization solution, and the reaction was carried out fully at room temperature. At the same time, a set of 2μL water without AFP was used as a blank experiment. And a group with 2μL of 1mM BSA was added as a control. Then respectively take 2μL of the above reaction solution and add 50μL of 3.5nM nano-gold solution. After the buffer solution is used to make up the total volume of 0.2mL, the fluorescence spectra of the experimental group and the control group are recorded.

[0041] Results: In a set of solutions containing AFP, AFP combined wit...

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Abstract

The invention discloses a method for detecting a tumor marker. The method comprises the steps of making a specific aptamer of the tumor marker react with a complementary sequence cDNA of the specific aptamer to prepare a double-stranded probe, and marking a fluorophore at a 5' end of the complementary sequence; adding a to-be-detected tumor marker target molecule to the double-stranded probe as an experimental group; adding water to the double-stranded probe as a blank group; adding a tumor marker which is not matched with the aptamer to the double-stranded probe as a control group; taking the experimental group, the blank group and the control group, adding nanogold and recording the fluorescence spectrum intensity of the three groups before and after adding the nanogold; and in the experimental group, specifically combining the to-be-detected tumor marker target molecule with the aptamer, releasing the complementary sequence and quenching fluorescent light of the complementary sequence through the nanogold, wherein the fluorescence intensity of the experimental group after the nanogold is added is significantly reduced, the fluorescence intensity of the blank group and the control group is not significantly changed after the nanogold is added. According to the method, fast detection of a prostate-specific antigen, a carcino-embryonic antigen, alpha fetoprotein and an epithelial cell adhesion molecule can be achieved.

Description

technical field [0001] The invention relates to a novel nanometer probe based on the interaction between gold nanoparticle and nucleic acid structure, and a rapid fluorescence detection method for tumor markers established on the basis. Background technique [0002] DNA mainly exists in the form of double helix in nature. However, with the establishment of in vitro rapid screening technology, people can obtain a large number of new nucleic acid structures such as nucleic acid aptamers, DNases, and RNases. Nucleic acid aptamer is a class of DNA or RNA with high affinity for ligands, while DNase / RNase is a structure with enzymatic activity such as protease. These in vitro screening techniques are becoming increasingly important as molecular tools in the field of biotechnology. For example, they play an important role in the design of biosensors, and are used in the detection of biology, environment, safety and other fields, including proteins (thrombin, growth factors, HIV-re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2525/205C12Q2563/137C12Q2565/525
Inventor 苏金荣陈宇彪俞强
Owner 浙江汉耀科技股份有限公司
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