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Novel protein-DNA complex for detecting carcino-embryonic antigen as well as synthetic method and application thereof

A carcinoembryonic antigen and complex technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of complex process and high production cost, and achieve the effect of high-specificity colorimetric detection.

Inactive Publication Date: 2019-03-01
QINGDAO UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

Although this method has good sensitivity and selectivity, the process is complicated, requiring two nucleic acid amplifications, and the production cost is high

Method used

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  • Novel protein-DNA complex for detecting carcino-embryonic antigen as well as synthetic method and application thereof
  • Novel protein-DNA complex for detecting carcino-embryonic antigen as well as synthetic method and application thereof
  • Novel protein-DNA complex for detecting carcino-embryonic antigen as well as synthetic method and application thereof

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preparation example Construction

[0037] In one or some typical embodiments of the present invention, the synthetic method of described novel protein-DNA complex is provided, and described synthetic method comprises the following steps:

[0038]S1. The padlock probe and the primer strand are subjected to a circular reaction to synthesize a circular template for rolling circle amplification; the nucleotide sequence of the padlock probe is shown in SEQ ID NO: 1, and the 5' end of the padlock probe is Modify the phosphate group; the nucleotide sequence of the primer chain is shown in SEQ ID NO:2;

[0039] S2. After mixing the circular template prepared in step S1 with horseradish peroxidase-labeled carcinoembryonic antigen secondary antibody and glucose oxidase solution, a rolling circle amplification reaction is triggered to obtain a HRP-Ab2 / GOD@DNA complex.

[0040] Further, the concentration of the circular template used in step S2 is 1.5-1.8 μM.

[0041] In one or some typical embodiments of the present inve...

Embodiment 1

[0054] Embodiment 1 A kind of novel protein-DNA complex

[0055] A novel protein-DNA complex, the novel protein-DNA complex comprises horseradish peroxidase-labeled carcinoembryonic antigen secondary antibody, glucose oxidase and DNA structure. Horseradish peroxidase-labeled carcinoembryonic antigen secondary antibody and glucose oxidase were encapsulated into the DNA structure to form HRP-Ab2 / GOD@DNA complex.

[0056] The DNA structure is synthesized by rolling circle replication and self-assembly. The synthesis method is as follows: the padlock probe and the primer chain are subjected to a circular reaction to synthesize a circular template for rolling circle amplification, and then trigger the rolling circle amplification reaction to form a long DNA single strand; the long DNA single strand is precipitated by anisotropic crystallization to form a micron-scale porous DNA structure due to excessive local concentration.

[0057] The nucleotide sequence of the padlock probe is...

Embodiment 2

[0064] Embodiment 2 A kind of test kit for detecting carcinoembryonic antigen and its application method

[0065] The kit includes the following components: carcinoembryonic antigen monoclonal antibody, padlock probe, primer strand, T4 DNA ligase, 1×T4 DNA ligase buffer, exonuclease I, 1× exonuclease buffer, phi29DNA Polymerase solution, 1×phi29 DNA polymerase buffer, dNTPs solution, horseradish peroxidase-labeled carcinoembryonic antigen secondary antibody solution, glucose oxidase solution, carcinoembryonic antigen standard, acetate buffer, glucose solution and ABTS solution ;

[0066] The nucleotide sequence of the padlock probe is shown in SEQ ID NO:1, and the 5' end of the padlock probe is modified with a phosphate group; the nucleotide sequence of the primer chain is shown in SEQ ID NO:2.

[0067] 1×T4 DNA Ligase Buffer: 40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 0.5mM ATP, pH7.8.

[0068] 1× Exonuclease Buffer: 67mM Glycine-KOH, 6.7mM MgCl 2 , 1 mM DTT, pH 9.5.

[0069...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a novel protein-DNA complex for detecting a carcino-embryonic antigen as well as a synthetic method and application thereof. The novel protein-DNA complex comprises a horseradish peroxidase-marked carcino-embryonic antigen secondary antibody, glucose oxidase and a DNA structure. The novel protein-DNAcomplex structure encapsulated with a great amount of enzyme-labeled secondary antibodies and glucose oxidase is established on the basis of a rolling circle replication technology and is used as enzyme cascade signal amplification probe. A 96-pore plate is used for fixing carcino-embryonic antigen monoclonal antibody molecules, after the to-be-detected carcino-embryonic antigen is added, a sandwiched structure is formed. The encapsulated glucose oxidase is used for catalyzing the glucose to be oxidized to generate H2O2, then the H2O2 is catalyzed by virtue of the horseradish peroxidase to oxidize ABTS, so that the high-sensitivity and high-specificity colorimetry detection of the carcino-embryonic antigen can be realized, and the novel protein-DNA complex has important significance for the early diagnosis and clinical research of cancers.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a novel protein-DNA complex for detecting carcinoembryonic antigen and its synthesis method and application. Background technique [0002] In addition to the function of storing and transmitting genetic information, DNA has the advantages of sequence designability, simple synthesis, easy functionalization, and good biocompatibility, so the molecules of DNA nanostructures can be precisely controlled during the DNA assembly process. Structural and dynamic functions to construct 2D and 3D DNA nanostructures. At present, the methods for constructing DNA structures mainly include single-strand tiling assembly, hybrid tiling assembly, DNA origami, etc., which have been widely used in biosensing, bioimaging, biomedicine and other fields. However, the DNA self-assembly technology based on the Watson-Crick base pairing principle generally has problems such as com...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531G01N33/574
CPCG01N33/577G01N33/531G01N33/574
Inventor 毕赛闫永存李园芳李娟
Owner QINGDAO UNIV
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