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A kind of dna nanostructure, electrochemical aptamer biosensor system and its preparation method and application

A technology of biosensors and nanostructures, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., to achieve the effect of increasing electrochemical surface area, improving signal amplification ability, and simplifying the experimental process

Active Publication Date: 2022-05-17
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a DNA nanostructure, electrochemical aptamer biosensor system and its preparation method and application, so as to solve many problems existing in the current detection of food-borne pathogens

Method used

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  • A kind of dna nanostructure, electrochemical aptamer biosensor system and its preparation method and application
  • A kind of dna nanostructure, electrochemical aptamer biosensor system and its preparation method and application
  • A kind of dna nanostructure, electrochemical aptamer biosensor system and its preparation method and application

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preparation example Construction

[0066] A kind of preparation method of DNA nanostructure of the present invention, mainly is to synthesize DNA nanostructure (RCA NCs) by rolling circle amplification reaction, comprises the following steps:

[0067] Step 1. Annealing

[0068] In T4 DNA ligase buffer (40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 0.5mM ATP, pH7.8), mix the 5'-phosphorylated linear template with the primer, heat the mixture and then cool it to room temperature to form a circular template-primer hybrid;

[0069] Wherein, the nucleic acid sequence of the 5'-phosphorylated linear template is 5'-(PO 4 3- )-ACCCGCCCTACCCAAAATATGCCCTCGGTTGTGGTATTATACGGCATTCCTCTCCGGACAACCCTCGCGGCATCTCGTCCACACTGCCTAAATTTTCCCA-3';

[0070] Wherein, the nucleic acid sequence of the primer is 5'-TTTTGGGTAGGGCGGGTTGGGAAAA-3';

[0071] Step 2. Connect

[0072] Mix the circular template-primer hybrid with T4 DNA ligase and incubate to ligate the circular template gap;

[0073] Step 3. Enzyme inactivation

[0074] After heati...

Embodiment 1

[0111] Synthesis and preparation of embodiment 1 DNA nanostructure

[0112] (1) Annealing

[0113] In T4 DNA ligase buffer (40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 0.5mM ATP, pH7.8), mixed 0.3μM 5'-phosphorylated linear template with 0.6μM primer, heated the mixture at 90°C for 5min, then gradually cooled to room temperature within 3h, to form a circular template-primer hybrid;

[0114] The nucleic acid sequence of the 5'-phosphorylated linear template is 5'-(PO 4 3- )-ACCCGCCCTACCCAAAATATGCCCTCGGTTGTGGTATTATACGGCATTCCTCTCCGGACAACCCTCGCGGCATCTCGTCCACACTGCCTAAATTTTCCCA-3';

[0115] The nucleic acid sequence of the primer is 5'-TTTTGGGTAGGGCGGGTTGGGAAAA-3'.

[0116] (2) connection

[0117] Mix the circular template-primer hybrid with 10 U of T4 DNA ligase and incubate at 16°C for 16 hours to ligate the circular template gap.

[0118] (3) Enzyme inactivation

[0119] Heat at 65°C for 10 min to inactivate T4 DNA ligase.

[0120] (4) Amplification

[0121] Add the 2mM clos...

Embodiment 2

[0126] Hemin / G-quadruplex DNAzyme functionalization of Example 2RCA NCs

[0127] (1) Treat 25 μL of TE buffer containing RCA NCs at 90°C for 1 min, cool to 4°C, and keep for 60 min.

[0128] (2) Add the product obtained in step (1) together with 100 μM Hemin (heme) solution to 50 μL Tris-HCl buffer, and incubate at 37°C for 60 min, so that Hemin (heme) can be embedded in the RCA NCs In the G-quadruplex unit, functionalized RCA NCs were formed, that is, functionalized Hemin / G-quadruplex DNAzyme.

[0129] (3) Store the prepared functionalized Hemin / G-quadruplex DNAzyme below 4°C. RCA NCs with peroxidase-mimicking DNAzyme activity can catalyze the substrate H 2 o 2 reduction, resulting in an electrochemical signal.

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Abstract

A DNA nanostructure, electrochemical aptamer biosensor system and its preparation method and application relate to the field of detection of food-borne pathogenic bacteria. The present invention synthesizes a stable DNA nanostructure through a rolling circle amplification reaction, and loads heme as an electrochemical signal label to amplify the signal; the single-stranded DNA capture probe is modified on the surface of the Au electrode through the Au-S bond; Pathogen-specific aptamers and capture probes form double-stranded DNA structures on the Au electrode; aptamers preferentially bind foodborne pathogens, resulting in dissociation of the aptamer-capture probe duplex and release of the captured Probes; Free capture probes hybridize to DNA nanostructures through complementary DNA sequences; Quantitative determination of foodborne pathogenic bacteria concentration through electrochemical signals of DPV method. The invention is simple, fast, easy to operate, low in experiment cost, sensitive and specific, and has good application prospects.

Description

technical field [0001] The invention relates to the technical field of detection of food-borne pathogenic bacteria, in particular to a DNA nanostructure, electrochemical aptamer biosensor system and its preparation method and application. Background technique [0002] Infectious diseases caused by pathogenic bacteria kill and hospitalize millions of people every year and are one of the leading causes of death worldwide. Pathogenic bacteria can be transmitted through food and drinking water, posing a serious threat to human health and causing serious economic losses. Therefore, it is very important to establish a rapid, reliable and effective detection method for pathogenic bacteria in the field of clinical microbiology. Standard culture and colony count methods used to identify pathogenic bacteria require several days for accurate detection; in addition, several traditional detection methods based on nucleic acid amplification (eg, PCR) or immunological reactions (eg, ELISA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3277G01N27/3278G01N27/48Y02A50/30
Inventor 万家余柏华松韩烨卜胜君刘文森
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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