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CRISPR/Cas-driven DNA hydrogel colorimetric sensor as well as preparation method and application thereof

A technology of colorimetric sensors and hydrogels, applied in biochemical equipment and methods, instruments, scientific instruments, etc., can solve the problem of inability to realize low-concentration MC-LR detection, increase operational professionalism, require detection costs, and colorimetric sensor sensitivity Low-level problems, to achieve the effect of high accuracy, low detection cost, and accurate detection results

Active Publication Date: 2021-12-14
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although biosensing strategies based on CRISPR / Cas systems have achieved good progress and effects, such as DETECTR and SHERLOCK, a series of sensors based on CRISPR / Cas systems that have been established so far have the following limitations: (1) Most of them use The integration of nucleic acid amplification technology and CRISPR / Cas system greatly increases the professional requirements of operation and detection cost; (2) Although sensors based on CRISPR / Cas system are gradually used for the detection of non-nucleic acid targets, CRISPR / Cas The potential of Cas sensors in ultrasensitive detection of non-nucleic acid targets such as biotoxins, proteins, and biological metabolites has yet to be discovered
However, colorimetric sensors usually have low sensitivity and high detection limit, and cannot detect low-concentration MC-LR.

Method used

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  • CRISPR/Cas-driven DNA hydrogel colorimetric sensor as well as preparation method and application thereof
  • CRISPR/Cas-driven DNA hydrogel colorimetric sensor as well as preparation method and application thereof
  • CRISPR/Cas-driven DNA hydrogel colorimetric sensor as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] A CRISPR / Cas-driven DNA hydrogel colorimetric sensor, including DNA hydrogel embedded with metal-organic framework materials, sgRNA-Cas protein complex and its target DNA, wherein:

[0048] The DNA hydrogel embedding the metal organic framework material is composed of Cu-TCPP(Fe) and DNA hydrogel composite, and the DNA hydrogel contains the DNA chain of the nucleotide sequence shown in SEQ ID NO: 1-3;

[0049] The sgRNA-Cas protein complex is composed of Cas14a protein and sgRNA, and the nucleotide sequence of the sgRNA is shown in SEQID NO:4.

[0050] The preparation method of the DNA hydrogel colorimetric sensor driven by this CRISPR / Cas comprises the following steps:

[0051] 1. Preparation of Cu-TCPP(Fe) (refer to Ultrathin 2D Metal-Organic Framework Nanosheets), the specific steps are as follows:

[0052] 2.4 mg Cu(NO 3 ) 2 ·3H 2 O (0.01 mmol), 10 μL of trifluoroacetic acid (1.0 M), and 10.0 mg of polyvinylpyrrolidone were dissolved in a mixture of 12 mL of DMF...

Embodiment 2

[0068] The application of the CRISPR / Cas-driven DNA hydrogel colorimetric sensor in the detection of MC-LR in Example 1 includes the method and principle verification of using the CRISPR / Cas-driven DNA hydrogel colorimetric sensor to detect MC-LR:

[0069] 1. Preparation of cDNA-MC-LR aptamer complex solution:

[0070] Add MC-LR aptamer solution with a final concentration of 100 nmol / L and cDNA solution with a final concentration of 100 nmol / L in 1×Bing Buffer, denature at 95°C for 5 min, cool to 25°C for 30 min, and form cDNA-MC-LR aptamer complex solution. The specific sequences of MC-LR aptamers and cDNA sequences are shown in Table 3.

[0071] Table 3: Sequence information of MC-LR aptamers and cDNA

[0072]

[0073] 2. Competition reaction of MC-LR aptamer / MC-LR / cDNA:

[0074] In the above cDNA-MC-LR aptamer complex solution, add MC-LR standard solution with a final concentration of 10 ng / mL, mix well and react at 25°C for 30 min to obtain a competition reaction sol...

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-driven DNA (deoxyribonucleic acid) hydrogel colorimetric sensor. The CRISPR / Cas-driven DNA hydrogel colorimetric sensor comprises DNA hydrogel and an sgRNA-Cas protein complex, wherein a metal organic framework material is embedded in the DNA hydrogel; the DNA hydrogel in the DNA hydrogel embedded with the metal organic framework material contains a DNA chain with a specific nucleotide sequence; and the sgRNA-Cas protein complex is formed by compounding Cas protein and sgRNA corresponding to the Cas protein, and a part of nucleotide sequence of the sgRNA is complementary with a nucleotide sequence of target DNA of a target object to be detected. According to the DNA hydrogel colorimetric sensor, the DNA hydrogel embedded with the metal organic framework material is introduced for signal amplification, a to-be-detected target does not need to be amplified or enriched, the sensitivity is high, and the accuracy is high. The invention further discloses a preparation method and application of the DNA hydrogel colorimetric sensor. The DNA hydrogel colorimetric sensor is easy to operate, short in time consumption, low in cost, high in sensitivity and accurate in detection result.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a CRISPR / Cas-driven DNA hydrogel colorimetric sensor and its detection of a non-nucleic acid target—microcystin-LR. Background technique [0002] The CRISPR / Cas system consists of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (CRISPR-associated, Cas), and is an adaptive immune system for archaea and bacteria. Due to its unique precise recognition ability, the CRISPR / Cas system is not only used for genome editing, but also applied in the field of biosensing. Although biosensing strategies based on CRISPR / Cas systems have achieved good progress and effects, such as DETECTR and SHERLOCK, a series of sensors based on CRISPR / Cas systems that have been established so far have the following limitations: (1) Most of them use The integration of nucleic acid amplification technology and CRISPR / Cas system greatly increases the professi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825G01N33/53G01N21/25G01N21/31
CPCC12Q1/6825G01N33/5308G01N21/25G01N21/31C12Q2521/327C12Q2525/151C12Q2525/161
Inventor 丁萍伍翩开天瀚陈翠梅王丹琪黄瑞雪张竞文
Owner CENT SOUTH UNIV
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