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Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method

A protein marker and gold nanotechnology, which is applied in the chemical and biological fields, can solve the problems of sample cross-contamination, complicated operation procedures, and increased reaction costs, and achieve the effects of preventing cross-contamination, simplifying the operation process, and widening the linear range of detection

Pending Publication Date: 2021-04-30
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, in order to improve the sensitivity of the detection, the activation strand is subjected to nucleic acid amplification before the CRISPR reaction. The commonly used amplification methods include polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal Amplification (LAMP), etc. However, these methods usually require the use of specialized instruments, are costly and easily lead to cross-contamination between samples
In addition, the conversion of the analyte recognition signal into the CRISPR bypass cleavage activity signal and the activation strand nucleic acid amplification are usually carried out step by step, which makes the operation procedure more complicated
Finally, in order to detect different analytes, it is usually necessary to design different activation strands and crRNAs, which makes the operation more complicated and increases the cost of the reaction

Method used

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  • Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method
  • Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method
  • Protein marker detection kit combining gold nanoprobe and CRISPR-Cas and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] In this example, CEA was used as an analyte for quantitative analysis.

[0050] 1. Preparation of gold nanoprobes

[0051] The preparation process of gold nanoprobes is as follows: figure 2 As shown, the CEA aptamer and the activated chain were activated with 10 mM TECP for 1 hour at room temperature, and then they were added to the gold nanoparticle solution and reacted with shaking at room temperature for 16 hours. Then add 1M NaCl solution six times to make the final concentration 0.1M, shake at room temperature for no less than 24 hours to obtain gold nanoprobes, centrifuge at 10000r / min for 10min to remove excess nucleic acid, and use PBS (0.01M, pH 7.4) buffer solution was washed three times, redispersed in PBS (0.01M, pH 7.4) buffer solution, and stored at 4°C in the dark for future use. like image 3 UV characterization, indicating the successful preparation of gold nanoprobes.

[0052] The sequence of the CEA aptamer is:

[0053] 5'-SH-C 6 -TTTTTTTTTTTAA...

Embodiment 2

[0072] In this example, PSA was used as an analyte for quantitative analysis.

[0073] The difference from Example 1 is that the CEA aptamer is replaced with the PSA aptamer, and the specific coating antibody of CEA is replaced with the specific coating antibody of PSA.

[0074] The sequence of the PSA aptamer is:

[0075] 5'-SH-C 6 -TTTTTAATTAAAGCTCGCCATCAAATAGC-3'

[0076] The PSA was purchased from Shanghai Lingchao Biotechnology Co., Ltd., diluted with PBS (0.01M, pH 7.4) buffer solution to different concentrations: 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 30, 50, 70, 100, 150ng / mL.

[0077] Figure 6 The standard curve of PSA is shown, and it can be seen from the figure that the signal of the detection system increases with the increase of PSA analyte, and there is a good linear relationship between 0.5-150 ng / mL.

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Abstract

The invention relates to the technical field of chemistry and biology, in particular to a protein marker detection method combining a gold nanoprobe and CRISPR-Cas. Immunoassay, a nanotechnology and a CRISPR detection technology are combined, and an aptamer and a CRISPR activation chain are covalently connected to gold nanoparticles to obtain the gold nanoprobe. Through the specific recognition effect of the antibody and the aptamer on the protein marker, a sandwich structure of the antibody-analyte-gold nanoprobe is formed in the 96-hole elisa plate. The activation chain on the gold nanoprobe can activate the para-cleavage activity of the CRISPR protein and cleave the fluorescence reporter molecule to generate a fluorescence signal, thereby quantitatively detecting the analyte. According to the present invention, with the gold nanometer probe, the conversion from the analyte identification signal to the CRISPR side cleavage activity signal is achieved, and the amplification of the activation chain signal is achieved; the detection method is simple to operate, high in sensitivity, strong in specificity and wide in detection linear range.

Description

technical field [0001] The invention relates to the fields of chemistry and biotechnology, in particular to a protein marker detection kit and detection method combined with gold nanoprobes and CRISPR-Cas. Background technique [0002] In recent years, the discovery and research of CRISPR systems have provided new methods for nucleic acid detection, which mainly rely on fluorescent signals to detect the concentration of target nucleic acid molecules in samples. The CRISPR proteins currently used for nucleic acid detection include Cas12a, Cas12b, Cas13a, Cas13b, Cas14, and Csm6, which have bypass cleavage activity. Among them, the Cas12a protein molecule combines with crRNA to form a crRNA-Cas12a complex, and the target single-stranded DNA molecule or double-stranded DNA molecule specifically binds to crRNA to activate the bypass nucleic acid cleavage activity of the CRISPR protein, and arbitrarily cut single-stranded DNA molecules; in addition, After the Cas13a protein form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/543
CPCC12N15/115G01N33/54313C12N2310/16
Inventor 王玉珍赵桥
Owner NANJING UNIV OF TECH
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