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A component, system and application for quantitative detection of prostate cancer biomarker miRNA-141

A technology of miRNA-141 and biomarkers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of miRNA blood background complex, complex and cumbersome process, etc., to achieve improved sensitivity, reliable analysis results, and convenient The effect of carrying and transporting

Active Publication Date: 2022-05-27
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can achieve the purpose of detecting miRNA, the process is very complicated and cumbersome, and requires trained professionals to detect
Meanwhile, developing a method for the quantitative detection of miRNAs directly in human blood samples remains a challenging issue due to the low content of miRNAs and the very complex background in blood.

Method used

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  • A component, system and application for quantitative detection of prostate cancer biomarker miRNA-141
  • A component, system and application for quantitative detection of prostate cancer biomarker miRNA-141
  • A component, system and application for quantitative detection of prostate cancer biomarker miRNA-141

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Experimental program
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Effect test

Embodiment 1

[0059] The preparation method of paper base, comprises the steps:

[0060] Gold nanoparticles were synthesized by the Turk evich method. Trisodium citrate reduction of HAuCl 4 ·3H 2 O to synthesize gold nanoparticles of citrate. The specific synthesis steps are as follows, 94mL of HAuCl 4 The aqueous solution (2 mM) was heated to 110°C, stirred until boiling vigorously, and then 19 mL of trisodium citrate solution (38.8 mM) was added immediately. The mixture was kept boiling for a certain time until the color of the solution gradually changed from yellow to wine red. The reaction was terminated when the suspension was cooled to room temperature. The concentration of as-synthesized gold nanoparticles was calculated to be 6 × 10 -8 M, and stored at 4°C for further use.

[0061] A PCs-sRNA probe containing gold nanoparticles and DNA strands was synthesized, wherein the gold nanoparticles were able to form Au-S bonds with the thiol-modified DNA strand SH-L, thus realizing t...

Embodiment 2

[0068] The specific steps to verify the strand displacement reaction are as follows (experimental results such as figure 2 shown):

[0069] (1) Prepare thiolated DNA strand (SH-L), signal strand (PCs-sRNA), SH-L (3.0 μM) and PCs-sRNA (6.2 μM) in 20 mM Tris-HCl buffer (100 mM Na + , pH=7.4) was heated to 90°C for 5 min and then cooled to room temperature for 2 hours to form a ternary DNA duplex probe. Based on Marker, SH-L, PCs-sRNA and ternary DNA were verified by PAGE gel. This step verifies the successful binding of ternary DNA double strands.

[0070] (2) Divide the remaining ternary DNA solution into two parts, add miRNA-141 (50 pM) to one solution, add an equal volume of water to the other solution, heat the two mixed solutions to 90°C for 5 min, and then cool them to 2 hours at room temperature. Based on Marker, SH-L, PCs-sRNA, miRNA-141, the mixed solution with water added in this step, and the mixed solution with miRNA-141 were used for gel running verification. ...

Embodiment 3

[0074] At 37°C, PCs-AuNPs nanoprobes were added dropwise to the thiol-modified paper substrate, then different concentrations of miRNA-141 (18 μL) were added dropwise and incubated for 60 min, and F chain (1 μM, 18 μL) was added dropwise and incubated for the same time. During , different matrix solutions (here, serum matrix solution and pH=7.4 ammonium acetate buffer solution) were used to keep the paper substrate wet, and then infiltrated into the lower paper substrate for paper spray mass spectrometry detection. Take lg c as the abscissa and the mass spectrum peak intensity ratio of the signal molecule to the internal standard as the ordinate to draw a standard curve, the specific concentrations are 50fM, 500fM, 1pM, 5pM, 10pM, 50pM, the results are as follows Figure 4 As shown, it can be seen that the method of the present invention has a good linear relationship in quantitative analysis.

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Abstract

The present application provides a component, system and application for quantitative detection of prostate cancer biomarker miRNA-141. The components include: PCs-AuNPs nanoprobes, thiol-modified paper substrates, removable detection paper substrates and DC power supplies; wherein, PCs-AuNPs nanoprobes can react with the prostate cancer marker miRNA-141, which is passed The Au-S bond is fixed on the thiol-modified paper base; the movable detection paper base is located on the lower layer of the paper base, and can be moved to contact the thiol-modified paper base; the DC power supply powers the movable detection paper base . The present invention can achieve high sensitivity, specificity and repeatability based on a small amount of blood sample.

Description

technical field [0001] The present application relates to the technical field of biological detection, in particular to a component, system and application for quantitative detection of prostate cancer biomarker miRNA-141. Background technique [0002] The disclosure of information in this Background section is only for enhancement of understanding of the general background of the application and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. [0003] miRNAs are small non-protein-coding single-stranded RNA fragments of 19-23 nucleotides, and miRNAs can be released from cells into the blood circulation in the form of protein-RNA complexes. miRNAs are involved in many basic cellular processes, including cell development, apoptosis, and metabolism, and have important research significance. In addition, various molecular biology experiments have dem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6834
CPCC12Q1/6886C12Q1/6834C12Q2600/158C12Q2600/178C12Q2525/207C12Q2563/137C12Q2563/155C12Q2565/627C12Q2537/1373C12Q2523/313
Inventor 唐波王为卿杨燕美陈蓁蓁刘慧敏
Owner SHANDONG NORMAL UNIV
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