MiRNA fluorescence sensor and method for detecting miRNA
A fluorescent sensor and target detection technology, applied in the field of biomedical engineering, can solve the problems of strict control, low content, undetectable miRNA, etc., and achieve the effect of mild reaction
Inactive Publication Date: 2022-06-03
JIHUA LAB
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Problems solved by technology
However, the efficient detection of miRNAs is difficult due to their short sequences, high homologous sequence similarity, low content, and lack of common features that allow them to be selectively amplified.
Early techniques used to detect miRNAs relied on Northern blotting, which has disadvantages including low throughput, low sensitivity, time-consuming, high sample consumption, etc.
In addition, the development of microarray technology is more effective for high-throughput analysis of multiple groups of miRNAs, but the limitation of this technology is that miRNAs that are too short and low in copy number cannot be detected within a limited concentration range, and the specificity is not strong
Another commonly used technique is real-time quantitative PCR, which has a large dynamic range, high sensitivity, and good selectivity, but it also has certain shortcomings, such as RNA extraction needs to be very complete, and steps such as cDNA synthesis and primer design need to be strictly controlled
Method used
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Embodiment 1
Using hsa-let-7a as the target miRNA, DNA probe A, probe B, and probe C with a dumbbell structure were synthesized, and hsa-let-7b~7g were used as the corresponding mismatch sequences for verification. Example 1 involved The gene sequence is shown in Table 2.
[0042] Table 2
(1) Preparation of gold nanoparticles:
Prepare 0.01% (w / v) chloroauric acid solution and 1% (w / v) trisodium citrate solution. Add 3.5 mL of trisodium citrate to 100 mL of condensed reflux chloroauric acid solution, stir and boil for 15 minutes; stop heating and stir the mixture again for 30 minutes; cool the solution to room temperature to obtain gold nanoparticles; Nanoparticles were purified by centrifugation at 12,000 rpm for 20 minutes. The prepared gold nanoparticles are bare gold nanoparticles.
[0043] In order to realize the functionalization of DNA probe A on the surface of gold nanoparticles, DNA probe A was directly added to the bare gold nanoparticles at a concentration of 4 μM. After...
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The invention relates to the field of biomedical engineering, and discloses a miRNA fluorescence sensor and a method for detecting miRNA. The miRNA fluorescence sensor at least comprises gold nanoparticles covalently linked with a DNA probe A, wherein the DNA probe A comprises a probe B and a first DNA sequence which can be hybridized with target miRNA to generate a DNA / RNA heterozygote; one end of the probe C with the dumbbell structure is modified with fluorescent molecules, the other end of the probe C with the dumbbell structure is modified with sulfydryl, and a stem part of the probe C is provided with a second DNA sequence capable of being hybridized with the probe B; the sequence at one end of the probe C with the dumbbell structure can be hybridized with the sequence at the other end. When the miRNA fluorescence sensor is used for detecting the target miRNA, the concentration of the target miRNA can be indicated by detecting the intensity of a signal emitted by a fluorescent molecule.
Description
technical field [0001] The present application relates to the field of biomedical engineering, and mainly relates to a miRNA fluorescence sensor and a method for detecting miRNA. Background technique [0002] miRNA is a class of single-stranded non-coding RNAs, about 18-25 nucleotides in length, derived from the hairpin precursor molecules encoded by the genomes of animals, plants, and microorganisms. Combined, and then regulate the expression of many genes after transcription. Studies have found that miRNAs can regulate cell development, differentiation, and apoptosis, and their abnormal expression is closely related to the occurrence and development of many diseases, including tumors, cardiovascular diseases, and neurodegenerative diseases. [0003] Considering the biological functions of miRNAs and their role in disease diagnosis, the specific and sensitive detection of miRNAs is becoming more and more important. However, the efficient detection of miRNAs is difficult d...
Claims
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Patent Timeline
Login to View More IPC IPC(8): C12Q1/6825C12N15/11
CPCC12Q1/6825C12Q2563/137C12Q2563/155C12Q2525/207C12Q2521/327C12Q2563/107C12Q2525/301
Inventor 缪鹏郭振振陈锡峰
Owner JIHUA LAB