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Alzheimer's disease related ApoE gene detection method based on entropy driving signal amplification and label-free fluorescent probe

An Alzheimer's disease and fluorescent probe technology, applied in the field of bioanalytical chemistry, can solve problems such as increasing synthesis cost, and achieve the effects of sensitive detection, reduction of non-specific interference, and simple synthesis method

Pending Publication Date: 2022-05-06
重庆医科大学国际体外诊断研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the entropy-driven signal amplification strategy, it is usually necessary to modify fluorescent molecules and quencher molecules at the ends of the two sequences, which undoubtedly increases the cost of synthesis.

Method used

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  • Alzheimer's disease related ApoE gene detection method based on entropy driving signal amplification and label-free fluorescent probe
  • Alzheimer's disease related ApoE gene detection method based on entropy driving signal amplification and label-free fluorescent probe
  • Alzheimer's disease related ApoE gene detection method based on entropy driving signal amplification and label-free fluorescent probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) Design of Alzheimer's disease-related ApoE gene detection method based on entropy-driven signal amplification and unlabeled fluorescent probes:

[0029] The nucleic acid sequences used in the present invention are all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the specific sequence design is as follows:

[0030] name Sequence(from 5'to 3') target ATG GAG GAC GTG TGC GGC CG CS Biotin-TTTTTTTTTT GGC CAC GTA GGG TAT TGC ACA CGT CCT CCA T TS CGA TTC AAT ACC CTA CGT GG CC TTTTTTT SS GGC CAC GTA GGG TAT TGA ATG CGA TTT CGT ATA GTC TGA CCC CCT AAT TCC C OS TTT TTT TTT TAT CGC ATT CAA TAC CCT A AS GGG GTG GGT GGG TGG GGT CGG GTC AGA CTA TAC GAA FS GGG TCA GAC TAT ACG AAA TCG CAT TCA ATA CCC TA M1 ATG GAG GAC GTG CGC GGC CG M2 ATG GAG GAC CTG CGC GGC CG NC ACT GCT AGA GAT TTT CCA CAT

[0031] (2) Preparation of magnetic bead-double strand recognition probe complex:

[0032...

Embodiment 2

[0041] Feasibility verification of Alzheimer's disease-related ApoE gene detection method based on entropy-driven signal amplification and unlabeled fluorescent probe:

[0042] (1) In this embodiment, electrophoresis is first used to verify the feasibility of the entropy-driven reaction, such as figure 2 As shown, a band with slower mobility appears in lane 5, which confirms the formation of a three-strand complex (SS+OS+AS); most of the triple-strand complex still exists in lane 6, because the activation chain In the absence of TS, the triple-strand complex structure cannot be completely destroyed; on the contrary, in lane 7, when TS and FS are added, the triple-strand complex band disappears. Taken together, the results confirmed that the entropy-driven mediated reaction proceeded successfully.

[0043] (2) In this embodiment, use fluorescence spectroscopy to verify the feasibility of the principle, as image 3 As shown, compared with the fluorescence intensity of pure silv...

Embodiment 3

[0045] Experimental condition optimization

[0046] (1) Optimization of fuel chain FS amount: Fuel chain FS is an important factor affecting the efficiency of entropy-driven reactions. From Figure 4 It can be obtained that (F0-F) / F0 (F0 is the fluorescence value when blank, F is the fluorescence value when the target exists) increases with the increase of FS volume, and starts to stabilize when the FS volume is 5 μL. Therefore, 5 μL was chosen for the next study.

[0047] (2) Optimization of the volume of the magnetic bead-recognition double-strand complex: the volume of the required optimal magnetic bead-recognition double-strand complex is optimized, Figure 5 As shown, when the volume was 10 μL, (F0-F) / F0 achieved the maximum, and the fluorescence ratio remained stable with the increase of the number of conjugates.

[0048] (3) Optimization of EXOⅢ concentration and reaction time: The signal amplification mediated by EXOⅢ is an important factor to improve the sensitivit...

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Abstract

The invention provides an Alzheimer's disease related ApoE gene detection method based on entropy driving signal amplification and an unmarked fluorescent probe, and belongs to the technical field of life analysis. The detection of the ApoE gene is realized by two-step signal amplification through target circulation mediated by exonuclease III (EXO III) on the surface of a magnetic bead and entropy driving and by taking an unmarked silver nano-cluster fluorescent probe as a signal output mode. The target gene is specifically combined with the recognition double-chain probe on the surface of the magnetic bead to form an enzyme cutting site of EXO III; under the action of EXO III, a large number of activation chains are generated; the supernatant is separated and extracted by the magnetic beads, and the existing activation chain triggers a three-chain compound template formed by the silver nanoclusters to generate branch migration and chain substitution of the chain, so that the structure of the silver nanoclusters is changed, and a fluorescence signal is reduced. The method has the advantages of no label and low cost, and can realize sensitive detection of the ApoE gene.

Description

technical field [0001] The invention belongs to the technical field of life analysis chemistry, and in particular relates to a method for detecting Alzheimer's disease-related ApoE genes based on entropy-driven signal amplification and unlabeled fluorescent probes. Background technique [0002] Alzheimer's disease (AD) is a severe neurodegenerative disease with insidious onset and slow progression. The main clinical manifestations are progressive memory decline accompanied by other cognitive decline. With the intensification of population aging situation, the number of patients with Alzheimer's disease is also increasing year by year. The etiology of AD is currently unknown, and there is no effective treatment to prevent or even delay the progression of dementia. In recent years, finding specific biomarkers for early diagnosis and prevention is an important goal in the field of AD research. Some related literature pointed out that the apolipoprotein E (apolipoprotein E, Ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/682C12Q1/6806
CPCC12Q1/6883C12Q1/682C12Q1/6806C12Q2600/158C12Q2563/143C12Q2563/149C12Q2563/107
Inventor 杨晓燕向华
Owner 重庆医科大学国际体外诊断研究院
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