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A self-assembled probe based on conformational change and its label-free detection method for exosomes

A technology of configuration change and detection method, applied in the field of molecular biology, can solve the problems of affecting the efficiency of identification, background interference and so on

Active Publication Date: 2022-07-26
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Patent CN201811548979.0 discloses an exosome detection method based on aptamer and rolling circle amplification, using an electrochemical sensor method to construct an aptamer probe based on exosome specificity, using G-quadruple Body-Hemin Mimetic Peroxidase Catalyzed H 2 o 2 The reaction generates signals; the patent synthesizes a large number of G-quadruplexes through rolling circle amplification for signal amplification to realize the quantitative detection of exosomes; patent CN201811173676.5 provides an aptamer group for detecting exosomes, lateral flow aptamer biosensor and its preparation method. The aptamer group for detecting exosomes in this patent includes CD63 aptamers and EpCam aptamers. CD63 aptamers can specifically bind to the CD63 protein of exosomes. EpCam aptamers The ligand can specifically bind to the EPCAM protein of exosomes. The invention also provides a lateral flow aptamer biosensor, which mainly sprays the CD63 aptamer on the binding pad based on the principle of chromatography test strips, and sprays the EpCam aptamer The detection line is formed, and the quality control line is sprayed with DNA probes that can be combined with nano-gold-labeled aptamers through base complementary pairing; the qualitative and quantitative detection of exosomes is realized through the lateral flow aptamer biosensor; now Some detections of exosomes are mostly based on the direct combination of antibodies or nucleic acid aptamers to exosomes surface general protein CD63, which will generate high background interference, and most of them need to label signal molecules , which will undoubtedly affect the efficiency of recognition, so it is imminent to develop an activatable, label-free fluorescent sensing platform to detect exosomes

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  • A self-assembled probe based on conformational change and its label-free detection method for exosomes
  • A self-assembled probe based on conformational change and its label-free detection method for exosomes
  • A self-assembled probe based on conformational change and its label-free detection method for exosomes

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Embodiment 1

[0036] In this embodiment, the self-assembly probe based on configuration change is used for the label-free detection method of exosomes, and the steps are as follows:

[0037] (1) Cell culture: CCRF-CEM, Ramos, Hela, B16F1 and HL-7702 cells were cultured in RPMI 1640 medium supplemented with 10% exosomes (fetal bovine serum removed) and 100IU / mL penicillin or streptomycin cultured at 37 °C with 5% wt / vol CO 2 Incubate in a humidified incubator for 48 hours, and collect the cell supernatant when the cells grow to 80-90%;

[0038] (2) Isolation of exosomes: The cell supernatant obtained in step (1) was centrifuged at 3,000g for 30 minutes at 4°C, and filtered through a syringe-driven filter unit to remove intact cells and cell debris, and the collected filter and concentrated using 100 KDa MWCO at 5,000 g for 30 min at 4 °C, then the filtered medium was ultracentrifuged at 160,000 g for 2 h at 4 °C, the supernatant was discarded, and the exosome pellet was resuspended in phospha...

Embodiment 2

[0042] In this example, in order to further study the hybridization and binding ability of G4-DNA and different blocker-DNAs, eight groups of Blocker DNA sequences were designed, such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO. .6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10; refer to the procedure of Example 1 for fluorescence detection. First, a Bare DNA strand was designed, which contained a complete base of the priming region to simulate the probe recognizing exosomes, which could be used to trigger a strand displacement reaction to release G4. Test results such as figure 2 shown, by figure 2 It can be seen that by using BlockerDNA-2, when Bare DNA is present, the maximum fluorescence enhancement can be obtained, which is significantly higher than that of blocker DNA-1, blockerDNA-3, blocker DNA-4, Blocker DNA-5, Blocker DNA-6 and Blocker DNA- 7. Therefore, blocker DNA-2 was selected as the best blocker DNA for subsequent experiments and named R-DN...

Embodiment 3

[0044] In this example, in order to prove the feasibility of the experimental principle, the fluorescence intensity of the system under different conditions was studied. The test results are shown in image 3 shown, by image 3 It can be seen that in the absence of target exosomes (red curve) and control exosomes (blue curve), there is no obvious fluorescence signal, indicating that the present invention has a lower fluorescence background. After the target exosomes were introduced into the above mixed solution, an obvious fluorescence enhancement could be observed at around 615 nm (green curve), which indicated that the G-rich sequence was exposed and folded into a quadruple structure. These results suggest that this experimental platform can be used for the specific detection of leukemia-related exosomes.

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Abstract

The invention relates to the technical field of molecular biology, in particular to a self-assembly probe based on configuration change and a label-free detection method for exosomes. In the presence of exosomes, the exosome surface marker, protein tyrosine kinase-7 (PTK7), specifically binds to the nucleic acid aptamer, changes the configuration of the recognition probe, and releases the trigger chain, thereby interacting with G-R DNA A strand displacement reaction occurs, exposing the G-rich sequence to form a G-quadruplex, and NMM intercalates into the G-quadruplex to generate a strong fluorescent signal, enabling label-free and sensitive detection of exosomes. Under optimized experimental conditions, the linear range is 5 × 10 5 to 5×10 7 particles / μL, the minimum detection limit is 3.4×10 5 particles / μL, this probe is used to prepare a kit for the highly sensitive and selective detection of exosomes in actual blood samples.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a self-assembly probe based on configuration change and a label-free detection method for exosomes. Background technique [0002] Exosomes are membrane-bound nanovesicles between 30 nm and 150 nm in diameter. They carry a large number of proteins, lipids, DNA, RNA and other biomolecules, making exosomes a non-invasive cancer marker for the preparation of early diagnostic reagents for tumors. Therefore, accurate quantification and classification of tumor exosomes is of great significance for the preparation of reagents for cancer diagnosis and prognosis assessment. [0003] The performance analysis of different existing methods for detecting exosomes is summarized in the following table: [0004] . [0005] Patent CN201811548979.0 discloses an exosome detection method based on aptamer and rolling circle amplification. The method of electrochemical sensor is used to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6876C12Q1/682C12N15/11
CPCC12Q1/6876C12Q1/682C12Q2531/119C12Q2563/107C12Q2525/205
Inventor 孟红敏陈娟李朝辉葛佳
Owner ZHENGZHOU UNIV
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