Nanoscale single exosome separation method

Abstract

The invention discloses a nanoscale single exosome separation method. The method comprises the following steps of fully diluting a cell culture solution containing exosome; adding a superparamagneticamino magnetic bead diluent, and carrying out low-frequency oscillation mixing until the nano magnetic beads are coupled with the exosome; transferring a coupling solution to a surface layer of a nanopore array microchip; placing the microchip on a shaking table, and oscillating in low speed and small angle, so that the coupling solution is uniformly distributed on the surface layer of the chip; placing a magnetic source on the lower surface of the microchip, and enabling a magnetic bead exosome couplet to enter a nanopore array by using a progressive magnetic adsorption mode; under the actionof larger magnetic force, maintaining stable adsorption of the magnetic bead exosome couplet, cleaning the upper surface of a substrate with a buffer solution, and standing to obtain the nano microchip containing the single exosome which can be directly used for subsequent detection. On the basis of the nanopore microarray microchip with the high-precision screening effect, high-purity single exosome can be obtained through magnetic bead coupling exosome and progressive magnetic adsorption.

Description

technical field [0001] The invention belongs to the technical field of single exosome analysis, and in particular relates to a nanoscale single exosome separation method. Background technique [0002] Exosomes refer to biologically active vesicles with a diameter of about 30-150 nm secreted by (most) cells. Exosomes have a typical lipid bilayer membrane structure, carrying important information such as various proteins, lipids, and nucleic acids secreted by the mother cell, and are important carriers and key messengers for information exchange between cells. It plays an important role in the physiological and pathological processes of various diseases. [0003] Currently, exosome isolation techniques mainly include ultracentrifugation, ultrafiltration centrifugation, microchannel method, magnetic bead immunoassay, etc. Ultracentrifugation is the most commonly used exosome purification method. Vesicle particles of different sizes are precipitated and purified from soluble m...

AI Technical Summary

Problems solved by technology

Ultracentrifugation is the most commonly used exosome purification method. Vesicle particles of different sizes are precipitated and purified from soluble molecules by high-speed centrifugation, but repeated centrifugation is likely to cause damage to exosome vesicles and reduce their quality.

The ultrafiltration centrifugation method uses the difference in relative molecular mass to selectively separate samples. This method is simple and efficient, and does not affect the biological activity of exosomes. However, the filter pores are easily blocked, resulting in shortened membrane life and low separation efficiency.

Based on the principles of viscoelastic flow and nanowire traps, the microchannel method realizes the effective separation of exosomes of different scales through acoustic, electrical and electromagnetic manipulation. This method has the advantages of minimal consumption, high resolution and low cost. , but the diameter of the microchannel is usually above the micron level, making it difficult to separate single exosomes at the nanoscale

[0004] Magnetic bead immunoassay is an immunoaffinity technique. The protein surface of exosomes has specific receptors, and different types of exosomes are captured from the sample by nanoparticles with corresponding modified antibodies, so as to achieve exosome Selective separation, this method has the advantages of high specificity and high-purity exosomes, but lacks appropriate carrier support to separate independent single exosomes, resulting in the efficiency and dispersion of single exosomes isolated not high

[0005] CN 109576210 A discloses a method for rapidly separating exosomes, which effectively improves the efficiency of separating exosomes from serum by constructing a flexible structural unit between superparamagnetic nanoparticles and ligands, but the method obtained The superparamagnetic nanoparticle-polyethylene glycol-ligand complex is a high-purity cluster, which requires further dispersion and processing to obtain a single independent exosome

CN 109266599 A discloses a high-efficiency and non-destructive exosome isolation method, which uses magnetic beads modified with nucleic acid aptamers to identify and capture exosome samples, and focuses on the replacement of complementary strands using nucleic acid aptamers Exosomes are released, but this method still uses static extraction and precipitation to obtain magnetic bead-exosome complexes after the co-incubation of magnetic beads and exosome samples, the dispersion efficiency is low, and it cannot be directly used for subsequent detection

[0006] At present, the isolation and detection of single exosomes at the nanometer scale is facing challenges and bottlenecks, and it is urgent to develop precise isolation methods for single exosomes. On the basis of the lack of appropriate carrier to separate and obtain independent single exosomes, the efficiency of separating and obtaining single exosomes is not high. Efficient and stable distribution of nanoscale single exosomes provides a feasible method

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