CN-116359493-B - Digital flow microsphere biochemical analysis method based on spherical nucleic acid signal amplification mechanism

Abstract

The invention discloses a digital flow microsphere biochemical analysis method based on a spherical nucleic acid signal amplification mechanism, and belongs to the technical field of detection of protein and exosome markers. According to the invention, antibodies or aptamers are loaded on the magnetic microspheres, when the concentration of the target is ultra-micro, the surface of one magnetic microsphere is specifically combined with a single target marker molecule, the conversion efficiency of protein or exosomes to nucleic acid is greatly improved by introducing functionalized spherical nucleic acid, so that a large amount of nucleic acid amplification and enrichment of fluorescent signals are induced on the surface of the single target marker molecule on the surface of the microsphere to lighten the single magnetic microsphere, namely the positive microsphere, while the magnetic microsphere without the target marker molecule cannot be combined with the functionalized spherical nucleic acid, the nucleic acid amplification is induced and the fluorescent signals are enriched, namely the negative microsphere. The positive microspheres and the negative microspheres can be obviously distinguished by adopting a common flow cytometer, and the digital analysis of the protein or exosome markers is realized by counting the number of the positive microspheres.

Inventors

• LIU CHENGHUI
• SHI JINGJING
• FAN WENJIAO
• REN WEI

Assignees

• 陕西师范大学

Dates

Publication Date

20260512

Application Date

20230328

Claims (8)

1. 1. A digital flow microsphere biochemical analysis method based on a spherical nucleic acid signal amplification mechanism is characterized by comprising the following steps: S1, preparing functionalized spherical nucleic acid simultaneously loaded with a recognition element and a nucleic acid probe, loading a capture antibody or an aptamer of a specific recognition target marker molecule on a magnetic microsphere to obtain the functionalized magnetic microsphere; S2, specifically combining a capture antibody or an aptamer on the surface of the S1 functionalized magnetic microsphere with target marker molecules to form a magnetic microsphere loaded with 1 or 0 target marker molecules; s3, combining the target marker molecule specificity on the S2 magnetic microsphere with the identification element of the S1 functionalized spherical nucleic acid to form a sandwich composite structure of the magnetic microsphere-single target marker molecule-functionalized spherical nucleic acid; S4, initiating rolling circle amplification reaction on a single target marker molecule of the sandwich composite structure of S3, and enriching fluorophores to lighten corresponding magnetic microspheres, namely positive fluorescent magnetic microspheres, wherein the magnetic microspheres loaded with 0 target marker molecules in S2 do not initiate rolling circle amplification reaction and enrich fluorophores, namely negative fluorescent magnetic microspheres; s5, distinguishing the positive fluorescent magnetic microspheres and the negative fluorescent magnetic microspheres in the S4 by using a flow cytometer, and realizing digital analysis of the target marker according to the number or the proportion of the positive fluorescent magnetic microspheres.
2. 2. The method for biochemical analysis of digital flow microsphere based on amplification mechanism of spherical nucleic acid signal according to claim 1, wherein in S1, the recognition element is a detection antibody or an aptamer.
3. 3. The digital flow microsphere biochemical analysis method based on the spherical nucleic acid signal amplification mechanism according to claim 2, wherein the detection antibody comprises a specific detection antibody of prostate specific antigen, alpha fetoprotein, carcinoembryonic antigen and cardiac troponin I, and the aptamer comprises an exosome membrane protein CD63 aptamer, a HER2 aptamer, an EpCAM aptamer and a PSA aptamer.
4. 4. The method for biochemical analysis of digital flow microsphere based on amplification mechanism of spherical nucleic acid signal according to claim 1, wherein in S1, the capture antibody or aptamer is loaded on the magnetic microsphere by a group reaction or binding between streptavidin and biotin.
5. 5. The method for biochemical analysis of digital flow microsphere based on amplification mechanism of spherical nucleic acid signal according to claim 4, wherein the group reaction is one of amino group reaction with carboxyl group reaction, amino group reaction with epoxy group reaction, amino group reaction with aldehyde group reaction, amino group reaction with ester group reaction.
6. 6. The method according to claim 4, wherein the capture antibody comprises a specific capture antibody of prostate specific antigen, alpha fetoprotein, carcinoembryonic antigen, cardiac troponin I protein, and the aptamer comprises exocrine membrane protein CD63 aptamer, HER2 aptamer, epCAM aptamer, and PSA aptamer.
7. 7. The method for biochemical analysis of digital flow microsphere based on amplification mechanism of spherical nucleic acid signal according to claim 1, wherein in S2, the target marker molecule is a protein molecule or exosome.
8. 8. The digital flow microsphere biochemical analysis method based on the spherical nucleic acid signal amplification mechanism according to claim 1, wherein in S2, when the number of target marker molecules is lower than that of the magnetic microspheres, 1 or 0 target marker molecules are loaded on each magnetic microsphere according to the poisson distribution principle.

Description

Digital flow microsphere biochemical analysis method based on spherical nucleic acid signal amplification mechanism Technical Field The invention relates to the technical field of detection of protein and exosome markers, in particular to a digital flow microsphere biochemical analysis method based on a spherical nucleic acid signal amplification mechanism. Background The abnormal expression of important protein or exosome markers in body fluid is directly and closely related to the generation and development of related diseases, and the high-sensitivity accurate analysis of the very low-abundance biomarkers is the biggest challenge faced by the current early-stage liquid biopsy of the important diseases, so that the realization of the high-sensitivity accurate analysis of the related protein or exosome markers has important significance for screening and diagnosis of the early diseases. Taking immune analysis of protein markers as an example, the high-sensitivity analysis of protein markers is mainly an enzyme-linked immunosorbent assay analysis method based on an analog signal reading mode, and the like, and the related method mainly reflects the concentration of target protein marker molecules according to the total signal output by a reaction system, so that the method has wide application, but is easy to be interfered and low in sensitivity, and cannot meet the requirement of ultra-sensitive accurate analysis of the target protein molecules with ultra-low abundance in body fluid and cannot reflect the difference among detection individuals. Aiming at the problems of the existing analysis method at present, a digital immunoassay method which can detect target protein molecules at a single molecule level has been raised in recent years, and therefore, the method has unique advantages in the ultra-sensitive detection of the target protein molecules with ultra-micro content. At present, a single-molecule array (Sioma) technology is mature and widely applied commercially, the method takes micropores as independent reaction units, utilizes the poisson distribution principle, loads 1 or 0 target protein molecules on the surface of each magnetic bead, then loads the magnetic beads into the micropores, and at the moment, the magnetic beads with the target protein molecules on the surface trigger enzymatic reaction to release fluorescence so as to lighten the micropores to present positive fluorescent signals, and the digital analysis of the target protein molecules is realized by counting the number of the positive micropores. This method has a lower detection limit than the conventional immunoassay method, but is liable to cause a large loss of magnetic beads when they are loaded into microwells, resulting in low sampling efficiency so that accuracy thereof is limited. In addition, the method needs to prepare a large number of micropores with uniform pore diameters as micro-reaction units to divide target protein molecules and perform signal generation reaction, and the micropores need oil sealing, so that the process is complicated in operation and needs special equipment or special reagents/consumables. However, the ideal detection method of the protein or exosome marker has the similar digital analysis principle, gets rid of the dependence on a high-end instrument and a micro chamber, and meets the characteristics of stability, simplicity, low price, high efficiency, sensitivity, wide application range and the like. Therefore, the invention provides a digital flow microsphere biochemical analysis method based on a spherical nucleic acid signal amplification mechanism. Disclosure of Invention Aiming at the technical problems, the invention provides a digital flow microsphere biochemical analysis method based on a spherical nucleic acid signal amplification mechanism, which uses microspheres which are not dependent on a micro chamber as completely opened independent reaction carriers, loads 1 or 0 target protein molecules or exosomes on the surfaces of the microspheres through poisson distribution, uses gold nanoparticles as carriers and simultaneously loads target protein or exosome specific recognition elements and high-density nucleic acid amplification primers to form functional spherical nucleic acids as recognition and protein/exosome-nucleic acid signal conversion and amplification elements, uses high-efficiency nucleic acid amplification to amplify signals of proteins and exosomes, overcomes the technical problem that the proteins and exosomes lack a nucleic acid-like signal amplification mechanism, uses the functional spherical nucleic acids as intermediates to greatly improve the conversion efficiency of the proteins/exosomes to the nucleic acids, and uses simple instruments and operations to realize digital analysis of the target proteins or exosomes by enough lighting the single microspheres. The invention provides a digital flow microsphere biochemical analysis method based on a spherical nucleic