Biological target molecule detection method based on multivalent capture and output signal amplification

Abstract

The invention discloses a biological target molecule detection method based on multivalent capture and output signal amplification, and belongs to the technical field of biological detection. The detection method comprises the following steps: adding an identification probe into a solution to be detected, identifying and combining targeted biological target molecules, releasing an initiating sequence, allowing the initiating sequence to open hairpin probes to generate a hybridization chain reaction, adding an auxiliary probe, sealing hairpin probes which do not participate in the reaction, placing a carrier which is self-assembled with a capture probe on a gold interface in the reaction solution, capturing a hybridization chain reaction product onto a gold sheet, and finally analyzing thesignal mark change on the gold interface to further calculate the content of the targeted biological target molecules in the sample to be detected. The method forms a plurality of hybridization chainreaction products are formed through the hybridization chain reaction, and then utilizes interface multivalent capture and signal output amplification, so that the detection of target molecules is realized; and under the help of the auxiliary probe, the efficiency of capturing hybridization chain reaction products by the sensing interface is improved, and the detection sensitivity is improved.

Description

technical field

[0001] The present invention relates to the technical field of biological detection, in particular to a biological target molecular detection method based on multivalent capture and amplified output signals and its application in liquid biopsy for exosomes, drugs / biological small molecules, DNA, RNA, metal ions in organisms, Precise analytical detection in antigens, antibodies, enzymes and cells. Background technique

[0002] Exosomes are an important carrier of intercellular communication. Exosomes secreted by tumor cells are closely related to the occurrence and development of tumors, immune escape, and the establishment of microenvironment. Exosomes play an important role in the treatment and diagnosis of tumors. The diagnostic research of exosomes is still in the early clinical stage. In recent years, the enrichment detection research based on exosomes has become one of the key areas of liquid biopsy. Exosomes secreted by tumor tissues carry abundant tum...

Examples

Embodiment 1

[0073] Detection of exosomes based on single-step multivalent capture and amplification

[0074] 1. Recognition probe design: design 4 probes A, B, C, and D that make up the nucleic acid framework, wherein A, B, C, and D contain sequences that hybridize with each other to form the nucleic acid framework structure FNAs / apt;

[0075] According to the detection of epithelial cell adhesion molecules on the surface of the target breast cancer-associated exosome membrane, A includes the nucleic acid aptamer sequence Aapt that can specifically recognize and bind epithelial cell adhesion molecules, and B includes Ba, where the full or partial sequence of Ba can Crossed with Aapt. DNA three-dimensional nanostructures such as nucleic acid frameworks have a certain ability to resist enzymatic degradation in complex biological environments, which is conducive to improving the stability of the carried recognition probes in complex biological samples and improving the ability to recognize a...

Embodiment 2

[0093] Based on single-step multivalent capture of exosomes and amplified fluorescent signal detection

[0094] The design of the detection probe: the modification of biotin on H2 was changed to the modification of fluorescent group, and the rest of the sequences were kept the same.

[0095] Figure 10 It is a flow chart of the principle of single-step multivalent capture of exosomes and fluorescence amplification detection, using fluorescence imaging to verify target recognition, multivalent capture and signal amplification output in one step. FNAs / apt with a concentration of 100nM (final concentration), H1 and H2-biotin were added to 10 10 In a target exosome (total volume 100 μL), after the chain hybridization reaction is initiated, each chain product has a multi-branched single-chain structure. With the aid of the blocker probe, a single-step multivalent The exosomes were captured, and the gold flakes were imaged by a fluorescence microscope to output corresponding fluor...

Embodiment 3

[0098] Single-step multivalent capture and amplified detection of exosomes in serum

[0099] When detecting exosomes in serum, DNase in serum will degrade the recognition probe and reduce the detection efficiency of biological targets. The stability of needles and the efficiency of binding to exosomes. In addition, the efficiency of the hybridization chain reaction is reduced due to the interference of a large number of non-target biomolecules and the reduced concentration of salt ions required for hybridization. In order to improve the hybridization chain reaction efficiency in serum, 25 μL 2*SPSC buffer solution was added to 75 μL serum. There is a large amount of albumin in the serum, which can play a blocking role, prevent the adsorption of horseradish peroxidase on the electrode interface, and reduce the background signal. comparable detection sensitivity.

[0100] Figure 12 The result is that the number of exosomes in the serum is directly proportional to the output...

technical field

[0001] The present invention relates to the technical field of biological detection, in particular to a biological target molecular detection method based on multivalent capture and amplified output signals and its application in liquid biopsy for exosomes, drugs / biological small molecules, DNA, RNA, metal ions in organisms, Precise analytical detection in antigens, antibodies, enzymes and cells. Background technique

[0002] Exosomes are an important carrier of intercellular communication. Exosomes secreted by tumor cells are closely related to the occurrence and development of tumors, immune escape, and the establishment of microenvironment. Exosomes play an important role in the treatment and diagnosis of tumors. The diagnostic research of exosomes is still in the early clinical stage. In recent years, the enrichment detection research based on exosomes has become one of the key areas of liquid biopsy. Exosomes secreted by tumor tissues carry abundant tum...

Examples

Embodiment 1

[0073] Detection of exosomes based on single-step multivalent capture and amplification

[0074] 1. Recognition probe design: design 4 probes A, B, C, and D that make up the nucleic acid framework, wherein A, B, C, and D contain sequences that hybridize with each other to form the nucleic acid framework structure FNAs / apt;

[0075] According to the detection of epithelial cell adhesion molecules on the surface of the target breast cancer-associated exosome membrane, A includes the nucleic acid aptamer sequence Aapt that can specifically recognize and bind epithelial cell adhesion molecules, and B includes Ba, where the full or partial sequence of Ba can Crossed with Aapt. DNA three-dimensional nanostructures such as nucleic acid frameworks have a certain ability to resist enzymatic degradation in complex biological environments, which is conducive to improving the stability of the carried recognition probes in complex biological samples and improving the ability to recognize a...

Embodiment 2

[0093] Based on single-step multivalent capture of exosomes and amplified fluorescent signal detection

[0094] The design of the detection probe: the modification of biotin on H2 was changed to the modification of fluorescent group, and the rest of the sequences were kept the same.

[0095] Figure 10 It is a flow chart of the principle of single-step multivalent capture of exosomes and fluorescence amplification detection, using fluorescence imaging to verify target recognition, multivalent capture and signal amplification output in one step. FNAs / apt with a concentration of 100nM (final concentration), H1 and H2-biotin were added to 10 10 In a target exosome (total volume 100 μL), after the chain hybridization reaction is initiated, each chain product has a multi-branched single-chain structure. With the aid of the blocker probe, a single-step multivalent The exosomes were captured, and the gold flakes were imaged by a fluorescence microscope to output corresponding fluor...

Embodiment 3

[0098] Single-step multivalent capture and amplified detection of exosomes in serum

[0099] When detecting exosomes in serum, DNase in serum will degrade the recognition probe and reduce the detection efficiency of biological targets. The stability of needles and the efficiency of binding to exosomes. In addition, the efficiency of the hybridization chain reaction is reduced due to the interference of a large number of non-target biomolecules and the reduced concentration of salt ions required for hybridization. In order to improve the hybridization chain reaction efficiency in serum, 25 μL 2*SPSC buffer solution was added to 75 μL serum. There is a large amount of albumin in the serum, which can play a blocking role, prevent the adsorption of horseradish peroxidase on the electrode interface, and reduce the background signal. comparable detection sensitivity.

[0100] Figure 12 The result is that the number of exosomes in the serum is directly proportional to the output...

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