▎ 摘　　要

NOVELTY - Combination of reagents comprises first detection probe formed by coupling first single-chain DNA and first antibody, where first single-stranded DNA comprises first pairing sequence and second pairing sequence, and first antibody is capable of specifically binding to a first epitope of the protein of interest. USE - Combination of reagents is used in target protein detection kit and target protein detection system for detecting protein of interest (all claimed). ADVANTAGE - The first antibody and second antibody are adopted to form a double-antibody sandwich structure with the target protein when a solution to be detected contains target protein, and complementary pairing of a first single-chain DNA, a second single-chain DNA and a third single-chain DNA enables an acceptor fluorescent molecule and a donor fluorescent molecule to generate fluorescence resonance energy transfer, so that the acceptor fluorescent molecule is excited to emit fluorescence, and the content of the target protein is calculated through the fluorescence intensity. The detection method is simple, small in background interference, high in sensitivity and small in measurement error. DETAILED DESCRIPTION - Combination of reagents comprises first detection probe formed by coupling first single-chain DNA and first antibody, where first single-stranded DNA comprises first pairing sequence and second pairing sequence, and first antibody is capable of specifically binding to a first epitope of the protein of interest. The second detection probe is formed by sequentially coupling a second antibody, a second single-stranded DNA and a receptor fluorescent molecule, where second single-stranded DNA has a third pairing sequence and a fourth pairing sequence, the third pairing sequence is complementary to the second pairing sequence, and second antibody is capable of specifically binding to a second antigenic epitope of the protein of interest. The third detection probe is formed by coupling donor fluorescent molecules and third single-stranded DNA, where third single-stranded DNA comprises a fifth pairing sequence and a sixth pairing sequence, fifth pairing sequence is complementary to the fourth pairing sequence, and sixth pairing sequence is complementary to the first pairing sequence. The donor fluorescent molecule emits first fluorescence under the condition that the donor fluorescent molecule is oxidized by an oxidizing agent, and the first fluorescence excites the acceptor fluorescent molecule to emit second fluorescence under the condition that the first single-stranded DNA, the second single-stranded DNA and the third single-stranded DNA are matched with each other, so that the content of the target protein is obtained based on the intensity of the second fluorescence. A fourth detection probe comprises antioxidant for inhibiting the first fluorescence from emitting by the donor fluorescent molecule. INDEPENDENT CLAIMS are included for: (1) a method for detecting a target protein, which involves: (a) adding first, second, third and fourth detection probe into solution to be detected, and mixing to form sample to be detected, where first detection probe is formed by coupling first single-chain DNA and first antibody, second detection probe is formed by sequentially coupling second antibody, second single-chain DNA and acceptor fluorescent molecule, and third detection probe is formed by coupling a donor fluorescent molecule and a third single-chain DNA, using first antibody, target protein and second antibody to form double-antibody sandwich structure under the condition that the solution to be detected comprises target protein, using first, second and third single-chain DNA to form neck ring structure, and positioning donor fluorescent molecule and acceptor fluorescent molecule on the same side of the neck ring structure; (b) removing the fourth detection probe for inhibiting the donor fluorescent molecule from getting oxidized from the sample to be detected, adding an oxidant for oxidizing the donor fluorescent molecule to emit first fluorescence, and collecting second fluorescence based on the maximum emission wavelength of the acceptor fluorescent molecule; (c) judging that the target protein is not contained in the solution to be detected under the condition that the second fluorescence is not collected, and obtaining the content of the target protein in the solution to be detected based on the intensity of the second fluorescence based on a functional relation between the fluorescence intensity and the protein content under the condition that the second fluorescence is collected; (2) a target protein detection kit, which comprises first container storing a conjugate of first single-stranded DNA and first antibody, second container storing a conjugate of second antibody, second single-stranded DNA, and acceptor fluorescent molecule, where first antibody and second antibody forms double-antibody sandwich structure with a target protein under the condition that the target protein exists, third container storing a conjugate of donor fluorescent molecule and third single-stranded DNA, where first, second and third single-stranded DNA forms a neck ring structure under the condition of forming double-antibody sandwich structure, and donor fluorescent molecule and acceptor fluorescent molecule excite the acceptor fluorescent molecule to emit second fluorescence when being positioned on the same side of the neck ring structure, so that content of target protein is obtained based on intensity of second fluorescence, fourth container storing an antioxidant capable of inhibiting oxidation of the donor fluorescent molecule, and fifth container storing an oxidizing agent capable of oxidizing the donor fluorescent molecule to emit a first fluorescence; and (3) a target protein detection system, which comprises reaction container provided with an accommodating chamber capable of accommodating a solution to be detected, micro-injection pump communicated with the accommodating cavity through an injection pipeline and injects a mixture of the first detection probe, the second detection probe, the third detection probe and the fourth detection probe into the accommodating cavity, optical filter arranged on the emergent light path of the first fluorescence and allows the second fluorescence with the same wavelength as the maximum emission wavelength of the acceptor fluorescent molecules to penetrate through, optical signal detection module arranged on an emergent light path of the first fluorescence and is positioned at the downstream side of the optical filter, and the optical signal detection module acquires the second fluorescence transmitted by the optical filter, and the calculation module for converting the second fluorescence into a digital signal and obtaining the content of the target protein in the solution to be detected based on a functional relation between fluorescence intensity and protein content.