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CN-122017218-A - Signal amplification system capable of controlling density biotin deposition and application thereof

CN122017218ACN 122017218 ACN122017218 ACN 122017218ACN-122017218-A

Abstract

The invention relates to a signal amplification system for controlled density biotin deposition and application thereof. The core of the system is that the system comprises a biotinylation tyramine derivative, the general formula of which is Biotin-S1-Tyr-Linker-R, wherein S1 is a non-cleavable stable spacer arm, linker is a quick response type degradable connecting arm, and R is a steric hindrance regulating group. After the molecule is covalently anchored to a target point through tyramine (Tyr) free radical reaction, linker breaks under mild conditions, only the steric hindrance group R is removed, and the signal core formed by biotin and tyramine is completely reserved in a solid phase. The design can realize the accurate control of the biotin deposition density and the remarkable inhibition of background signals, is favorable for controlling the variation coefficient among batches, improves the detection sensitivity and the detection linear range, and provides a preferred scheme for high-sensitivity quantitative immune detection.

Inventors

  • CUI YUFENG
  • CHEN XIAMIN
  • WANG ZHIGANG

Assignees

  • 上海北昂医药科技股份有限公司

Dates

Publication Date
20260512
Application Date
20251226

Claims (10)

  1. 1. A signal amplification system for controlled density biotin deposition, said system comprising: (1) A horseradish peroxidase-labeled detection antibody; (2) A biotinylated tyramine derivative having the general formula: Biotin--S1--Tyr--Linker--R, Wherein, the S1 is a non-cleavable stable spacer comprising polyethylene glycol of 4-12 ethylene glycol units; linker is a quick response degradable linking arm selected from the group consisting of an o-hydroxybenzoate, a p-hydroxybenzoate, a carbonate or a carbamate structure; R is a steric hindrance regulating group, and the molecular weight is not less than 1000 Da; (3) Streptavidin-labeled enzyme complex.
  2. 2. The system according to claim 1, wherein the sterically hindered group R is a linear or branched polyethylene glycol of molecular weight 1000-5000Da, or a low branched polysaccharide.
  3. 3. The system of claim 1, wherein the system further comprises: (4) Radical scavenger comprising catechol, gallic acid or ascorbic acid derivatives; (5) The surface pre-sealing liquid contains 0.5-2% bovine serum albumin and 0.1-0.5M mannitol or trehalose.
  4. 4. A system according to claim 3, wherein the free radical scavenger concentration is in the range of 0.0005% to 0.002% (w/v).
  5. 5. A system according to claim 3, characterized in that the surface pre-blocking solution is preferably 1% bovine serum albumin with 0.3M mannitol or trehalose.
  6. 6. A method of performing a chemiluminescent immunoassay using the system of claims 1-5 wherein the method comprises the steps of: (1) Fixing the capture antibody on a solid phase carrier, and sealing by using a pre-sealing liquid; (2) Adding a sample to be detected, and incubating to form a capture complex; (3) Adding an HRP-labeled detection antibody, and incubating to form a sandwich immune complex; (4) After washing, adding a deposition solution comprising the biotinylated tyramine derivative and H 2 O 2 to deposit the biotinylated tyramine derivative around the immune complex; (5) After washing, incubating to break Linker, removing steric hindrance group R, exposing Biotin-S1-Tyr signal core; (6) Adding streptavidin-alkaline phosphatase complex, and incubating for 5-10 min; (7) After washing, alkaline phosphatase chemiluminescent substrate was added for detection reading.
  7. 7. The method of claim 6, wherein the biotinylated tyramine derivative is used in the deposition solution at a concentration of 10-50 μg/ml.
  8. 8. The method of claim 6, wherein the H 2 O 2 is used at a concentration of 0.01% to 0.05%.
  9. 9. The method according to claim 6, characterized in that the Linker is capable of cleavage at pH 8-10, 35-38 ℃, preferably in pH 9.5 Tris-HCl buffer, under incubation conditions at 37 ℃.
  10. 10. The method of claim 6, wherein the minimum limit of detection is up to 0.0051pg/ml.

Description

Signal amplification system capable of controlling density biotin deposition and application thereof Technical Field The invention relates to the technical field of biology, in particular to an improved biotinylation tyramine derivative for Tyramine Signal Amplification (TSA) technology and application thereof. Background The Tyramine Signal Amplification (TSA) technology is a high-sensitivity signal amplification technology widely applied to immune detection methods such as immunohistochemistry, immunofluorescence, in situ hybridization and the like. The basic principle is that horseradish peroxidase catalyzes tyramine molecules marked with reporter molecules (such as fluorescent dyes, biotin and enzymes) in the presence of hydrogen peroxide to convert the tyramine molecules into high-activity free radical intermediates, and the intermediates can be covalently bound with protein tyrosine residues around antigen-antibody binding sites on a solid phase carrier, so that a large amount of deposition of signals at original sites is realized. Due to its high sensitivity, TSA technology is widely used in the field of immunodetection. Although TSA technology has extremely high sensitivity, it has several inherent drawbacks in practical applications, which limit further improvement of its performance. First, steric hindrance is a critical issue. Because the signal deposition process is extremely efficient, the reporter molecules (e.g., fluorochromes, biotin, enzymes) are labeled too densely in localized areas. When a large molecular detection complex (such as streptavidin-alkaline phosphatase complex or streptavidin-horseradish peroxidase complex) is used subsequently, the large complex cannot effectively access and bind to each deposited biotin molecule due to physical size limitation, so that the signal amplification efficiency cannot reach the theoretical maximum value, and a signal saturation phenomenon occurs, which greatly reduces the linear detection range for quantitative detection reagents. Second, diffusion artifacts are another major challenge. After formation, highly reactive tyramine radicals, if not covalently bound in time to nearby tyrosine residues, may diffuse to regions remote from the original antigen-antibody binding site, resulting in ambiguous signal localization, reduced resolution, and the potential for false positive signals. The prior art alleviates the problems by optimizing the reaction time, concentration and enhancing the blocking of tyramine, but the methods are always compromise between sensitivity, resolution and signal to noise ratio, and cannot fundamentally solve the problems. In addition, the prior art solves the steric hindrance effect by adding a long arm of biotin, to which a spacer arm such as an extended carbon chain or polyethylene glycol (PEG) is attached between a molecule of biotin and its reactive group (e.g., NHS ester). This extra "arm" "supports the biotin molecule" from the surface of the labeled molecule, allowing it to be fully exposed to the solution, thereby reducing steric hindrance, improving the sensitivity of detection, and allowing the larger avidin/streptavidin molecule to bind biotin more easily and efficiently. However, although biotin long arms can improve steric effects to some extent, it still does not solve the problem of tyramine radical diffusion, which is particularly evident when signals are deposited at high density. The sensitivity and resolution of tyramine signal amplification techniques are still limited by steric hindrance and diffuse shading, resulting in a reduced linear detection range and reduced signal resolution. Therefore, there is an urgent need in the art for a new TSA solution that can break through the steric hindrance constraint, while having both high positioning accuracy and low background noise. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a signal amplification system capable of controlling the density of biotin deposition and an application method thereof in chemiluminescence immunoassay, and realizes accurate regulation and control of the deposition density, remarkable inhibition of background signals and comprehensive improvement of detection performance. In a first aspect, the present invention provides a signal amplification system for controlled density biotin deposition, the system comprising: (1) The horseradish peroxidase-labeled detection antibody is used as an active core for catalyzing deposition of tyramine derivatives, and provides an initial catalytic site for signal amplification; (2) A biotinylated tyramine derivative, a system core functional molecule, having the general formula: Biotin--S1--Tyr--Linker--R, Wherein each structural unit is defined as follows: biotin (Biotin) as a final reporter molecule for specific binding to streptavidin-labeled enzyme complex, initiating signal output; Tyr (tyramine group) generates a high-activity free radical intermediate unde