Signal amplification system for density-controllable biotin deposition and application of signal amplification system

Abstract

The invention relates to a signal amplification system for density-controllable biotin deposition and application thereof. The core of the system is that the biotinylated tyramine derivative is included, the general formula of the biotinylated tyramine derivative is Biotin-S1-Tyr-Linker-R, S1 is a stable spacer arm which cannot be cracked, Linker is a quick response type degradable connecting arm, and R is a steric hindrance adjusting group. After the molecule is covalently anchored to a target spot through tyramine (Tyr) free radical reaction, Linker breaks under a mild condition, only a steric hindrance group R is removed, and a signal core formed by biotin and tyramine is completely retained in a solid phase. The design can realize accurate control of biotin deposition density and significant inhibition of background signals, is beneficial to control of inter-batch variable coefficients, improves detection sensitivity and detection linear range, and provides an optimal scheme for high-sensitivity quantitative immunodetection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a modified biotinylated tyramine derivative for tyramine signal amplification (TSA) technology and its applications. Background Technology

[0002] Tyramine signal amplification (TSA) is a highly sensitive signal amplification technique widely used in immunoassay methods such as immunohistochemistry, immunofluorescence, and in situ hybridization. Its basic principle is that horseradish peroxidase, in the presence of hydrogen peroxide, catalyzes the conversion of tyramine molecules labeled with reporter molecules (such as fluorescent dyes, biotin, or enzymes) into highly reactive free radical intermediates. These intermediates can covalently bind to tyrosine residues of proteins surrounding the antigen-antibody binding site on a solid-phase support, thereby achieving a large-scale deposition of the signal at the original site. Due to its high sensitivity, TSA technology has been widely used in the field of immunoassay.

[0003] Despite the extremely high sensitivity of TSA technology, several inherent limitations hinder its further performance improvement in practical applications. First, steric hindrance is a critical issue. Due to the highly efficient signal deposition process, reporter molecules (such as fluorescent dyes, biotin, and enzymes) are labeled too densely in localized areas. When subsequent detection complexes (such as streptavidin-alkaline phosphatase or streptavidin-horseradish peroxidase complexes) are used, these large complexes, due to their physical size limitations, cannot effectively approach and bind to each deposited biotin molecule. This results in signal amplification efficiency failing to reach the theoretical maximum, leading to signal saturation, which significantly reduces the linear detection range for quantitative assays. Second, diffusion artifacts are another major challenge. Highly reactive tyramine radicals, after generation, may diffuse to areas far from the original antigen-antibody binding site if they fail to covalently bind to nearby tyrosine residues in time. This leads to blurred signal localization, reduced resolution, and potentially false positives.

[0004] Existing technologies mitigate these problems by optimizing tyramine reaction time and concentration, and enhancing blocking. However, these methods often involve trade-offs between sensitivity, resolution, and signal-to-noise ratio, failing to fundamentally solve the problem. Furthermore, existing technologies address steric hindrance by adding a long biotin arm. This involves connecting an extended carbon chain or polyethylene glycol (PEG) spacer arm between a biotin molecule and its reactive group (such as NHS ester). This extra "arm" supports the biotin molecule from the surface of the labeled molecule, ensuring its full exposure to the solution, thereby reducing steric hindrance and improving detection sensitivity. This allows larger avidin / streptavidin molecules to bind to biotin more easily and efficiently. However, while the long biotin arm can improve steric hindrance to some extent, it still cannot solve the problem of tyramine radical diffusion, which is particularly noticeable with high-density deposition signals. The sensitivity and resolution of tyramine signal amplification techniques remain limited by steric hindrance and diffusion shadowing, leading to a reduced linear detection range and decreased signal resolution.

[0005] Therefore, there is an urgent need in this field for a new TSA technology solution that can overcome spatial steric hindrance limitations while also possessing high positioning accuracy and low background noise. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a signal amplification system for controllable density biotin deposition and its application in chemiluminescence immunoassay, achieving precise control of deposition density, significant suppression of background signals, and a comprehensive improvement in detection performance.

[0007] In a first aspect, the present invention provides a signal amplification system for controllable density biotin deposition, the system comprising: (1) Horseradish peroxidase-labeled detection antibody serves as the active core for catalyzing the deposition of tyramine derivatives, providing an initial catalytic site for signal amplification; (2) Biotinylated tyramine derivatives, the core functional molecules of the system, have the general formula: Biotin--S1--Tyr--Linker--R The structural units are defined as follows: Biotin: As a final reporter molecule, it is used to specifically bind to the streptavidin-labeled enzyme complex and initiate signal output; Tyr (tyramine group): under HRP / H2O2 catalysis, a highly active free radical intermediate is generated, which realizes the covalent anchoring of the molecule around the immune complex; S1 is a non-degradable stable spacer arm, containing 4-12 ethylene glycol units of polyethylene glycol, specifically 4, 5, 6, 7, 8, 9, 10, 11, or 12 ethylene glycol units of polyethylene glycol. Linker is a fast-response biodegradable linker arm selected from o-hydroxybenzoate, p-hydroxybenzoate, carbonate or carbamate structures; R is a steric hindrance regulating group with a molecular weight of not less than 1000 Da; more preferably, it is a linear or branched polyethylene glycol or a low-branched polysaccharide with a molecular weight of 1000-5000 Da, specifically 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 4500 Da, or 5000 Da; (3) Streptavidin-labeled enzyme complex, used to specifically bind exposed biotin molecules to achieve signal cascade amplification.

[0008] According to an embodiment of the present invention, the signal amplification system further includes: (4) Free radical scavenger, which can quench tyramine free radicals that diffuse into the solution bulk or far from the specific binding site, inhibit non-specific deposition, and does not affect the efficient deposition of the target site.

[0009] (5) Surface pre-sealing solution, containing 0.5%-2% bovine serum albumin and 0.1-0.5M mannitol or trehalose.

[0010] According to an embodiment of the present invention, the free radical scavenger includes catechol, gallic acid, or ascorbic acid derivatives, with a concentration range of 0.0005%-0.002% (w / v), specifically 0.0005% (w / v), 0.001% (w / v), 0.0015% (w / v), or 0.002% (w / v).

[0011] According to an embodiment of the present invention, the surface pre-sealing solution is preferably 1% bovine serum albumin and 0.3M mannitol or trehalose.

[0012] Secondly, the present invention provides a method for performing chemiluminescent immunoassay using the above-mentioned signal amplification system, comprising the following steps: (1) Coating: The capture antibody is immobilized on a solid support; (2) Antigen binding: Add the sample to be tested and incubate to form a capture complex; (3) Detection of antibody binding: Add HRP-labeled detection antibody and incubate to form a sandwich immune complex; (4) Controlled density deposition: After washing, a deposition solution containing the biotinylated tyramine derivative and H2O2 is added to deposit the biotinylated tyramine derivative around the immune complex. (5) Removal of steric hindrance groups: After washing, incubate to break the Linker, remove the steric hindrance group R, and expose the Biotin--S1--Tyr signal core; (6) Signal labeling: Add streptavidin-alkaline phosphatase complex and incubate for 5-10 minutes; (7) Signal detection: After washing, alkaline phosphatase chemiluminescent substrate is added for detection and reading; According to an embodiment of the present invention, the concentration of the biotinylated tyramine derivative used in the deposition solution is 10-50 μg / ml, specifically 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, or 50 μg / ml.

[0013] According to the embodiments of the present invention, the concentration of H2O2 used is 0.01%-0.05%, specifically 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%.

[0014] According to embodiments of the present invention, the Linker can break down at pH 8-10 and 35-38°C, preferably in a pH 9.5 Tris-HCl buffer solution under constant temperature incubation conditions at 37°C.

[0015] According to the embodiments of the present invention, the lowest detection limit can reach 0.0051 pg / ml, and the linear range can cover 0-2500 pg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects: Existing TSA technologies, due to the small size of tyramine molecules, tend to deposit as many reporter molecules as possible per unit area to achieve high signal strength, thus preventing the subsequent large-molecule detection complex from reaching them. This invention fundamentally changes the molecular design by introducing a removable steric hindrance group R. During the signal deposition stage, each tyramine molecule carries a large R group. These R groups generate steric repulsion between each other during deposition, creating a "physical no-go zone" around each deposition point, naturally preventing other tyramine-biotin-R molecules from depositing at close range. This intermolecular repulsion forces the deposition events to be spatially uniformly distributed, actively preventing excessive crowding of biotin at the nanoscale from the very beginning of deposition. This creates a prerequisite for the efficient binding of the subsequent detection complex, breaking through the theoretical ceiling of signal saturation caused by disordered and dense deposition in traditional TSA.

[0017] Because excessive biotin density was avoided during the deposition stage, and the binding channels were thoroughly cleared by removing the R group before detection, the subsequently added streptavidin-enzyme complex could unimpededly approach and bind to each biotin molecule at its pre-designated location. This significantly improved the binding efficiency of the reporter molecule, making the relationship between the final detection signal and the antigen quantity more linear, and significantly enhancing the sensitivity and dynamic range of the detection.

[0018] The large R group not only inhibits dense intermolecular deposition but also effectively restricts the diffusion of tyramine radicals themselves. Its steric hindrance reduces the diffusion rate and range of the radicals, more strictly confining their reactivity to the very short range of the horseradish peroxidase active site. A small number of tyramine radicals that diffuse into the bulk solution or far from their specific binding sites are quenched by the added radical scavenger. This synergistic effect significantly reduces signal diffusion and localization bias caused by long-range radical diffusion, thus lowering the background signal.

[0019] The entire technical process, including the removal of steric hindrance groups, employs mild biochemical conditions and is seamlessly compatible with existing chemiluminescence immunoassay platforms. Attached Figure Description

[0020] Figure 1 This is the pyrolysis kinetic curve of target product 1 in verification example 1.

[0021] Figure 2 This is the RLU curve of NFL calibrator concentration versus test light value in Example 6. Detailed Implementation

[0022] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0023] Example 1: Synthesis of Biotinylated Tyramine Derivative Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG2000 1. Synthetic route This embodiment employs a stepwise synthesis strategy, sequentially preparing the intermediate Biotin-PEG4-Tyr and the activated intermediate PEG2000-o-hydroxybenzoate-active ester, and then obtaining the target product Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG2000 via a coupling reaction. The reaction route is shown below: (1) PEG2000-OH + p-nitrophenyl chloroformate → PEG2000-O-CO-ONp (2) Biotin-PEG4-Tyr + PEG2000-O-CO-ONp → Biotin-PEG4-Tyr-O-CO-PEG2000 (i.e., Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG2000) 2. Specific experimental steps Step 1: Preparation of PEG2000-o-hydroxybenzoate-active ester Weigh PEG2000-OH (polyethylene glycol with a molecular weight of 2000, 0.25 mmol) and dissolve it in 10 mL of anhydrous dichloromethane (DCM). Add salicylic acid (0.30 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 0.30 mmol), and a catalytic amount of 4-dimethylaminopyridine (DMAP, 0.02 mmol). Stir magnetically at room temperature (20-25℃) for 12 hours. After the reaction, filter. Concentrate the filtrate under reduced pressure and pour it into 50 mL of ice-cold diethyl ether to precipitate. Collect the solid by centrifugation, wash with diethyl ether 2-3 times, and dry under vacuum to obtain a white waxy solid HO-PEG2000-salicylic acid ester. Dissolve the obtained solid in 10 mL of anhydrous acetonitrile, add N,N'-disuccinimidyl carbonate (DSC, 0.24 mmol), and a catalytic amount of DMAP. Stir the reaction at room temperature in the dark for 4 hours. After the reaction, 50 mL of ice-cold diethyl ether was added to precipitate the product. The product was collected by centrifugation and vacuum dried to obtain PEG2000-o-hydroxybenzoate-NHS intermediate.

[0024] Step 2: Synthesis of the target product Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG2000 Weigh out Biotin-PEG4-Tyr (0.30 mmol of biotinylate purchased from Qiyue Biotechnology, where PEG4 refers to the polyethylene glycol chain) and dissolve it in 10 mL of anhydrous N,N-dimethylformamide (DMF). Add DMAP (0.03 mmol, 0.1 eq) and DIPEA (0.45 mmol, 1.5 eq), then slowly add PEG2000-o-hydroxybenzoate-NHS (0.33 mmol) obtained in step two to the reaction system. Under a nitrogen atmosphere, stir the reaction at 30-35 °C in the dark for 16-24 hours. After the reaction is complete, pour the system into 50 mL of ice-cold diethyl ether, where a white precipitate forms. The precipitate was collected by centrifugation and washed three times with cold ether. It was then dissolved in an appropriate amount of deionized water, and small molecule impurities were removed by dialysis using a dialysis bag (molecular cutoff 1000 Da). After lyophilization, the target product Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG2000 was obtained as a white powder.

[0025] Example 2: Synthesis of Biotin-PEG4-Tyr-carbonate-PEG1000, a biotinylated tyramine derivative 1. Synthetic route In this embodiment, p-nitrophenyl chloroformate is used to activate the terminal hydroxyl group of PEG1000 into a highly reactive carbonate intermediate. Subsequently, this reactive intermediate undergoes a nucleophilic substitution reaction with the phenolic hydroxyl group of Biotin-PEG4-Tyr to form the carbonate linker arm in the target product. The reaction route is as follows: (1) PEG1000-OH + p-nitrophenyl chloroformate → PEG1000-O-CO-O-Ph-pNO2 (2) Biotin-PEG4-Tyr + PEG1000-O-CO-O-Ph-pNO2 → Biotin-PEG4-Tyr-O-CO-O-PEG1000 (i.e., Biotin-PEG4-Tyr-carbonate-PEG1000) 2. Specific experimental steps Step 1: Preparation of PEG1000-carbonate-p-nitrophenol ester (active intermediate) In a dry 100 mL round-bottom flask, PEG1000-monomethyl ether (average molecular weight 1000 Da, 1.0 mmol) was dissolved in 20 mL of anhydrous dichloromethane (DCM), and the solution was cooled to 0–5 °C in an ice-water bath. While stirring, 10 mL of anhydrous DCM solution containing p-nitrophenyl chloroformate (p-NPC, 1.2 mmol) and a catalytic amount of anhydrous pyridine (0.1 mmol) was slowly added dropwise to the system. After the addition was complete, the ice bath was removed, and the reaction system was allowed to warm naturally to room temperature (20–25 °C), and the reaction was continued with stirring under nitrogen protection for 6–8 hours. The reaction progress could be monitored by thin-layer chromatography (developing solvent: ethyl acetate / petroleum ether = 1:1). After the reaction was complete, the reaction solution was washed twice with 50 mL of ice water, and then once with saturated saline solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure at 30°C to obtain a pale yellow viscous oily substance, PEG1000-carbonate-p-nitrophenol ester, which could be used directly in the next reaction without further purification.

[0026] Step 2: Synthesis of the target product Biotin-PEG4-Tyr-carbonate-PEG1000 The active intermediate PEG1000-carbonate-p-nitrophenol ester (approximately 1.0 mmol) obtained in the previous step was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF). In another container, Biotin-PEG4-Tyr (0.9 mmol, purchased from Qiyue Biotechnology) was dissolved in 10 mL of anhydrous DMF, and N,N-diisopropylethylamine (DIPEA, 1.8 mmol, 2.0 eq) was added. Under ice bath cooling and stirring, the DMF solution of Biotin-PEG4-Tyr was slowly added dropwise to the DMF solution of the active intermediate. After the addition was complete, the ice bath was removed, and the reaction was carried out at room temperature in the dark for 16–20 hours under nitrogen protection.

[0027] After the reaction was complete, the reaction mixture was added dropwise to 200 mL of rapidly stirred, ice-cold anhydrous diethyl ether, resulting in the formation of a white flocculent precipitate. After standing, the supernatant was discarded, and the precipitate was washed three times with cold diethyl ether. The obtained crude product was dissolved in an appropriate amount of ultrapure water, and dialyzed against ultrapure water at 4°C for 48 hours (with water changes 6-8 times) using a dialysis bag with a molecular weight cutoff of 500 Da to thoroughly remove small molecule impurities such as DMF, DIPEA, and p-nitrophenol. Finally, the solution in the dialysis bag was freeze-dried to obtain the white powdered target product, Biotin-PEG4-Tyr-carbonate-PEG1000.

[0028] Example 3: Synthesis of Biotin-PEG4-Tyr-aminobenzoate-PEG5000, a biotinylated tyramine derivative 1. Synthetic route This synthesis employs a more efficient and direct strategy, utilizing the bifunctional linker N,N'-disuccinimidyl carbonate (DSC) to activate the amino group at the end of linear PEG5000 into a urethane-NHS active ester intermediate, which then reacts with the phenolic hydroxyl group of Biotin-PEG4-Tyr to form a urethane linker arm in one step.

[0029] The reaction route is as follows: (1) PEG5000-NH2+ DSC → PEG5000-NH-CO-O-NHS (2) Biotin-PEG4-Tyr + PEG5000-NH-CO-O-NHS → Biotin-PEG4-Tyr-O-CO-NH-PEG5000 (i.e., Biotin-PEG4-Tyr-carbamate-PEG5000) 2. Specific experimental steps Step 1: Preparation of bPEG5000-isocyanate (bPEG5000-NCO) intermediate Step 1: Preparation of the intermediate PEG5000-carbamate-NHS active ester (PEG5000-NH-CO-O-NHS) In a dry 50 mL round-bottom flask, terminal amino-modified polyethylene glycol (PEG5000-NH2, average molecular weight 5000 Da, 0.1 mmol) was dissolved in 10 mL of anhydrous dichloromethane (DCM). N,N'-disuccinimidyl carbonate (DSC, 0.12 mmol) and a catalytic amount of anhydrous pyridine (0.01 mmol) were added to the solution. The reaction flask was cooled in an ice-water bath, and then the reaction was stirred at 0–5 °C for 2 hours under nitrogen protection, followed by a further reaction at room temperature (20–25 °C) in the dark for 4–6 hours. After the reaction was complete, the reaction solution was added dropwise to 100 mL of vigorously stirred, ice-cold anhydrous diethyl ether, precipitating a white solid. The solid was collected by centrifugation and washed three times with cold diethyl ether. The obtained solid was dried overnight in a vacuum desiccator to obtain a white powdery intermediate PEG5000-NH-CO-O-NHS.

[0030] Step 2: Synthesis of the target product Biotin-PEG4-Tyr-carbamate-PEG5000 In another dry 50 mL round-bottom flask, Biotin-PEG4-Tyr (0.12 mmol, purchased from Qiyue Biotechnology) was dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (DIPEA, 0.18 mmol, 1.5 eq) was added. Under ice bath cooling and stirring, the PEG5000-NH-CO-O-NHS intermediate prepared in step one (0.10 mmol based on amino groups) was dissolved in 5 mL of anhydrous DMF and slowly added dropwise to the above reaction system. After the addition was complete, the ice bath was removed, and the reaction was carried out at room temperature in the dark for 18–24 hours under nitrogen protection.

[0031] After the reaction was complete, the reaction mixture was added dropwise to 200 mL of ice-cold methyl tert-butyl ether (MTBE) under vigorous stirring, producing a large amount of white precipitate. After standing, the supernatant was discarded, and the precipitate was washed three times with MTBE (50 mL each time). The resulting crude product was dissolved in an appropriate amount of ultrapure water, and dialyzed against ultrapure water at 4°C for 48 hours (changing the water 6-8 times during this period) using a dialysis bag with a molecular weight cutoff of 3500 Da to thoroughly remove small molecule impurities such as DMF, DIPEA, and N-hydroxysuccinimide. Finally, the solution in the dialysis bag was filtered through a 0.22 μm filter membrane and freeze-dried to obtain a white solid, which was the target product Biotin-PEG4-Tyr-carbamate-PEG5000.

[0032] Example 4: Synthesis of Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG750, a biotinylated tyramine derivative The synthesis of the target compound Biotin-PEG4-Tyr-o-hydroxybenzoate-PEG750 was performed in the same manner as step two of Example 1, except that PEG750-OH was used instead of PEG2000-OH as the starting material. The product was obtained after dialysis purification.

[0033] Verification Example 1: Linker Decomposability Verification The linkers of the target products in Examples 1-4 are all linked by ester bonds, and their chemical cleavage mechanisms are common. Therefore, the target product of Example 1 was selected as a representative for product verification and functional testing. The verification results can be extended to the target products of Examples 2, 3, and 4.

[0034] Target product 1 (50 μg / mL) was incubated in pH 9.5 Tris-HCl buffer at 37°C. Samples were taken at different time points (0, 5, 10, 15, 20, 30 min) and immediately terminated with trifluoroacetic acid (TFA) for HPLC analysis. The remaining percentage content was expressed as the ratio of the HPLC peak area (A) of target product 1 to the initial peak area (A0) (A / A0), and a fragmentation kinetic curve was plotted. Assuming first-order reaction kinetics, the reaction rate constant (k) and half-life (t1 / 2) were calculated by linearly fitting the relationship between ln(A / A0) and t.

[0035] The results show that the fitted equation is ln(A / A0) = -0.1471t + 4.6257, R0 2 =0.997, rate constant K=0.147, half-life t 1 / 2 =ln(2) / k=4.72min, indicating that the connecting arm is a fast-response degradable connecting arm that can basically complete the breakage (>90%) within 10-15 minutes, meeting the operational requirements of rapid incubation in immunoassay.

[0036] Table 1. Pyrolysis kinetic data of target product 1 Verification Example 2: Biotin Binding Capacity Verification The target products from Examples 1 and 4 were used to prepare samples before lysis (dissolved in pH 7.4 PBS) and after lysis (incubated in pH 9.5 Tris-HCl buffer at 37°C for 30 minutes to completely break the linker arms, followed by neutralization). All samples were adjusted to the same theoretical biotin molar concentration (50 µM).

[0037] The HABA (4'-hydroxyazobenzene-2-carboxylic acid) / streptavidin competitive colorimetric method was used for determination. Each sample was mixed with SA-HABA working solution, and after the reaction reached equilibrium at room temperature, the absorbance decrease (ΔA500) was measured at 500 nm. A standard curve was constructed using known concentrations of free biotin, and the apparent effective biotin concentration in each sample was calculated accordingly. The result was calculated using the formula: Biotin availability (%) = (apparent concentration / theoretical concentration) × 100%. This value directly reflects the potential binding capacity of biotin to streptavidin.

[0038] Table 2. Results of HABA assay for different biotinylated tyramine derivatives The results showed that the biotin accessibility of the target product in Example 1 increased by approximately 6.3 times after cleavage, reaching a level comparable to that of free biotin. This indicates that the steric hindrance group was successfully removed after Linker cleavage, and the exposed biotin could fully participate in streptavidin binding. In Example 4, the target product R had a molecular weight of less than 1000 Da, and the biotin accessibility increased by only 1.3 times after cleavage, showing a significant difference compared to the control group. A possible reason is that the low molecular weight R group has insufficient space volume, resulting in weak intermolecular repulsion and an inability to effectively control the biotin deposition density during the deposition stage, leading to localized overcrowding of biotin molecules. Therefore, in this invention, the molecular weight of the R group is not less than 1000 Da.

[0039] Example 5: Detection and performance comparison of neurofilament light chain protein (NFL) in chemiluminescent immunoassay. 1. Experimental Materials and Grouping Target protein for detection: human neurofilament light chain protein.

[0040] Solid support: 1.5µm carboxyl magnetic beads, purchased from JSR.

[0041] Capture antibody: Anti-NFL monoclonal antibody, purchased from Hytest.

[0042] Detection antibody: Anti-NFL monoclonal antibody, purchased from Hytest and HRP-labeled.

[0043] 2. Experimental steps: (1) JSR 1.5µm carboxyl magnetic beads were used. The NFL capture antibody was conjugated to the magnetic beads according to the instructions. During the conjugation process, the magnetic beads were blocked with 1% BSA solution. After the magnetic beads were conjugated, they were diluted to a working concentration of 0.2mg / ml with PBS (0.05M, containing 1% BSA and 0.3M trehalose). (2) HRP-labeled detection antibody: The detection antibody was purchased from Hytest. HRP was labeled onto the antibody according to the instructions. The working concentration of the labeled antibody was 0.1 ug / ml. (3) Prepare standards. The standards are NFL antigen concentration gradient solutions. The antigens were purchased from Beijing Deoping Biotechnology and diluted with PBS (0.05M, pH=7.4) to 0 pg / ml, 0.05 pg / ml, 0.25 pg / ml, 1 pg / ml, 5 pg / ml, 20 pg / ml, 100 pg / ml, 500 pg / ml and 2500 pg / ml.

[0044] (4) Preparation of biotinylated tyramine working solution: Dilute the biotinylated tyramine of Example 1 to a concentration of 20 μg / ml with PBS (0.01M, pH=6), and then add 0.01wt / %H2O2 and 0.0015% (w / v) propyl gallate (free radical scavenger). (5) Preparation of dissociation working solution: Prepare a 0.1M pH=9.5 Tris-HCl buffer solution as the dissociation working solution. (6) Optical value testing was performed using a BEION T100C fully automated chemiluminescence immunoassay analyzer. The reaction mode was as follows: 50 μL of magnetic bead working solution was mixed with 100 μL of standard and 50 μL of HR-labeled detection antibody and incubated at 37°C for 10 min; the magnetic beads were washed with washing buffer, and then 100 μL of biotinylated tyramine working solution was added and incubated at 37°C for 5 min; the magnetic beads were washed with washing buffer, and then 100 μL of dissociation working solution was added and incubated at 37°C for 20 min; after washing again, streptavidin-alkaline phosphatase complex was added and incubated for 5 min. Finally, after washing, CDP-Star chemiluminescent substrate was added, and the relative optical units were read on the chemiluminescence analyzer.

[0045] Comparative Example 1 The difference between Comparative Example 1 and Example 5 is that the biotinylated tyramine working solution in this example does not contain the trapping agent propyl gallate. Other operations are the same as in Example 5.

[0046] Comparative Example 2 The difference between Comparative Example 2 and Example 5 lies in the biotinylate working solution and the reaction process. In this example, the biotinylate working solution used was commercially available ordinary biotinylate (purchased from Qiyue Biotechnology), which does not contain sterically hindered groups that can be dissociated. No dissociation working solution was needed during the photoluminescence measurement process in this example. The specific reaction mode was as follows: 50 μL of magnetic bead working solution was mixed with 100 μL of standard and 50 μL of HR-labeled detection antibody and incubated at 37°C for 10 min; the magnetic beads were washed with washing buffer, then 100 μL of biotinylated tyramine working solution was added, and the mixture was incubated at 37°C for 5 min; the magnetic beads were washed again with washing buffer, and the streptavidin-alkaline phosphatase complex was added and incubated for 5 minutes. Finally, the mixture was washed, CDP-Star chemiluminescent substrate was added, and the relative photon units were read on a chemiluminescence analyzer.

[0047] Example 6 Each sample was tested three times, and the average luminescence value (RLU) was recorded in the table. Table 3 shows the luminescence value records of the tested standards in Example 5 and Comparative Examples 1-2: Table 3 The results are shown in Table 3 and Figure 2 As shown, at the zero concentration point (0 pg / mL blank sample), the average RLU value of Example 5 was 2350, and the signal-to-noise ratio at the 0.05 pg / mL concentration point was 1.5; the average RLU value of Comparative Example 1 was 3829, and the signal-to-noise ratio at the 0.05 pg / mL concentration point was 1.1; the average RLU value of Comparative Example 2 was 7892, and the signal-to-noise ratio at the 0.05 pg / mL concentration point was 0.99.

[0048] Combining the results of Example 5 and Comparative Example 2, it can be seen that although the conventional biotinylate-tyramine signal amplification process can exhibit a certain signal amplification effect and a relatively high absolute RLU value for low-value samples, the background is relatively high, the signal-to-noise ratio for low-value samples is low, and the linear range is narrow. At low concentrations, the signal increases with the increase of antigen concentration, but after reaching 500 pg / mL, the signal no longer increases, and the RLU value tends to stabilize, i.e., "signal saturation". In contrast, the present invention in Example 5 uses a biotinylated tyramine derivative with a steric hindrance regulating group. Although the absolute luminescence value of low-concentration samples is not as good as that of the conventional biotinylate-tyramine signal amplification process, its background luminescence value is lower, the limit of detection and the signal-to-noise ratio of low-concentration samples are better than the conventional tyramine signal amplification process, and the linear range is wider, covering 0-2500 pg / mL, while the conventional TSA can only cover 0-500 pg / mL.

[0049] Combining the results of Example 5 and Comparative Example 1, the reaction group using a low concentration of free radical scavenger exhibited a lower background light value, indicating that a low concentration of free radical scavenger during the biotinylate deposition process can significantly inhibit the non-specific deposition of tyramine free radicals.

[0050] The above data demonstrate that the present invention (Example 5) has a higher signal-to-noise ratio at low concentrations (1.5 for 0.05 pg / mL vs. 0.99 for conventional TSA); and a significantly reduced background signal (2350 for blank RLU vs. 7892 for conventional TSA). This indicates that the complete system of the present invention can significantly reduce background and improve the signal-to-noise ratio.

[0051] Example 7 Reagent Performance Verification (1) Minimum detection limit The limit of detection (LOD) is defined as the mean signal value (M) of the 0 pg / ml standard plus twice its standard deviation (S). The mean signal value (LLU) is obtained by repeating the 0 pg / ml standard 20 times in the same experiment. A curve equation is obtained by fitting a straight line between the concentration values ​​and LLU values ​​of the 0 pg / ml standard and adjacent standards. Substituting M+2S into this curve equation, the corresponding concentration value is calculated, which is the LOD.

[0052] According to the data in the table above, M+2S=2469.84, and the limit of detection after substituting into the fitted curve is 0.0051pg / ml.

[0053] (2) Precision Precision was tested for standards at concentrations of 1 pg / mL and 100 pg / mL. The specific experimental method involved repeating the tests for both concentrations 1 pg / mL and 100 pg / mL 10 times each. The concentration values ​​were calculated based on the RLU values ​​and the fitted calibration curve. The mean (M) and standard deviation (S) of all 10 data sets were calculated, and the coefficient of variation (CV) was calculated, where CV = S / M * 100%.

[0054] According to the data in the table, the repeatability CV was 5.2% when the standard at a concentration of 1 pg / mL was tested 10 times; the repeatability CV was 3.7% when the standard at a concentration of 100 pg / mL was tested 10 times. The results meet the requirement that the repeatability CV of conventional chemiluminescent reagents is less than 8%, indicating that the results meet the repeatability requirements.

[0055] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the components of the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0056] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0057] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A signal amplification system with controllable density biotin deposition, characterized in that, The system includes: (1) Detection antibody labeled with horseradish peroxidase; (2) Biotinylated tyramine derivatives, which have the general formula: Biotin--S1--Tyr--Linker--R in, S1 is a non-degradable stable spacer arm, containing 4-12 ethylene glycol units of polyethylene glycol; Linker is a fast-response biodegradable linker arm selected from o-hydroxybenzoate, p-hydroxybenzoate, carbonate or carbamate structures; R is a steric hindrance regulating group with a molecular weight of not less than 1000 Da; (3) Streptavidin-labeled enzyme complex.

2. The system according to claim 1, characterized in that, The steric hindrance group R is a linear or branched polyethylene glycol or a low-branched polysaccharide with a molecular weight of 1000-5000 Da.

3. The system according to claim 1, characterized in that, The system also includes: (4) Free radical scavengers, including catechols, gallic acid or ascorbic acid derivatives; (5) Surface pre-sealing solution, containing 0.5%-2% bovine serum albumin and 0.1-0.5M mannitol or trehalose.

4. The system according to claim 3, characterized in that, The concentration range of the free radical scavenger is 0.0005%-0.002% (w / v).

5. The system according to claim 3, characterized in that, The preferred surface pre-sealing solution is 1% bovine serum albumin and 0.3M mannitol or trehalose.

6. A method for chemiluminescent immunoassay using the system described in claims 1-5, characterized in that, The method includes the following steps: (1) The capture antibody was immobilized on a solid support and blocked using a pre-blocking solution; (2) Add the sample to be tested and incubate to form a capture complex; (3) Add HRP-labeled detection antibody and incubate to form a sandwich immune complex; (4) After washing, a deposition solution containing the biotinylated tyramine derivative and H2O2 is added to deposit the biotinylated tyramine derivative around the immune complex. (5) After washing, incubate to break the Linker, remove the steric hindrance group R, and expose the Biotin--S1--Tyr signal core; (6) Add streptavidin-alkaline phosphatase complex and incubate for 5-10 minutes; (7) After washing, add alkaline phosphatase chemiluminescent substrate for detection and reading.

7. The method according to claim 6, characterized in that, The concentration of the biotinylated tyramine derivative used in the deposition solution is 10-50 μg / ml.

8. The method according to claim 6, characterized in that, The concentration of H2O2 used is 0.01%-0.05%.

9. The method according to claim 6, characterized in that, The linker is capable of breaking down at pH 8-10 and 35-38°C, preferably in a pH 9.5 Tris-HCl buffer solution under constant temperature incubation conditions at 37°C.

10. The method according to claim 6, characterized in that, The lowest detection limit is 0.0051 pg / ml.

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