tyramide signal amplification kit

Abstract

The present application relates to the field of biological detection, and particularly relates to a tyramide signal amplification kit. The tyramide signal amplification kit has higher detection sensitivity and can detect low-expression markers.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically a tyramine signal amplification kit. Background Technology

[0002] In the field of disease diagnosis, the detection and analysis of biomarkers in target cells with high specificity and sensitivity are of great significance. Fluorescence analysis methods have extremely high spatiotemporal resolution, enabling the effective detection of biomolecules such as nucleic acids and proteins in target cells, and have a wide range of biomedical applications. However, the concentrations of certain disease-related biomolecules (such as circulating epithelial cells, circulating endothelial cells, immune cells, and other tumor biomarkers) in organisms are very low, and conventional fluorescence detection techniques cannot accurately analyze them. Therefore, there is still a need to design and construct fluorescent biosensors with better selectivity and higher sensitivity.

[0003] Currently, fluorescent antibodies used for immunofluorescence detection generally have low detection sensitivity, especially when used for fluorescence microscopy scanning analysis. For some samples with low expression levels, conventional fluorescent antibodies are difficult to detect.

[0004] Tyramine signal amplification (TSA) is an enzyme-mediated amplification technique. In the presence of hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the conversion of tyramine into oxygen-reactive free radicals. This intermediate can rapidly covalently bind to proteins or HRP-linked antibodies. A fluorescent dye is then bound to the tyramine molecule, forming a covalent binding site, thereby stably and efficiently enriching the fluorescein around and depositing it onto the target cell, resulting in a geometric amplification of the detection signal. However, the detection sensitivity of the currently used fluorescent substrate TSA-FITC (FITCT) is still relatively low, requiring further improvement. This is mainly because the fluorescence signal intensity of FITC fluorescein itself is insufficient. Other fluoresceins such as Alexa Fluor488, Dylight488, and iFluor488 are available, but these are all imported reagents, resulting in higher costs. Summary of the Invention

[0005] This invention provides a tyramine signal amplification kit with higher detection sensitivity, capable of detecting low-expressed biomarkers.

[0006] On one hand, the present invention provides a tyramine signal amplification kit comprising an enzyme-conjugated antibody and a fluorescent substrate, wherein the fluorescent substrate has a compound of formula I or II or a stereoisomer thereof: (I) or (II); Where R is the dye; Ring A and ring B are each independently aryl, heteroaryl, cycloalkyl, or heterocyclic. E is -O-, -S-, -NH- or -C≡C-; L does not exist, or it is a chain consisting of 1-20 atoms; Each R 1 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; Each R 2 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; R 3 It is either hydroxyl or amino; n can be 0, 1, 2, 3, or 4; m can be 0, 1, 2, 3, or 4.

[0007] In some embodiments, the enzyme is selected from any one of dehydrogenase, oxidase, peroxidase, and oxygenase.

[0008] In some embodiments, peroxidase activity may be associated with one or more portions of the peroxidase bound to one or more binders.

[0009] In some embodiments, the enzyme is any one of horseradish peroxidase, catalase, and superoxide dismutase.

[0010] In some embodiments, the enzyme is horseradish peroxidase.

[0011] In some embodiments, the antibody is one or more of the following: anti-HER2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-HER3 antibody, anti-MUC1 antibody, anti-PD-L1 antibody, anti-EpCAM antibody, anti-EGFR antibody, anti-c-met antibody, anti-FR (folate receptor) antibody, anti-CEA antibody, anti-PMSA antibody, anti-AR-V7 antibody, anti-CK antibody, anti-PLAP antibody, anti-GPC3 antibody, anti-CD31 antibody, anti-CD44 antibody, anti-Vimentin antibody, and anti-cadherin antibody.

[0012] In some embodiments, the kit further includes a blocking agent and a buffer solution.

[0013] In some embodiments, the sealing agent is a peroxide.

[0014] In some embodiments, the peroxide is H2O2.

[0015] In some embodiments, the buffer solution is a PBS solution, a Tris-HCl solution, or a Hepes solution.

[0016] In some embodiments, the buffer solution is a PBS solution containing FBS or BSA, or a Tris-HCl solution or a Hepes solution.

[0017] In some embodiments, the fluorescent substrate is 40-60 parts, the buffer solution is 30-45 parts, the enzyme-conjugated antibody is 15-30 parts, and the blocking solution is 50-70 parts.

[0018] In some embodiments, the kit includes HRP blocking buffer, antibody dilution and blocking buffer, tyrosine luminescent buffer, HRP polymer-conjugated secondary antibody, a fluorescent substrate having a compound of formula I or II or a stereoisomer thereof, and DAPI.

[0019] In some embodiments, the dye is AF350, AF405, AF425, AF430, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF640, AF647, AF660, AF680, CF405M, CF450, CF488A, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF680, CF680R, CF750, CF770, CF790, or CF820; Ring A and ring B are each independently phenyl, naphthyl, pyrrolyl, furanyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyranonel, indolyl, benzofuranyl, benzothiophene, quinolinyl, isoquinolinyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothiophene, tetrahydropyranyl, piperidinyl, tetrahydrothiophene, dioxane, piperazinyl, hexahydropyrazinyl, or morpholinyl. Each R 1 and R 2Independently, it is hydroxyl, amino, F, Cl, Br, methyl, ethyl, propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylamino, ethylamino, propylamino, isopropylamino, butylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclopentylmethyl, oxecyclopropyl, azicyclopropyl, oxecyclobutyl, azicyclobutyl, tetrahydrofuranyl, tetrahydropyrrolyl, piperidinyl, tetrahydropyranyl, oxecyclopropylmethyl, azicyclopropylmethyl, oxecyclobutylmethyl, azicyclobutylmethyl, oxecyclopropylethyl, azicyclopropylethyl, oxecyclobutylethyl, or azicyclobutylethyl.

[0020] In some embodiments, L is absent, or is a chain consisting of 1-20 atoms; L serves a connecting function, and its selection depends on the size of the dye molecule. If the dye molecule is too large, L is selected from a relatively long chain; if the dye molecule is small and hinders the interaction site, L may also be selected from a relatively short chain or L may not be required.

[0021] In some embodiments, the chain consisting of 1-20 atoms is selected from: , , , , , , , ; t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is selected from 1, 2, 3, or 4; Each q is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0022] In some embodiments, the fluorescent substrate of the present invention has the structure shown in Formula II: (I') or (II'), R, R 1 R 2 R 3 A, B, n, and m have the definitions described herein.

[0023] L 1 It is a C1-10 straight-chain carbon or -CH2-(CH2OCH2) p -CH2-; p is selected from 1, 2, 3 or 4.

[0024] In some embodiments, the compound represented by Formula I or II has one of the following structures: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (twenty one), (twenty two), (twenty three).

[0025] On the other hand, the present invention provides a method for detecting a target in a sample, wherein the method includes the following steps: (i) Sealing the sample: Take the sample to be tested, add sealing agent, incubate, and obtain the sealed sample; (ii) Antibody incubation: Mix the enzyme-labeled antibody with the blocked sample obtained in step (i) and incubate to obtain an antibody-sample complex solution. (iii) Incubation substrate: The fluorescent substrate having the structure of Formula I or Formula II or its stereoisomer is contacted with the incubation antibody-sample complex solution obtained in step (ii) and incubated to obtain the fluorescent substrate-incubation antibody-sample complex solution; (iv) Imaging: The fluorescent substrate-antibody-sample complex was dropped onto a glass slide, dried, stained with DAPI, and then scanned and analyzed using a fluorescence microscope.

[0026] In some embodiments, the sample is a sample containing cells or cell debris; the concentration of the fluorescent substrate having the structure of Formula I or Formula II or its stereoisomer is 0.15 μg / mL to 2.5 μg / mL.

[0027] In some embodiments, the targets that can be detected by the kit or method are peptides, nucleic acids, carbohydrates, lipids or their derivatives, molecular complexes, particles, eukaryotic or prokaryotic cells or microorganisms.

[0028] In some embodiments, the sample may be a biological sample, an environmental sample, or a chemical sample.

[0029] In some embodiments, the target or sample may be fixed to a solid support.

[0030] In some embodiments, the detection of deposited compounds may include an enzyme-linked immunosorbent assay (ELISA) step. In one embodiment, the method is used for immunohistochemical detection of a target. The method can be performed manually, automatically, or semi-automatically. The methods of the present invention can be successfully plasticized with many of the different reporter molecules described herein, retaining their advantageous characteristics such as detection speed, sensitivity, and specificity.

[0031] In one embodiment, the peroxidase portion is a part of HRP; for example, the whole HRP molecule is a fragment capable of having HRP enzymatic activity, or it can be a recombinant protein containing the enzymatically active portion of HRP, etc. In another embodiment, the peroxidase can be soybean peroxidase.

[0032] The site of the solid support containing peroxidase activity is referred to herein as a "target site". In one embodiment, the target site contains peroxidase activity, such as a portion of peroxidase directly immobilized on or within the solid support. In another embodiment, the target site contains peroxidase activity indirectly immobilized on or within the solid support, i.e., a portion of the peroxidase is linked to a binding agent capable of directly or indirectly binding to a target immobilized on or within the solid support. A robust support is provided. The latter is a non-limiting example of a target site of the present invention.

[0033] The method of the present invention can be performed in a variety of assay formats. The above-described compositions and methods can be used to detect target molecules comprising cell suspensions in any suitable assay format, such as flow cytometry (FC), ELISA, immunocytochemistry (IHC), or in situ hybridization (ISH). In one embodiment, the biological sample can be a cell suspension. FC, ELISA, IHC, or ISH can be used to detect target molecules or cellular structures in the suspension. When using ELISA, IHC, or ISH, the cells in the suspension are attached to a solid support, such as an ELISA plate or an IHC slide. In another embodiment, the biological sample can be a section of body tissue. IHC or ISH is typically used to detect target molecules or cellular structures in such samples.

[0034] IHC and ISH assays typically require a series of processing steps on tissue sections mounted on suitable solid supports for microscopic examination or the creation of photomicrographs, such as on slides or other planar supports, to highlight certain morphological indicators of disease states or biomarkers through selective staining. Therefore, in IHC, for example, samples are taken from individuals, fixed, and exposed to antibodies that specifically bind to the biomarkers of interest.

[0035] Histological samples can be preparations containing fresh tissue and / or cells, typically not fixed with aldehyde fixatives, or fixed and embedded tissue specimens, usually archived material. A pre-detection procedure will be performed before the target assay is performed in the form of an IHC assay. It may involve the following steps: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, thin-section cutting, mounting onto a glass slide, baking, deparaffining, fluid replenishment, antigen retrieval, blocking step, application of primary antibody, washing, application of secondary antibody-enzyme conjugate, and washing.

[0036] In ISH, samples are taken from individuals, fixed, and exposed to a nucleic acid binding agent that hybridizes with the nucleic acid of interest through complementary base pairing. Biological samples typically contain detectable nucleic acids, such as DNA and RNA, including messenger RNA. Detection of DNA / RNA levels can indicate the expression level of a specific gene and can therefore be used to detect the condition of cells, tissues, organs, or organisms (e.g., disease conditions). The nucleic acids in the sample are typically denatured to expose binding sites. The binding agent is usually a double-stranded or single-stranded nucleic acid, such as DNA or RNA, or a nucleic acid analog, such as PNA. The amount of the relevant target protein or nucleic acid detected by such techniques is then assessed to determine whether it is above a predetermined minimum threshold or compared to known standards, thus determining its diagnostic relevance. If necessary, appropriate treatment can be planned for the individual.

[0037] Many methods for fixing and embedding tissue specimens are known, such as alcohol fixation and formalin fixation followed by paraffin embedding (FFPE). Fixatives are needed to preserve cells and tissues in a reproducible and realistic manner. For this purpose, tissue blocks, sections, or smears are immersed in a fixative solution, or, in the case of smears, dried. Fixatives stabilize cells and tissues, thus protecting them from the effects of harsh handling and staining techniques.

[0038] Any suitable fixative can be used, such as ethanol, acetic acid, picric acid, 2-propanol, 3,3'-diaminobenzidine tetrahydrochloride dihydrate, acetoin (a mixture of monomers) and dimers, acrolein, crotonaldehyde, formaldehyde, glutaraldehyde, glyoxal, potassium dichromate, potassium permanganate, osmium tetroxide, paraformaldehyde, mercuric chloride, toluene-2,4-diisocyanate, trichloroacetic acid, and tungstic acid. Other examples include formalin (aqueous formaldehyde solution) and neutral buffered formalin (NBF), glutaraldehyde, acrolein, carbodiimide, imine salts, benzoquinone, osmium tetroxide, and osmium tetroxide.

[0039] Fresh biopsy specimens, cytological specimens (including touch specimens and blood smears), frozen sections, and tissues used for immunohistochemical analysis are typically fixed in organic solvents, including ethanol, acetic acid, methanol, and / or acetone.

[0040] Abbreviation: PPh3: Triphenylphosphine TBSCl: tert-butyldimethylchlorosilane DMF: N,N-Dimethylformamide t-BuOK: Potassium tert-butoxide DCM: Dichloromethane -OTBS: tert-butyldimethylsilyloxy TBAF: Tetrabutylammonium fluoride TBTU: O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate TSTU: O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate DIPEA: N,N-Diisopropylethylamine CF3COOH: Trifluoroacetic acid Et3N: Triethylamine THF: Tetrahydrofuran NaOH: Sodium hydroxide MeOH: Methanol K2CO3: Potassium carbonate H2SO4: sulfuric acid Boc: tert-Butoxycarbonyl Attached Figure Description

[0041] Figure 1 Immunofluorescence staining of CD31 in mouse kidney tissue is shown. (A) Staining group of compound 1; (B) Staining group of control compound.

[0042] Figure 2 Immunofluorescence staining of rat lung tissue with α-Actin. (A) Staining group of compound 12; (B) Staining group of control compound. Detailed Implementation

[0043] I. Synthesis of Compounds Example 1: Synthesis of Compound 1:

[0044] Step 1: Synthesis of Compounds 1-2 Methyl 4-bromomethylphenylacetate (11.50 g, 47.32 mmol) and triphenylphosphine (12.40 g, 47.32 mmol) were weighed and added to toluene (100 mL). The mixture was refluxed at 120 °C for about 5 hours. After cooling to room temperature, the mixture was filtered, and the filter cake was washed with a small amount of ethyl acetate. The filtrate was concentrated under reduced pressure to give compounds 1-2 (white solid, 15.7 g, yield 78.07%).

[0045] LC-MS [M-Br] + = 425.17.

[0046] Step 2: Synthesis of compounds 1-4 Weigh 10.00 g (58.14 mmol) of 6-hydroxy-2-naphthaldehyde and 11.86 g (174.41 mmol) of imidazole into DMF. After cooling in an ice-water bath for 30 min, add TBSCl (13.14 g, 87.02 mmol) in portions. After the addition is complete, react at room temperature for about 24 hours. Add 250 mL of water and extract with ethyl acetate (150 mL x 2). Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, purify by silica gel column chromatography, elute with petroleum ether:ethyl acetate = 95:5, and concentrate to give compounds 1-4 (orange-red oily liquid, 12.8 g, yield 76.98%).

[0047] LC-MS [M+H] + = 287.14.

[0048] Step 3: Synthesis of compounds 1-5 Compounds 1-2 (11.50 g, 27.06 mmol) were weighed and dissolved in dichloromethane (10 mL). The mixture was purged with nitrogen three times and cooled to -80 °C. Potassium tert-butoxide (7.6 g, 67.73 mmol) was added, followed by the addition of a dichloromethane solution of compound 1-4 (7.74 g, 27.06 mmol). The mixture was then reacted at -80 °C for approximately 24 hours. The pH was adjusted to 6 by adding 1 mol / L dilute hydrochloric acid at 0 °C. Water (5 V) was added, and the mixture was extracted with dichloromethane (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with petroleum ether:ethyl acetate = 98:2. The purified compound 1-5 was then concentrated to obtain a white solid (3.5 g, yield 47.49%).

[0049] LC-MS [M+H] + = 433.21.

[0050] Step 4: Synthesis of compounds 1-6 Compounds 1-5 (2.10 g, 4.86 mmol) were weighed and added to THF (5 mL), followed by TBAF (1.30 g, 4.86 mmol). The reaction was allowed to proceed at room temperature for approximately 24 hours. The mixture was then extracted with water (5 V) and ethyl acetate (3 V x 2), washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate = 80:20, and concentrated to give compounds 1-6 (pale yellow solid, 1.45 g, yield 93.82%).

[0051] LC-MS [M+H] + = 319.13.

[0052] Step 5: Synthesis of compounds 1-7 Compounds 1-6 (1.8 g, 5.66 mmol) were weighed and added to 15 mL of methanol. An aqueous solution of sodium hydroxide (0.60 g, 15.00 mmol) (1.5 mL) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for approximately 24 hours. The mixture was concentrated under reduced pressure, and 10 mL of pure water was added. The pH was adjusted to 4 by adding 1 mol / L dilute hydrochloric acid. The mixture was extracted with ethyl acetate (3 V x 2), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compounds 1-7 (a grayish-white solid, 1.7 g, yield 98.80%).

[0053] LC-MS [M+H] + = 305.11.

[0054] Step 6: Synthesis of compounds 1-8 Weigh compounds 1-7 (1.8 g, 5.92 mmol) and add DMF (15 mL). Under ice-water bath cooling, add TsTu (2.14 g, 7.11 mmol), DIPEA (2.29 g, 17.75 mmol), and mono-Boc ethylenediamine. After the addition is complete, react at room temperature for about 24 hours.

[0055] Post-treatment: Water (5 V) was added and extracted with ethyl acetate (3 V x 2); the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by silica gel column chromatography, eluted with dichloromethane:methanol = 95:5, and concentrated to give compounds 1-8 (pale yellow oil, 2.35 g, yield 88.90%).

[0056] LC-MS [M+H] + = 447.22.

[0057] Step 7: Synthesis of compounds 1-9 Compounds 1-8 (3.35 g, 7.50 mmol) were weighed and added to dichloromethane (30 mL). Trifluoroacetic acid (6 mL) was added under ice-water bath cooling. After the addition was complete, the reaction was carried out at room temperature for about 24 hours. The mixture was concentrated under reduced pressure and extracted with saturated sodium bicarbonate (5 V) and dichloromethane (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compounds 1-9 (grayish-white solid, 1.56 g, yield 60.11%).

[0058] LC-MS [M+H] + = 347.17.

[0059] Step 8: Synthesis of Compound 1 Weigh 5-AF488-NHS (CAS: 1374019-99-4, 120 mg, 0.19 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (38 mg, 0.38 mmol), followed by compounds 1-9 (66 mg, 0.19 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 1 (orange solid, 24 mg, yield 15.00%).

[0060] LC-MS [M+H] + = 863.16; 1H NMR (600 MHz, deuterated water) δ 7.72 (q, J = 8.2 Hz, 2H), 7.36 (d, J = 8.8Hz, 1H), 7.16 (s, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.80 (s, 1H), 6.77 – 6.72 (m, 4H), 6.70 (d, J = 9.1 Hz, 1H), 6.59 (d, J = 9.3Hz, 2H), 6.43 (d, J = 9.3 Hz, 2H), 6.14 (d, J = 12.3 Hz, 1H), 6.02 (d, J =12.3 Hz, 1H), 3.39 (s, 2H), 3.34 – 3.31 (m, 2H), 3.27 (s, 2H).

[0061] Example 2: Synthesis of Compound 8:

[0062] Weigh AF430-NHS (CAS: 467233-94-9, 100 mg, 0.17 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (34 mg, 0.33 mmol), followed by compounds 1-9 (66 mg, 0.19 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 8 (grayish-white solid, 35 mg, yield 24.77%).

[0063] LC-MS [M+H] + = 832.28.

[0064] Example 3: Synthesis of Compound 9:

[0065] Weigh AF555-NHS (CAS: 407627-69-4, 110 mg, 0.12 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol), followed by compounds 1-9 (45 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 9 (red solid, 46 mg, yield 33.04%).

[0066] LC-MS [M+H] += 1161.33.

[0067] Example 4: Synthesis of Compound 10:

[0068] Weigh AF647-NHS (CAS: 1620475-28-6, 110 mg, 0.12 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol) and NAP (45 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 10 (blue solid, 51 mg, yield 35.82%).

[0069] LC-MS [M+H] + = 1187.34 Example 5: Synthesis of Compound 11:

[0070] Weigh AF568-NHS (CAS: 247145-38-6, 100 mg, 0.13 mmol) and add it to DMF (3 mL). While cooling in an ice-water bath, add triethylamine (26 mg, 0.26 mmol), followed by compounds 1-9 (48 mg, 0.14 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, prepare and purify, and lyophilize to obtain compound 11 (red solid, 44 mg, yield 33.11%).

[0071] LC-MS [M+H] + = 1023.29.

[0072] Example 6: Synthesis of Compound 12:

[0073] Step 1: Synthesis of Compound 12-1 Weigh p-methoxyphenol (5.00 g, 40.32 mmol), p-fluoroacetophenone (5.56 g, 40.32 mmol), and potassium carbonate (6.68 g, 48.38 mmol) and add them to DMF (40 mL). React at 130 °C for approximately 12 hours. Cool to room temperature, extract with water (5 V), extract with ethyl acetate (3 V x 2), wash with saturated brine, dry to anhydrous sodium sulfate, concentrate under reduced pressure, purify by silica gel column chromatography, eluent with petroleum ether:ethyl acetate = 95:5, and concentrate to give compound 12 (white solid, 6.8 g, yield 69.69%).

[0074] LC-MS [M+H]+ = 243.09.

[0075] Step 2: Synthesis of Compound 12-2 Compound 12-1 (6.00 g, 24.79 mmol) and sulfur (1.98 g, 61.98 mmol) were weighed and added to morpholine (4.31 g, 49.58 mmol). The mixture was reacted at 120 °C for approximately 2 hours. After cooling to room temperature, the mixture was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 70:30. The purified product was concentrated to give compound 12-2 (yellow oily liquid, 3.8 g, yield 44.67%).

[0076] LC-MS [M+H] + = 344.12.

[0077] Step 3: Synthesis of Compound 12-3 Compound 12-2 (3.50 g, 10.20 mmol) was weighed and dissolved in glacial acetic acid (6.01 g, 100.20 mmol). Pure water (1.29 g, 71.40 mmol) and concentrated sulfuric acid (1.50 g, 15.30 mmol) were added. After the addition was complete, the mixture was reacted at 150 °C for approximately 12 hours. The mixture was cooled to room temperature and extracted with water (10 V) and ethyl acetate (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 12-3 (colorless oil, 2.2 g, yield 83.57%).

[0078] LC-MS [M+H] + = 259.09.

[0079] Step 4: Synthesis of compound 12-4 Compound 12-3 (2.00 g, 7.75 mmol) was weighed and dissolved in 48% hydrobromic acid (20 mL). After the addition was complete, the mixture was reacted at 120 °C for about 12 hours. The mixture was then cooled to room temperature, concentrated under reduced pressure, and extracted with water (5 V) and ethyl acetate (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 12-4 (colorless oil, 1.7 g, yield 89.87%).

[0080] LC-MS [M+H] + = 245.07.

[0081] Step 5: Synthesis of Compound 12-5 Compound 12-4 (1.5 g, 6.15 mmol) was weighed and added to DMF (15 mL). TsTu (2.03 g, 6.76 mmol), DIPEA (1.98 g, 15.38 mmol), and mono-Boc pentanediamine (1.37 g, 6.76 mmol) were added under ice-water bath cooling. The reaction was allowed to proceed for approximately 12 hours at room temperature. The mixture was extracted with water (5 V) and ethyl acetate (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography. Elution was performed with dichloromethane:methanol = 95:5, and the concentrate yielded compound 12-5 (pale yellow oil, 1.91 g, yield 72.52%).

[0082] LC-MS [M+H] + = 429.23.

[0083] Step 6: Synthesis of Compound 12-6 Compound 12-5 (1.80 g, 4.20 mmol) was weighed and added to dichloromethane (20 mL). Trifluoroacetic acid (4 mL) was added under ice-water bath cooling. After the addition was complete, the reaction was carried out at room temperature for about 24 hours. The mixture was concentrated under reduced pressure and extracted with saturated sodium bicarbonate (5 V) and dichloromethane (3 V x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 12-6 (a grayish-white solid, 1.22 g, yield 88.54%).

[0084] LC-MS [M+H] + = 329.18.

[0085] Step 7: Synthesis of Compound 12 Weigh 5-AF488-NHS (CAS: 1374019-99-4, 120 mg, 0.19 mmol) and add it to DMF (3 mL), then add ice water. Triethylamine (38 mg, 0.38 mmol) was added under bath cooling, followed by compound 12-6 (69 mg, 0.21 mmol). The reaction was carried out at room temperature for approximately 4 hours after the addition was complete. Post-treatment: The mixture was concentrated under reduced pressure, purified by high performance liquid chromatography, and lyophilized to obtain compound 12 (orange solid, 57 mg, yield 35.53%).

[0086] LC-MS [M+H] + = 845.17.

[0087] Example 7: Synthesis of Compound 16:

[0088] Weigh AF430-NHS (CAS: 467233-94-9, 100 mg, 0.17 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (34 mg, 0.33 mmol) and compound 12-6 (62 mg, 0.19 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Concentrate under reduced pressure, purify by preparative high-performance liquid chromatography, and lyophilize to obtain compound 16 (a grayish-white solid, 38 mg, yield 27.48%).

[0089] LC-MS [M+H] + = 814.29.

[0090] Example 8: Synthesis of Compound 17:

[0091] Weigh AF555-NHS (CAS: 407627-69-4, 110 mg, 0.12 mmol) and add it to DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol) and then compound 12-6 (43 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, purify by high-performance liquid chromatography (HPLC), and lyophilize to obtain compound 17 as a red solid (51 mg), yield 37.20%.

[0092] LC-MS [M+H] + = 1143.34.

[0093] Example 9: Synthesis of Compound 18:

[0094] Weigh AF647-NHS (CAS: 1620475-28-6, 100 mg, 0.13 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (26 mg, 0.26 mmol) and compound 12-6 (46 mg, 0.14 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-processing: Concentrate under reduced pressure, purify by high-performance liquid chromatography (HPLC), and lyophilize to obtain compound 18 (red solid, 56 mg, yield 42.89%).

[0095] LC-MS [M+H] + = 1005.30.

[0096] Example 20: Synthesis of Compound 19:

[0097] Weigh AF568-NHS (CAS: 247145-38-6, 110 mg, 0.12 mmol) and add DMF (3 mL). While cooling in an ice-water bath, add triethylamine (24 mg, 0.24 mmol) and compound 12-6 (43 mg, 0.13 mmol). After the addition is complete, react at room temperature for approximately 4 hours. Post-treatment: Concentrate under reduced pressure, prepare and purify, and lyophilize to obtain compound 19 (blue solid, 61 mg, yield 43.43%).

[0098] LC-MS [M+H] + = 1170.36.

[0099] II. Fluorescent Staining Test Example 21: Immunofluorescence staining of compound 1 and control compound 1. Experimental Materials Experimental sample: Paraffin-embedded sections of rat lung tissue.

[0100] Experimental reagents: ethanol, xylene, sodium citrate antigen retrieval solution (pH 6.0), HRP Blocking Buffer (#BUF0102), Antibody Dilution & Blocking Buffer (#BUF0103), DAPI (#BUF0105), Tyramide LUMO Buffer (#BUF0101), and anti-fluorescence quenching mounting medium.

[0101] Antibodies: α-Actin (#23660-1-AP), HRP-polymer conjugated Secondary Antibody (Goat Anti-Rabbit IgG) (#BUF0106).

[0102] Tyramine dyes: Compound 1, control compound.

[0103] Compare the structures of the compounds:

[0104] 2. Experimental Procedure (1) Dewaxing and hydration: Immerse the tissue sections in 100% xylene I and II for 10 minutes each; then immerse in 100% ethanol I and II for 10 minutes each; finally immerse in 95%, 85%, 75%, and 50% ethanol for 5 minutes each. Rinse the sections with ddH2O for 5 minutes, repeating 3 times.

[0105] (2) Antigen retrieval: Immerse the tissue sections in sodium citrate antigen retrieval solution (pH 6.0), microwave on high (100% power) for 10 minutes, then microwave on medium (50% power) for 15 minutes. After cooling to room temperature, rinse the sections with 1×PBS for 5 minutes. Repeat 3 times.

[0106] (3) Tissue permeability: Add an appropriate amount of cell permeability solution to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0107] (4) Quenching endogenous HRP: Add an appropriate amount of HRP Blocking Buffer to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0108] (5) Non-specific blocking: Add an appropriate amount of Antibody Dilution & Blocking Buffer to cover the tissue and incubate at room temperature for 30 minutes. (6) Peroxidase labeling: Add an appropriate amount of antibody dilution solution to cover the tissue and incubate overnight at 4°C. After the primary antibody incubation is complete, allow the sections to return to room temperature, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times. Then add HRP-polymer conjugated secondary antibody (Goat Anti-Rabbit IgG) to cover the tissue, incubate at room temperature for 30-60 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0109] (7) TSA staining: Dilute 200× tyramine dye to 1× with Tyramide LUMO Buffer. Add an appropriate amount of 1× tyramine dye to cover the tissue and incubate at room temperature for 10-15 minutes. Rinse the sections with 1× PBS for 5 minutes, repeat 3 times.

[0110] (8) DAPI staining: Add an appropriate amount of DAPI to cover the tissue, rinse the section with ddH2O for 5 minutes, and repeat 3 times.

[0111] (9) Mounting: Add anti-fluorescence quenching mounting medium to mount the slide.

[0112] (10) Microscopic imaging: Select the FITC fluorescence channel for imaging and taking pictures.

[0113] 3. Experimental Results Figure 1 It can be seen that the compound 1 staining group (A) has a higher signal-to-noise ratio and stronger sensitivity than the control compound staining group (B).

[0114] Example 22: Immunofluorescence staining of compound 12 and control compound 1. Experimental Materials Experimental sample: Paraffin-embedded sections of mouse kidney tissue.

[0115] Experimental reagents: ethanol, xylene, sodium citrate antigen retrieval solution (pH 6.0), cell permeation solution, HRPB locking buffer (#BUF0102), antibody dilution & blocking buffer (#BUF0103), DAPI (#BUF0105), tyramide LUMO buffer (#BUF0101), and antifluorescence quenching mounting medium.

[0116] Antibodies: CD31 (#ab182981), HRP-polymer conjugated Secondary Antibody (GoatAnti-Rabbit IgG) (#BUF0106).

[0117] Tyramine dyes: Compound 12 (200×), control compound (200×).

[0118] Compare the structures of the compounds:

[0119] 2. Experimental Procedure 1) Dewaxing and hydration: Immerse tissue sections in 100% xylene I and II for 10 minutes each; then immerse in 100% ethanol I and II for 10 minutes each; finally immerse in 95%, 85%, 75%, and 50% ethanol sequentially for 5 minutes each. Rinse sections with ddH2O for 5 minutes, repeating 3 times.

[0120] 2) Antigen retrieval: Immerse tissue sections in sodium citrate antigen retrieval solution (pH 6.0), microwave on high (100% power) for 10 minutes, then microwave on medium (50% power) for 15 minutes. After cooling to room temperature, rinse the sections with 1×PBS for 5 minutes. Repeat 3 times.

[0121] 3) Tissue permeability: Add an appropriate amount of cell permeability solution to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0122] 4) Quenching endogenous HRP: Add an appropriate amount of HRP Blocking Buffer to cover the tissue, incubate at room temperature for 10-15 minutes, rinse the sections with 1×PBS for 5 minutes, and repeat 3 times.

[0123] 5) Non-specific blocking: Add an appropriate amount of Antibody Dilution & Blocking Buffer to cover the tissue and incubate at room temperature for 30 minutes. 6) Peroxidase labeling: Add an appropriate amount of antibody dilution solution to cover the tissue and incubate overnight at 4°C. After primary antibody incubation, allow the sections to return to room temperature, rinse with 1×PBS for 5 minutes, and repeat 3 times. Then add HRP-polymer conjugated secondary antibody (Goat Anti-Rabbit IgG) to cover the tissue, incubate at room temperature for 30-60 minutes, rinse with 1×PBS for 5 minutes, and repeat 3 times.

[0124] 7) TSA staining: Dilute 200× tyramine dye to 1× with Tyramide LUMO Buffer. Add an appropriate amount of 1× tyramine dye to cover the tissue and incubate at room temperature for 10-15 minutes. Rinse the sections with 1× PBS for 5 minutes, repeating 3 times.

[0125] 8) DAPI staining: Add an appropriate amount of DAPI to cover the tissue, rinse the section with ddH2O for 5 minutes, and repeat 3 times.

[0126] 9) Mounting: Add anti-fluorescence quenching mounting medium to mount the slide.

[0127] 10) Microscopic imaging: Select the FITC fluorescence channel for imaging and taking pictures.

[0128] 3. Experimental Results Figure 2 It can be seen that the compound 12 staining group (A) has a higher signal-to-noise ratio and stronger sensitivity than the control compound staining group (B).

Claims

1. A tyramine signal amplification kit comprising an enzyme-conjugated antibody and a fluorescent substrate, wherein the fluorescent substrate has a compound of formula I or II or a stereoisomer thereof: (I) or (II); in, R is a dye; Ring A and ring B are each independently aryl, heteroaryl, cycloalkyl, or heterocyclic. E is -O-, -S-, -NH- or -C≡C-; L does not exist, or it is a chain consisting of 1-20 atoms; Each R 1 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; Each R 2 Independently hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, cycloalkylalkyl, heterocyclic or heterocyclic alkyl; R 3 It is either hydroxyl or amino; n can be 0, 1, 2, 3, or 4; m can be 0, 1, 2, 3, or 4.

2. The tyramine signal amplification kit according to claim 1, characterized in that, The enzyme is selected from any one of dehydrogenase, oxidase, peroxidase, and oxygenase; preferably, the enzyme is any one of horseradish peroxidase, catalase, and superoxide dismutase; more preferably, the enzyme is horseradish peroxidase. The antibody is one or more of the following: anti-HER2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-HER3 antibody, anti-MUC1 antibody, anti-PD-L1 antibody, anti-EpCAM antibody, anti-EGFR antibody, anti-c-met antibody, anti-FR (folate receptor) antibody, anti-CEA antibody, anti-PMSA antibody, anti-AR-V7 antibody, anti-CK antibody, anti-PLAP antibody, anti-GPC3 antibody, anti-CD31 antibody, anti-CD44 antibody, anti-Vimentin antibody, and anti-cadherin antibody.

3. The tyramine signal amplification kit according to claim 1, characterized in that, The kit also includes a blocking agent and a buffer solution.

4. The tyramine signal amplification kit according to claim 3, characterized in that, The sealing agent is a peroxide; preferably, the peroxide is H2O2. The buffer solution is a PBS solution, a Tris-HCl solution, or a Hepes solution; preferably, the buffer solution is a PBS solution, a Tris-HCl solution, or a Hepes solution containing FBS or BSA.

5. The tyramine signal amplification kit according to claim 3, characterized in that, The fluorescent substrate is 40-60 parts, the buffer solution is 30-45 parts, the enzyme-conjugated antibody is 15-30 parts, and the blocking solution is 50-70 parts.

6. The tyramine signal amplification kit according to claim 1, characterized in that, The kit includes HRP blocking buffer, antibody dilution and blocking buffer, tyrosine luminescent buffer, HRP polymer-conjugated secondary antibody, a fluorescent substrate having a compound of formula I or II or a stereoisomer thereof, and DAPI.

7. The tyramine signal amplification kit according to any one of claims 1 to 6, characterized in that, The dye is AF350, AF405, AF425, AF430, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF640, AF647, AF660, AF680, CF405M, CF450, CF488A, CF532, CF543, CF555, CF568, CF594, CF620R, CF633, CF640R, CF647, CF660C, CF680, CF680R, CF750, CF770, CF790, or CF820; Ring A and ring B are each independently phenyl, naphthyl, pyrrolyl, furanyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyranonel, indolyl, benzofuranyl, benzothiophene, quinolinyl, isoquinolinyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothiophene, tetrahydropyranyl, piperidinyl, tetrahydrothiophene, dioxane, piperazinyl, hexahydropyrazinyl, or morpholinyl. Each R 1 and R 2 Independently, it is hydroxyl, amino, F, Cl, Br, methyl, ethyl, propyl, isopropyl, butyl, vinyl, propenyl, butenyl, ethynyl, propynyl, butynyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylamino, ethylamino, propylamino, isopropylamino, butylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclopentylmethyl, oxecyclopropyl, azicyclopropyl, oxecyclobutyl, azicyclobutyl, tetrahydrofuranyl, tetrahydropyrrolyl, piperidinyl, tetrahydropyranyl, oxecyclopropylmethyl, azicyclopropylmethyl, oxecyclobutylmethyl, azicyclobutylmethyl, oxecyclopropylethyl, azicyclopropylethyl, oxecyclobutylethyl, or azicyclobutylethyl.

8. The tyramine signal amplification kit according to any one of claims 1 to 6, characterized in that, The compounds represented by Formula I or II have one of the following structures: （1）、 （2）、 （3）、 （4）、 （5）、 （6）、 （7）、 （8）、 （9）、 （10）、 （11）、 （12）、 （13）、 （14）、 （15）、 （16）、 （17）、 （18）、 （19）、 （20）、 （21）、 （22）、 （23）。 9. A method for detecting a target in a sample, wherein the method includes the following steps: (i) Sealing the sample: Take the sample to be tested, add sealing agent, incubate, and obtain the sealed sample; (ii) Antibody incubation: Mix the enzyme-labeled antibody with the blocked sample obtained in step (i) and incubate to obtain an antibody-sample complex solution. (iii) Incubation substrate: The fluorescent substrate having the structure of Formula I or Formula II or its stereoisomer is contacted with the incubation antibody-sample complex solution obtained in step (ii) and incubated to obtain the fluorescent substrate-incubation antibody-sample complex solution; (iv) Imaging: The fluorescent substrate-antibody-sample complex was dropped onto a glass slide, dried, stained with DAPI, and then scanned and analyzed using a fluorescence microscope.

10. The method according to claim 9, characterized in that, The sample is a sample containing cells or cell fragments; the concentration of the fluorescent substrate or its stereoisomer having the structure of Formula I or Formula II is 0.15 μg / mL to 2.5 μg / mL.

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