A fluorescence-enhancing composition and a fluorescence-enhancing solution for a tyramide signal amplification system
By introducing a fluorescence enhancement composition and a fluorescence enhancement solution into the tyrosine signal amplification system, the problems of low signal amplification efficiency and easy quenching of fluorescent dyes are solved, resulting in stronger fluorescence signal display and cost savings, making it suitable for a variety of biomedical experiments.
Patent Information
- Application Number
- CN202311375717.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-23
- Publication Date
- 2026-05-15
- Estimated Expiration
- 2043-10-23
AI Technical Summary
Existing tyrosine signal amplification systems have low signal amplification efficiency, fluorescent dyes are easily quenched, and fluorescence enhancement solutions are not suitable for TSA systems.
A fluorescence-enhancing composition is used, comprising an enhancer and a clustering agent. The enhancer is 4,4-bis(5-methyl-2-benzoxazolyl)stilbene, 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene, 4,4'-bis(2-sulfonate styryl)biphenyl, or 1,2-bis(5-methyl-benzoxazolyl)-ethylene, and the clustering agent is alginate, povidone, methyl vinyl ether-maleic anhydride copolymer, and carrageenan. Combined with a buffer, an oxidant, an emulsifier, and a preservative, a fluorescence-enhancing solution is formed.
It significantly enhances fluorescence signal, reduces non-specific staining, lowers antibody usage, improves detection sensitivity, is easy to operate, and is suitable for experiments such as immunohistochemistry and in situ hybridization, with significant cost-effectiveness.
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Figure CN117417737B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a fluorescence enhancement composition and fluorescence enhancement liquid for a tyrosine signal amplification system, belonging to the field of biomedical technology. Background Technology
[0002] Tyramide signal amplification (TSA) is a detection method that utilizes antibody- or probe-labeled horseradish peroxidase (HRP) to perform high-density in-situ labeling of target antigens. In the presence of hydrogen peroxide (H2O2), tyramine molecules, labeled with a fluorescent dye, are converted into a transiently active intermediate state by the HRP labeled on the antibody or probe. The activated intermediate molecule then rapidly and stably covalently binds to the electron-rich regions (tyrosine residues) of adjacent protein molecules, while unlabeled tyramine molecules are washed away, thereby achieving specific staining of the antigen. Because the target antigen contains numerous tyrosine binding sites, a large number of labeled molecules accumulate at the target antigen site, effectively amplifying the signal.
[0003] Tyramine fluorescent dyes play a crucial role in TSA (Transient Acid Subtraction Angiography) technology. However, when proteins are coupled with fluorescent dyes, the fluorescence emission of the dye molecules typically decreases, sometimes by 50-70%, a phenomenon known as fluorescence quenching. Fluorescence quenching refers to the irreversible destruction of fluorescent molecules caused by the combined effects of internal and external factors. Internal factors primarily involve molecules releasing energy through non-radiative transitions from an excited state to the ground state. External factors are multifaceted, including excited-state reactions, resonance energy transfer, the formation of non-fluorescent complexes between fluorescent molecules and external molecules or ions, molecular collisions, pH changes, solvent type, temperature changes, and pressure changes. Prolonged exposure to excitation light is the most common cause of fluorescence quenching. While fluorescence generation requires excitation light, this promotes interactions and collisions between excited-state molecules, leading to quenching. Currently, there is no simple and practical method to control this quenching phenomenon. Furthermore, fluorescent dyes typically require dilution during use, and the use of different solvents for dilution significantly impacts their staining effectiveness.
[0004] Therefore, in order to improve the efficiency of signal amplification in the TSA system, reduce the quenching of fluorescent dyes, and improve the conversion rate of enzymes from substrates to products, it is particularly important to develop a fluorescence enhancement solution specifically for TSA. Summary of the Invention
[0005] The first objective of this invention is to provide a fluorescence enhancement composition for a tyrosine signal amplification system, thereby addressing the problems of low efficiency and poor performance of signal amplification in existing TSA systems.
[0006] The second objective of this invention is to provide a fluorescence enhancement liquid for a tyrosine signal amplification system, thereby solving the problem that fluorescence enhancement liquids in the prior art are not suitable for TSA systems.
[0007] To achieve the above objectives, the technical solution of the present invention for a fluorescence enhancement composition for a tyrosine signal amplification system is as follows:
[0008] A fluorescence enhancement composition for a tyrosine signal amplification system, the fluorescence enhancement composition comprising an enhancer and a clustering agent; the molar ratio of the enhancer and the clustering agent is (0.1-1) mol:(1-10) mmol; the enhancer is one or a combination of two or more of 4,4-bis(5-methyl-2-benzoxazolyl)stilbene, 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene, 4,4'-bis(2-sulfonate styryl)biphenyl, and 1,2-bis(5-methyl-benzoxazolyl)-ethylene; the clustering agent is one or a combination of two or more of alginate, povidone, methyl vinyl ether-maleic anhydride copolymer, and carrageenan.
[0009] The beneficial effects of the above scheme are as follows: The fluorescence enhancement composition for the tyrosine signal amplification system of the present invention uses 4,4-bis(5-methyl-2-benzoxazolyl)stilbene, 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene, 4,4'-bis(2-sulfonate styryl)biphenyl, or 1,2-bis(5-methyl-benzoxazolyl)-ethylene as fluorescence enhancers. These enhancers can absorb invisible ultraviolet light and excite incident light to produce fluorescence, resulting in stronger fluorescence and extended sample preservation time. Furthermore, a clustering agent is creatively added for the first time. This clustering agent has a high viscosity when dissolved in water. Increased system viscosity leads to aggregation and condensation of substances in the system, resulting in better fluorescence signal aggregation and greater system stability, thus allowing for the observation of a stronger fluorescence signal. The two components work synergistically to significantly enhance the fluorescence signal.
[0010] To achieve the above objectives, the technical solution of the fluorescence enhancement liquid for a tyrosine signal amplification system of the present invention is as follows:
[0011] A fluorescence enhancement solution for a tyrosine signal amplification system, comprising the above-mentioned fluorescence enhancement composition and a base solution; the base solution includes a buffer solution; the pH of the fluorescence enhancement solution is 7.0-10.0.
[0012] The beneficial effects of the above solution are as follows: The fluorescence enhancement liquid for the tyrosine signal amplification system of the present invention, with the above-mentioned fluorescence enhancement composition as the main component, can further enhance the fluorescence signal amplification effect, and can reduce non-specific staining, making the stained fluorescence signal brighter and the background cleaner.
[0013] As a further improvement, the concentration of the enhancer in the fluorescence enhancement solution is 0.1-1M.
[0014] The beneficial effect of the above scheme is that the fluorescence enhancement effect can be achieved when the concentration of the enhancer in the fluorescence enhancement solution is 0.1-1M, which helps to reduce the production cost.
[0015] As a further improvement, the buffer is one of Tris buffer, PBS buffer, and amino acid buffer.
[0016] The beneficial effect of the above scheme is that the buffer solution provides a stable reaction system for the fluorescence enhancement solution.
[0017] To improve detection sensitivity and reduce non-specific staining, the buffer solution is preferably a PBS buffer or an amino acid buffer.
[0018] As a further improvement, the base liquid includes an oxidant.
[0019] As a further improvement, the concentration of the oxidant in the fluorescence enhancement solution is 0.01-0.1%; the oxidant is H2O2.
[0020] The beneficial effect of the above scheme is that the oxidant can activate the HRP labeled on the antibody or probe, providing the reaction conditions.
[0021] As a further improvement, the base liquid includes an emulsifier.
[0022] The beneficial effects of the above scheme are that the emulsifier helps stabilize the system and can reduce the surface tension of each component in the solution.
[0023] As a further improvement, the concentration of the emulsifier in the fluorescence enhancement solution is 0.01%-1%; the emulsifier is one or a combination of two or more of Tween-20, DMSO and glycerol.
[0024] As a further improvement, the base liquid includes a preservative.
[0025] The beneficial effects of the above scheme are that the preservative can effectively control the growth of microorganisms in the reagents, so that the fluorescence enhancement solution can be stored and used stably for a long time under both refrigeration and room temperature conditions, reducing the increase in experimental costs caused by reagent contamination.
[0026] Preferably, the preservative is one of thimerosal, gentamicin sulfate, cinnamaldehyde, and Proclin 300.
[0027] More preferably, the preservative is Proclin 300.
[0028] ProCin300 is a broad-spectrum, highly effective, stable, and low-toxicity antibacterial and preservative agent. At certain concentrations, it can broadly inhibit the growth of bacteria, fungi, and other microorganisms for a relatively long period of time, with very little drug resistance, and it has good biocompatibility with the entire system.
[0029] The beneficial effects of this invention are as follows:
[0030] 1. The fluorescence enhancement solution of the tyrosine signal amplification system of the present invention can be used with a higher antibody dilution ratio, reducing the amount of antibody or probe used and greatly saving costs;
[0031] 2. The fluorescent enhancement liquid used in the tyrosine signal amplification system of the present invention has high sensitivity, can reduce non-specific binding, reduce background staining, and improve the staining index. When performing multicolor labeling and co-localization detection, the fluorescent signal is better presented.
[0032] 3. The fluorescence enhancement solution of the tyrosine signal amplification system of the present invention is easy to operate and has a wide range of applications. It is suitable for experiments such as IHC, ICC and FISH. A stronger fluorescence signal can be obtained by processing the sample through a simple incubation step. Attached Figure Description
[0033] Figure 1 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 9 in Experiment 1 of the present invention (wherein, from left to right, AF488, Cy3, and BDP TR-X; 20×).
[0034] Figure 2 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 10 in Experiment 1 of the present invention (wherein, from left to right: AF488, Cy3, BDP TR-X; 20×).
[0035] Figure 3 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 11 in Experiment 1 of the present invention (wherein, from left to right: AF488, Cy3, BDP TR-X; 20×).
[0036] Figure 4 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 12 in Experiment 1 of the present invention (wherein, from left to right, AF488, Cy3, and BDP TR-X; 20×).
[0037] Figure 5The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 13 in Experiment 1 of the present invention (wherein, from left to right, AF488, Cy3, and BDP TR-X; 20×).
[0038] Figure 6 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 14 in Experiment 1 of the present invention (wherein, from left to right: AF488, Cy3, BDP TR-X; 20×).
[0039] Figure 7 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 15 in Experiment 1 of the present invention (wherein, from left to right: AF488, Cy3, BDP TR-X; 20×).
[0040] Figure 8 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Example 16 in Experiment 1 of the present invention (wherein, from left to right, AF488, Cy3, and BDP TR-X; 20×).
[0041] Figure 9 The image shows the staining results of AF488, Cy3 and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Comparative Example 1 in Experimental Example 1 of this invention (wherein, from left to right, AF488, Cy3, BDP TR-X; 20×).
[0042] Figure 10 The image shows the staining results of AF488, Cy3, and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Comparative Example 2 in Experimental Example 1 of this invention (wherein, from left to right, AF488, Cy3, and BDP TR-X; 20×).
[0043] Figure 11 The image shows the staining results of AF488, Cy3 and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Comparative Example 3 in Experimental Example 1 of the present invention (wherein, from left to right, AF488, Cy3, BDP TR-X; 20×).
[0044] Figure 12 The image shows the staining results of AF488, Cy3 and BDP TR-X dyes in the CKpan project after using the fluorescence enhancement solution of Comparative Example 4 in Experimental Example 1 of this invention (wherein, from left to right, AF488, Cy3, BDP TR-X; 20×). Detailed Implementation
[0045] In the experimental examples of this invention, a tyrosine signal amplification system was used for immunohistochemical staining. The specific operating steps are as follows:
[0046] 1) Dewaxing and hydration treatment of tissue sections.
[0047] Paraffin sections were placed in fresh xylene and soaked twice, 10 minutes each time.
[0048] After removing excess liquid, place it in anhydrous ethanol and soak it twice, 5 minutes each time;
[0049] After removing excess liquid, place in 95% ethanol and soak for 5 minutes;
[0050] After removing excess liquid, place in 85% ethanol and soak for 5 minutes;
[0051] Rinse three times with tap water, each time for 3 minutes.
[0052] 2) Antigen retrieval
[0053] Place the sample slides in EDTA (pH=9.0) immunohistochemical antigen retrieval buffer, autoclave the tissue sections for 2.5 min, and then allow them to cool naturally to room temperature. Rinse and soak three times with washing buffer, 3 min each time.
[0054] 3) Add peroxidase blocking agent
[0055] Remove the washing solution, add 100 μL of endogenous peroxidase inhibitor to the sample area, and incubate at room temperature for 5 min. Then rinse and soak three times with the washing solution, 3 min each time.
[0056] 4) Primary antibody incubation
[0057] Remove the washing solution, add 100 μL of antibody to the sample area, incubate at room temperature for 60 min, rinse and soak with washing solution 3 times, 3 min each time.
[0058] 5) Secondary antibody incubation
[0059] Remove the washing solution, add 100 μL of enzyme-labeled secondary antibody polymer reagent to the sample area, incubate at room temperature for 30 min, rinse with washing solution and soak 3 times, 3 min each time.
[0060] 6) Fluorescent staining
[0061] Tyramine fluorescent dye and enhancement solution were mixed at a ratio of 1:100 (all tyramine fluorescent dyes used in the following experimental examples were liquids with an effective content of 1.57 μmol / L) to prepare the immunofluorescence staining solution. After removing the washing solution, 100 μL of immunofluorescence staining solution was added to the sample area and incubated at room temperature for 30 min. The sample was then rinsed and soaked three times with the washing solution, for 3 min each time.
[0062] 7) Nuclear re-infection
[0063] Remove the cleaning solution, add DAPI staining solution to the sample area, and incubate at room temperature for 5 minutes. Rinse and soak three times with the cleaning solution, 3 minutes each time.
[0064] 8) Covering
[0065] Remove the cleaning solution, add an anti-fluorescence quenching mounting medium, and seal with a coverslip, avoiding air bubbles. For long-term storage, please seal the edges of the coverslip with clear nail polish.
[0066] 9) Film viewing
[0067] The stained tissue slides were observed and analyzed under a fluorescence microscope.
[0068] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.
[0069] I. Specific Embodiments of a Fluorescence Enhancement Composition for a Tyrosamide Signal Amplification System of the Present Invention
[0070] Example 1
[0071] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 0.1 mol: 10 mmol; the enhancer is 4,4-bis(5-methyl-2-benzoxazolyl)stilbene; and the clusterer is alginate (AA).
[0072] Example 2
[0073] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 1 mol: 1 mmol; the enhancer is 4,4-bis(5-methyl-2-benzoxazolyl)stilbene; and the clusterer is alginate (AA).
[0074] Example 3
[0075] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 0.1 mol: 10 mmol; the enhancer is 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene; and the clusterer is povidone (CAS: 9003-39-8, molecular weight 111.14).
[0076] Example 4
[0077] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 0.5 mol: 1 mmol; the enhancer is 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene; and the clusterer is povidone (CAS: 9003-39-8, molecular weight 111.14).
[0078] Example 5
[0079] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 0.2 mol: 10 mmol; the enhancer is 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene and 4,4'-bis(2-sulfonate styryl)biphenyl, and the molar ratio of 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene to 4,4'-bis(2-sulfonate styryl)biphenyl is 1:1; the clusterer is a methyl vinyl ether-maleic anhydride copolymer (CAS: 9011-16-9; molecular weight: 156.14).
[0080] Example 6
[0081] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 1 mol: 2 mmol; the enhancer is 4,4'-bis(2-sulfonate styryl)biphenyl; the clusterer is methyl vinyl ether-maleic anhydride copolymer and alginate (AA), and the molar ratio of methyl vinyl ether-maleic anhydride copolymer and alginate is 1:1.
[0082] Example 7
[0083] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 0.2 mol: 10 mmol; the enhancer is 1,2-bis(5-methyl-benzoxazolyl)ethylene and 4,4-bis(5-methyl-2-benzoxazolyl)stilbene, and the molar ratio of 1,2-bis(5-methyl-benzoxazolyl)ethylene and 4,4-bis(5-methyl-2-benzoxazolyl)stilbene is 1:1; the clusterer is carrageenan.
[0084] Example 8
[0085] The fluorescence enhancement composition for the tyrosine signal amplification system of this embodiment consists of an enhancer and a clusterer; the molar ratio of the enhancer to the clusterer is 1 mol: 1 mmol; the enhancer is 1,2-bis(5-methyl-benzoxazolyl)-ethylene; and the clusterer is carrageenan.
[0086] II. Specific Embodiments of the Fluorescence Enhancement Liquid for a Tyroamide Signal Amplification System of the Present Invention
[0087] Example 9
[0088] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 1 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is DMSO; the preservative is Proclin 300; the buffer solution is PBS buffer; the pH of the fluorescence enhancement solution is adjusted to 7 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0089] Reagent Name concentration disodium hydrogen phosphate 0.1M Potassium dihydrogen phosphate 0.1M <![CDATA[H2O2]]> 0.01％ 4,4-Bis(5-methyl-2-benzoxazolyl)stilbene 0.1M DMSO 0.01％ AA 10mM Proclin300 0.50％ Deionized water 100mL
[0090] Example 10
[0091] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 2 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is DMSO; the preservative is Proclin 300; the buffer solution is PBS buffer; the pH of the fluorescence enhancement solution is adjusted to 8.5 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0092] Reagent Name concentration disodium hydrogen phosphate 1M Potassium dihydrogen phosphate 1M <![CDATA[H2O2]]> 0.10％ 4,4-Bis(5-methyl-2-benzoxazolyl)stilbene 1M DMSO 0.05％ AA 1mM Proclin300 0.10％ Deionized water 100mL
[0093] Example 11
[0094] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 3 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is Tween-20; the preservative is Proclin 300; the buffer solution is PBS buffer; the pH of the fluorescence enhancement solution is adjusted to 9.5 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0095] Reagent Name concentration disodium hydrogen phosphate 1M Potassium dihydrogen phosphate 1M <![CDATA[H2O2]]> 0.05％ 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene 0.1M Tween-20 0.01％ Povidone 10mM Proclin300 0.01％ Deionized water 100mL
[0096] Example 12
[0097] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 4 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is Tween-20; the preservative is Proclin 300; the buffer solution is PBS buffer; the pH of the fluorescence enhancement solution is adjusted to 8.5 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0098] Reagent Name concentration disodium hydrogen phosphate 0.5M Potassium dihydrogen phosphate 0.5M <![CDATA[H2O2]]> 0.05％ 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene 0.5M Tween-20 0.01％ Povidone 1mM Proclin300 1.00％ Deionized water 100mL
[0099] Example 13
[0100] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 5 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is glycerol; the preservative is Proclin 300; the buffer solution is glycine buffer; the pH of the fluorescence enhancement solution is adjusted to 9 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0101] Reagent Name concentration glycine 0.1M NaOH 0.1M <![CDATA[H2O2]]> 0.05％ 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene 0.1M 4,4'-Bis(2-Sylenic sodium sulfonate)biphenyl 0.1M glycerin 0.01％ MVE-MAC 10mM Proclin300 0.05％ Deionized water 100mL
[0102] Example 14
[0103] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 6 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is DMSO; the preservative is Proclin 300; the buffer solution is glycine buffer; the pH of the fluorescence enhancement solution is adjusted to 10 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0104] Reagent Name concentration glycine 1M NaOH 1M <![CDATA[H2O2]]> 0.10％ 4,4'-Bis(2-Sylenic sodium sulfonate)biphenyl 1M DMSO 1.00％ MVE-MAC 1mM AA 1mM Proclin300 0.05％ Deionized water 100mL
[0105] Example 15
[0106] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 7 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is glycerol; the preservative is Proclin 300; the buffer solution is Tris buffer; the pH of the fluorescence enhancement solution is adjusted to 10 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0107] Reagent Name concentration Tris(hydroxymethyl)aminomethane 0.1M <![CDATA[H2O2]]> 0.01％ 1,2-Bis(5-methyl-benzoxazolyl)-ethylene 0.1M 4,4-Bis(5-methyl-2-benzoxazolyl)stilbene 0.1M Tween-20 0.03％ glycerin 0.03％ Carrageenan 10mM Proclin300 0.01％ Deionized water 100mL
[0108] Example 16
[0109] The fluorescence enhancement solution used in the tyrosine signal amplification system of this embodiment is composed of the fluorescence enhancement composition of Example 8 and a base solution; the base solution is composed of a buffer solution, a strong oxidant, an emulsifier, and a preservative; the strong oxidant is H2O2; the emulsifier is Tween-20; the preservative is Proclin 300; the buffer solution is Tris buffer; the pH of the fluorescence enhancement solution is adjusted to 8.5 using 1M NaOH solution or HCl solution. The specific formulation is as follows:
[0110] Reagent Name concentration Tris(hydroxymethyl)aminomethane 1M <![CDATA[H2O2]]> 0.05％ 1,2-Bis(5-methyl-benzoxazolyl)-ethylene 1M Tween-20 0.01％ Carrageenan 1mM Proclin300 0.05％ Deionized water 100mL
[0111] III. Comparative Example
[0112] Comparative Example 1
[0113] The fluorescence enhancement solution in this comparative example does not contain any enhancement agent. The specific formula is as follows:
[0114]
[0115] Comparative Example 2
[0116] The fluorescence enhancement solution in this comparative example does not contain clustering agents, and the specific formulation is as follows:
[0117]
[0118] Comparative Example 3
[0119] The fluorescence enhancement solution in this comparative example contains an enhancer and a clustering agent. The clustering agent is sodium polyacrylate (CAS: 9003-04-7, molecular weight: 72.06), and the specific formulation is as follows:
[0120]
[0121]
[0122] Comparative Example 4
[0123] The fluorescence enhancement solution in this comparative example does not contain any enhancers or clustering agents. The specific formulation is as follows:
[0124]
[0125] IV. Experimental Examples
[0126] Experimental Example 1
[0127] The subjects of this experimental study are the fluorescence enhancement solutions of Examples 9-16 and Comparative Examples 1-4. Breast tissue was sequentially sectioned and subjected to fluorescence immunohistochemical staining, with CKpan as the detection index. AF series dyes (AF488), anthocyanin fluorescent dye (Cy3), and Bodipy boron fluoride dipyrrole fluorescent dye (BDP TR-X) were used for fluorescence immunohistochemical staining. The specific procedures for fluorescence staining are as described in the detailed implementation method. After staining, photographs were taken for fluorescence effect comparison. Figures 1-12 The staining effects of fluorescence immunohistochemistry using the fluorescence enhancement solutions of Examples 9-16 and Comparative Examples 1-4 are shown respectively.
[0128] Depend on Figures 1-6 It can be seen that the positive sites show strong fluorescence signals, clear and transparent staining, and no nonspecific staining; Figure 7 , Figure 8 It can be seen that the positive sites show strong fluorescence signals, clear and transparent staining, and very slight nonspecific staining. From Figures 9-11 It can be seen that the fluorescence signal in the positive areas is weak, the staining is dark, and there is slight nonspecific staining; Figure 12 It can be seen that the fluorescence signal in the positive area is weak, the staining is dark, and there is severe nonspecific staining.
[0129] The above description is merely an illustrative embodiment of the present invention and is not intended to limit the scope of the invention. Any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present invention should fall within the scope of protection of the present invention.
Claims
1. A fluorescence enhancement composition for a tyrosine signal amplification system, characterized in that: The fluorescence-enhancing composition comprises an enhancer and a clusterer; the molar ratio of the enhancer and the clusterer is (0.1-1) mol: (1-10) mmol; the enhancer is one or more of 4,4-bis(5-methyl-2-benzoxazolyl)stilbene, 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene, 4,4'-bis(2-sulfonate styryl)biphenyl and 1,2-bis(5-methyl-benzoxazolyl)ethylene; the clusterer is one or more of alginate, povidone, methyl vinyl ether-maleic anhydride copolymer and carrageenan.
2. A fluorescence enhancement solution for a tyrosine signal amplification system, characterized in that: The composition comprises the fluorescence enhancement composition of claim 1 and a base solution; the base solution includes a buffer and an oxidant; the pH of the fluorescence enhancement solution is 7.0-10.0; the buffer is one of Tris buffer, PBS buffer and amino acid buffer; and the oxidant is H2O2.
3. The fluorescence enhancement solution for the tyrosine signal amplification system according to claim 2, characterized in that: The concentration of the enhancer in the fluorescence enhancement solution is 0.1-1M.
4. The fluorescence enhancement solution for the tyrosine signal amplification system according to claim 2, characterized in that: The buffer solution is either PBS buffer or amino acid buffer.
5. The fluorescence enhancement solution for the tyrosine signal amplification system according to claim 2, characterized in that: The concentration of the oxidant in the fluorescence enhancement solution is 0.01-0.1%.
6. The fluorescence enhancement solution for the tyrosine signal amplification system according to any one of claims 2 to 4, characterized in that: The base liquid includes an emulsifier.
7. The fluorescence enhancement solution for the tyrosine signal amplification system according to claim 6, characterized in that: The concentration of the emulsifier in the fluorescence enhancement solution is 0.01%-1%; the emulsifier is one or a combination of two or more of Tween-20, DMSO and glycerol.
8. The fluorescence enhancement solution for the tyrosine signal amplification system according to any one of claims 2 to 4, characterized in that: The base liquid includes a preservative.