A special mounting glue for FISH and its preparation method

Abstract

The present application relates to a kind of FISH special mounting glue and its preparation method, belong to the technical field of biological material for fluorescence in situ hybridization detection.The purpose is to solve the problems such as fluorescence quenching and insufficient adhesion of existing mounting glue.The mounting glue is composed of modified acrylic resin, fluorescent protective agent, tackifier and other components, the fluorescent protective agent is 3:2 compound of ultraviolet absorber and anti-quenching agent, the solvent is 7:3 compound of propylene glycol methyl ether acetate and ethanol.The preparation method includes the preparation of free radical polymerization of modified acrylic resin, and step-by-step feeding, temperature control stirring, filtering and defoaming process of mounting glue.The mounting glue has the characteristics of excellent fluorescence protection effect, strong adhesion, acid and alkali resistance, low autofluorescence, moderate drying speed, good long-term stability, good biocompatibility, high transparency and suitable viscosity, suitable for clinical detection and scientific research, and the process is stable and easy to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology for fluorescence in situ hybridization detection, specifically relating to a FISH-specific mounting adhesive and its preparation method. Background Technology

[0002] Fluorescence in situ hybridization (FISH) is a molecular biology technique that uses complementary base pairing to bind nucleic acid probes to target nucleic acid sequences in a sample, utilizing fluorescence signals to detect and quantify the target sequences. With its high specificity and sensitivity, it has been widely applied in chromosomal abnormality detection, gene amplification / deletion analysis, and pathogen identification. Mounting adhesive, a key component of FISH detection, has core functions including: fixing coverslips and slides, protecting fluorescent probes from photo-oxidation and environmental interference, maintaining a stable hybridization environment, ensuring optical transparency during observation, and preventing the generation of autofluorescence that could interfere with the detection signal.

[0003] However, existing FISH mounting adhesives still have many technical bottlenecks, which seriously affect the accuracy of detection and experimental efficiency:

[0004] Fluorescence quenching is a prominent issue: existing mounting adhesives lack an efficient anti-quenching system, and fluorescent probes are prone to photo-oxidation under excitation light, making long-term dynamic observation impossible, which is particularly unfavorable for the detection of low-abundance target sequences;

[0005] Insufficient adhesion and environmental stability: The adhesion to glass substrates (glass slides, coverslips) is weak. In subsequent washing, staining or acidic / alkaline environments (pH=2-12 buffer solution is commonly used in FISH detection), the slides are prone to detachment and peeling. They are also prone to aging and cracking after long-term storage.

[0006] Significant interference from autofluorescence: Some components of the mounting adhesive (such as ordinary resin and plasticizer) have inherent autofluorescence, which leads to increased background noise in the detection and affects the identification of weak fluorescence signals;

[0007] The imbalance between drying speed and ease of use: drying too quickly can easily cause bubbles and uneven application, while drying too slowly will prolong the experimental cycle. Existing products cannot balance both.

[0008] Poor long-term stability and resistance to mold: It is prone to oxidation and deterioration during storage. Its performance deteriorates significantly after being stored at 4℃ for 3 months. Furthermore, it is prone to mold growth in humid environments, which can lead to the failure of the sealing adhesive.

[0009] Poor biocompatibility: Some components are toxic to cell samples and may damage the structure of target nucleic acids, affecting hybridization efficiency and the accuracy of detection results.

[0010] To address the aforementioned technical deficiencies, this invention optimizes component design and preparation process to develop a FISH-specific mounting adhesive with low fluorescence quenching rate, strong adhesion, good environmental stability, low autofluorescence, moderate drying speed, and excellent long-term stability, meeting the high standards required for clinical testing and scientific research experiments. Summary of the Invention

[0011] The purpose of this invention is to provide a FISH-specific sealing adhesive and its preparation method, which has the characteristics of excellent fluorescence protection, strong and stable adhesion, low autofluorescence, moderate drying speed, good long-term stability, good biocompatibility, high transparency, and suitable viscosity.

[0012] The objective of this invention can be achieved through the following technical solutions:

[0013] A FISH-specific sealing adhesive is composed of the following components in parts by weight: 35-45 parts modified acrylic resin, 4-6 parts fluorescent protectant, 6-10 parts tackifier, 3-5 parts crosslinking agent, 1-2 parts stabilizer, 0.5-1 part defoamer, 30-40 parts solvent, and 0.5-1 part mildew inhibitor;

[0014] The fluorescent protective agent is composed of an ultraviolet absorber and an anti-quenching agent in a mass ratio of 3:2.

[0015] The solvent is a mixture of propylene glycol methyl ether acetate and ethanol in a mass ratio of 7:3.

[0016] As a preferred embodiment of the present invention, the modified acrylic resin is a copolymer of hydroxyethyl methacrylate and methyl methacrylate, with a molar ratio of 3:7.

[0017] As a preferred embodiment of the present invention, the ultraviolet absorber is 2-hydroxy-4-methoxybenzophenone, and the antiquenching agent is ascorbyl palmitate.

[0018] As a preferred embodiment of the present invention, the thickener is hydrogenated rosin glycerol ester.

[0019] As a preferred embodiment of the present invention, the crosslinking agent is 1,6-hexanediol diacrylate.

[0020] As a preferred embodiment of the present invention, the stabilizer is 2,6-di-tert-butyl-4-methylphenol.

[0021] As a preferred embodiment of the present invention, the defoamer is an organosiloxane defoamer, model BYK-066N.

[0022] As a preferred embodiment of the present invention, the antifungal agent is an isothiazolinone derivative, model MIT-10.

[0023] As a preferred embodiment of the present invention, the preferred mass proportions of each component are: 40 parts modified acrylic resin, 5 parts fluorescent protectant, 8 parts thickener, 4 parts crosslinking agent, 1.5 parts stabilizer, 0.8 parts defoamer, 35 parts solvent, and 0.7 parts mildew inhibitor.

[0024] A method for preparing the FISH-specific sealing adhesive as described above includes the preparation of modified acrylic resin and the preparation of the sealing adhesive, with the specific steps as follows:

[0025] S1. Preparation of modified acrylic resin:

[0026] W1. Weigh out hydroxyethyl methacrylate and methyl methacrylate in a molar ratio of 3:7. The amount of azobisisobutyronitrile (AIB) initiator is 0.8-1.2 parts of the total mass of the monomers, and the amount of ethyl acetate solvent is 1.5 times the total mass of the monomers.

[0027] W2. Add hydroxyethyl methacrylate, methyl methacrylate and ethyl acetate to a four-necked flask, stir at 400 r / min and 60℃ for 30 min to form a uniform prepolymer solution;

[0028] W3. Dissolve azobisisobutyronitrile in a small amount of ethyl acetate and add it dropwise to the prepolymer solution at a rate of 1 mL / min. Heat the solution to 70-75℃ and react at a constant temperature of 400 r / min for 6-8 hours.

[0029] W4. Add 0.5 parts of hydroquinone by total mass of monomers, stir for 10 min to terminate the reaction, and let it cool naturally to 30℃;

[0030] W5. Pour the reaction solution into 3 times the volume of anhydrous ethanol, stir at 300 r / min to precipitate, filter and collect the precipitate;

[0031] W6. The precipitate was washed three times with anhydrous ethanol and dried for 12 hours under vacuum of -0.09 MPa and 50°C to obtain the modified acrylic resin.

[0032] S2. Preparation of sealing adhesive:

[0033] X1. Weigh propylene glycol methyl ether acetate and ethanol according to the mass fraction, add them to the reaction vessel, stir at 25℃ and stirring speed of 300r / min for 15min, mix evenly, and obtain a mixed solvent;

[0034] X2. Heat the mixed solvent to 40±2℃, and slowly add the modified acrylic resin prepared in step S1 in 3 portions, with an interval of 10 min between each addition. Maintain a stirring speed of 500 r / min and stir for 60±5 min until the resin is completely dissolved to obtain a transparent and homogeneous resin solution.

[0035] X3. Cool the resin solution to 30±2℃, first add the fluorescent protective agent that has been mixed evenly in a mass ratio of 3:2, stir for 20 min, then add the thickener, crosslinking agent and stabilizer in sequence, adjust the stirring speed to 600 r / min, stir for 90±10 min, so that the components are fully dispersed.

[0036] X4. Add defoamer and mildew inhibitor, reduce the stirring speed to 400 r / min, and stir for 30 ± 5 min;

[0037] X5. Filter the above mixture through a 200-mesh filter cloth, then transfer it to a vacuum degassing tank and let it stand for 30 minutes at a vacuum of -0.08 to -0.09 MPa and 25°C to obtain FISH special sealing adhesive.

[0038] The beneficial effects of this invention are:

[0039] (1) This scheme uses a UV-9 and AP compound fluorescence protection system, with BHT stabilizer, to achieve a fluorescence intensity retention rate of ≥90% after 24 hours, which can support long-term dynamic observation for more than 24 hours and meet the requirements for low abundance target sequence detection.

[0040] (2) The modified acrylic resin in this solution forms a dense network structure with HDDA crosslinking agent and hydrogenated rosin glycerol ester tackifier, and the adhesion force reaches level 1 in the crosslinking test. After soaking in hydrochloric acid solution with pH=2 and sodium hydroxide solution with pH=12 for 24 hours, the adhesion force still remains at level 1, effectively solving the problems of peeling and cracking.

[0041] (3) All components are made of low autofluorescence materials, which have low autofluorescence intensity, effectively reducing background noise and improving the recognition accuracy of weak fluorescence signals.

[0042] (4) BHT stabilizer inhibits oxidative aging, MIT-10 antifungal agent has high antibacterial effect, fluorescence retention rate changes ≤3% after 6 months of storage at 4℃, no mold growth, and shelf life is extended to more than 12 months.

[0043] (5) The combination solvent of PMA and ethanol optimizes the drying rate. It dries completely in 2-3 hours at room temperature, which avoids the generation of bubbles too quickly and ensures experimental efficiency. The viscosity is controlled at 500-800 mPa·s, which facilitates uniform application and prevents dripping.

[0044] (6) All components meet the standards for biomedical materials, do not damage the structure of sample cells and target nucleic acids, and do not affect hybridization efficiency;

[0045] (7) High transparency, allowing clear observation of fluorescence signals without optical interference;

[0046] (8) The modified acrylic resin is prepared by free radical polymerization process, and the parameters are stable and easy to control; the sealing adhesive adopts step feeding, precise temperature control and vacuum degassing process, and the product batch stability is good, which is suitable for industrial production. Detailed Implementation

[0047] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0048] Example 1

[0049] Preparation of modified acrylic resin:

[0050] W1. Raw material preparation: Weigh 30 mol (3420 g) of hydroxyethyl methacrylate (HEMA) and 70 mol (7350 g) of methyl methacrylate (MMA) in a molar ratio of 3:7, with a total monomer mass of 10770 g; the amount of initiator azobisisobutyronitrile (AIBN) is 1.0 part of the total monomer mass, i.e., 107.7 g; the amount of solvent ethyl acetate is 1.5 times the total monomer mass, i.e., 16155 g.

[0051] W2. Construction of the prepolymer system: Add the above HEMA and MMA monomers and ethyl acetate solvent to a 50L four-necked flask, start the stirrer, set the stirring speed to 400r / min, heat to 60℃, and continue stirring for 30min. Observe the state of the system until the monomers are completely dissolved and a uniform and transparent prepolymer solution is formed.

[0052] W3. Initiation of polymerization reaction: Dissolve 107.7g AIBN in 500mL ethyl acetate and slowly add it dropwise to the prepolymer solution at a rate of 1mL / min through a constant pressure dropping funnel. After the addition is completed (about 8.3 hours), raise the reaction temperature to 72℃, maintain the stirring speed at 400r / min, and react at a constant temperature for 7 hours. During the reaction, take a sample for testing at 6 hours. The viscosity of the system reaches 920mPa·s. Continue the reaction for 1 hour to terminate the reaction.

[0053] W4. Termination of reaction and cooling: Add 0.5 parts of hydroquinone (53.85 g) of total monomer mass to the four-necked flask, stir for 10 min to terminate the polymerization reaction, turn off the heating device, and allow the system to cool down naturally to 30°C;

[0054] W5. Precipitation and purification: Slowly pour the cooled reaction solution into 3 times the volume of anhydrous ethanol, start the magnetic stirrer, set the stirring speed to 300 r / min, and gradually precipitate a white flocculent precipitate during the stirring process. Continue stirring for 30 min and then stop. Let stand for 30 min, filter with a Buchner funnel, and collect the precipitate.

[0055] W6. Washing and Drying: The precipitate was washed with anhydrous ethanol, with the volume of ethanol used each time being twice the mass of the precipitate (approximately 20 L of ethanol for the first wash, 18 L for the second, and 15 L for the third), for a total of 3 washes to remove unreacted monomers and residual solvents. The washed precipitate was then transferred to a vacuum drying oven, and dried at a vacuum of -0.09 MPa and a temperature of 50 °C for 12 hours to obtain powdered modified acrylic resin (HEMA-MMA copolymer). GPC analysis showed that the number average molecular weight was 6200 and the PDI was 1.35.

[0056] Preparation of FISH-specific sealing adhesive:

[0057] X1. Solvent pretreatment: Weigh 24.5 parts of propylene glycol methyl ether acetate (PMA) and 10.5 parts of ethanol, add them to a 500 mL stainless steel reactor, set the temperature to 25℃, the stirring speed to 300 r / min, and continue stirring for 15 min to obtain a uniform mixed solvent.

[0058] X2. Dissolving the base resin: Raise the temperature of the reactor to 40°C, and add 40 parts of the modified acrylic resin prepared above to the mixed solvent in three batches (13 parts for the first time, 13 parts for the second time after a 10-minute interval, and 14 parts for the third time after a 10-minute interval). Maintain a stirring speed of 500 r / min and continue stirring for 60 minutes. Observe every 15 minutes during this period to ensure that the resin is completely dissolved and obtain a transparent resin solution without particles.

[0059] X3. Mixing of functional components: Cool the resin solution to 30℃, first add 5 parts of pre-mixed fluorescent protective agent (3 parts of 2-hydroxy-4-methoxybenzophenone (UV-9) and 2 parts of ascorbyl palmitate (AP), stir for 20 min, then add 8 parts of hydrogenated rosin glycerol ester (tackifier), 4 parts of 1,6-hexanediol diacrylate (HDDA, crosslinking agent) and 1.5 parts of 2,6-di-tert-butyl-4-methylphenol (BHT, stabilizer) in sequence, adjust the stirring speed to 600 r / min, and continue stirring for 90 min to fully disperse the functional components and make the system uniform and transparent;

[0060] X4. Defoaming and mildew prevention treatment: Add 0.8 parts of organosiloxane defoamer (BYK-066N) and 0.7 parts of isothiazolinone derivative (MIT-10, mildew inhibitor), reduce the stirring speed to 400 r / min, stir for 30 min, observe the bubble situation of the system during the period, and ensure that the bubbles are completely eliminated;

[0061] X5. Filtration and Degassing: The above mixture is filtered through a 200-mesh nylon filter cloth to remove trace mechanical impurities and incompletely dissolved fine particles. The filtered liquid is then transferred to a vacuum degassing tank, where a vacuum of -0.085 MPa and a temperature of 25°C are set. The mixture is allowed to stand for 30 minutes to degas, resulting in a uniform, transparent, and bubble-free FISH-specific sealing adhesive.

[0062] Example 2

[0063] Preparation of modified acrylic resin:

[0064] W1. Raw material preparation: Weigh 27 mol (3078 g) of hydroxyethyl methacrylate (HEMA) and 63 mol (6615 g) of methyl methacrylate (MMA) in a molar ratio of 3:7, with a total monomer mass of 9693 g; the amount of initiator azobisisobutyronitrile (AIBN) is 0.9 parts of the total monomer mass, i.e., 87.24 g; the amount of solvent ethyl acetate is 1.5 times the total monomer mass, i.e., 14539.5 g.

[0065] W2. Prepolymer system construction: Add HEMA, MMA monomers and ethyl acetate solvent to a 50L four-necked flask, start the stirrer, stir at 400r / min and 60℃ for 30min until the monomers are completely dissolved and a homogeneous prepolymer solution is formed;

[0066] W3. Initiating the polymerization reaction: Dissolve 87.24g of AIBN in 400mL of ethyl acetate and add it dropwise to the prepolymer solution at a rate of 1mL / min. After the addition is complete, raise the temperature to 70℃ and react at a constant temperature of 400r / min for 6.5 hours. After 6 hours of reaction, take a sample and test the viscosity, which is 880mPa·s. Continue the reaction for another 0.5 hours.

[0067] W4. Termination of reaction and cooling: Add 48.47g of hydroquinone (0.5 parts of total monomer mass), stir for 10min to terminate the reaction, and allow to cool naturally to 30℃;

[0068] W5. Precipitation and purification: Pour the reaction solution into 3 times the volume of anhydrous ethanol, stir at 300 r / min to precipitate, let stand, filter and collect the precipitate;

[0069] W6. Washing and drying: The precipitate was washed three times with anhydrous ethanol (approximately 15L, 14L, and 13L of ethanol each time), and dried in a vacuum drying oven at -0.09MPa and 50℃ for 12 hours to obtain modified acrylic resin. The number-average molecular weight was 5800 and the PDI was 1.42, as determined by GPC.

[0070] Preparation of FISH-specific sealing adhesive:

[0071] X1. Solvent pretreatment: Weigh 21 parts of propylene glycol methyl ether acetate and 9 parts of ethanol, add them to a 500 mL reaction vessel, stir at 25 °C and 300 r / min for 15 min, and mix evenly to obtain a mixed solvent;

[0072] X2. Dissolving the base resin: Heat the mixed solvent to 38°C, and add 38 parts of the above modified acrylic resin in 3 portions (13 parts for the first time, 12 parts for the second time after a 10-minute interval, and 13 parts for the third time after a 10-minute interval). Stir at 500 r / min for 55 minutes until the resin is completely dissolved to form a transparent resin solution.

[0073] X3. Mixing of functional components: Cool the resin solution to 28°C, add 4 parts of pre-mixed fluorescent protective agent (2.4 parts of UV-9 and 1.6 parts of AP), stir for 20 min, then add 7 parts of hydrogenated rosin glycerol ester, 3.5 parts of HDDA and 1.2 parts of BHT in sequence, stir at 600 r / min for 85 min, and ensure that each component is fully dispersed.

[0074] X4. Defoaming and mildew prevention treatment: Add 0.6 parts of BYK-066N and 0.6 parts of MIT-10, stir at 400r / min for 28min to eliminate bubbles;

[0075] X5. Filtration and Degassing: After filtration through a 200-mesh filter cloth, the mixture is transferred to a vacuum degassing tank and allowed to stand at -0.08MPa and 25℃ for 30 minutes to degas, yielding FISH-specific sealing adhesive.

[0076] Example 3

[0077] Preparation of modified acrylic resin:

[0078] W1. Raw material preparation: Weigh 33 mol (3762 g) of hydroxyethyl methacrylate (HEMA) and 77 mol (8085 g) of methyl methacrylate (MMA) in a molar ratio of 3:7, with a total monomer mass of 11847 g; the amount of initiator azobisisobutyronitrile (AIBN) is 1.1 parts of the total monomer mass, i.e., 130.32 g; the amount of solvent ethyl acetate is 1.5 times the total monomer mass, i.e., 17770.5 g;

[0079] W2. Prepolymer system construction: Add the above monomers and solvents to a 50L four-necked flask, stir at 400r / min and 60℃ for 30min to form a homogeneous prepolymer solution;

[0080] W3. Initiating the polymerization reaction: AIBN was dissolved in 600 mL of ethyl acetate and added dropwise to the prepolymer solution at 1 mL / min. After the addition was completed, the temperature was raised to 75 °C and the reaction was carried out at a constant temperature of 400 r / min for 7.5 hours. After 7 hours of reaction, the viscosity was measured to be 980 mPa·s. The reaction was continued for another 0.5 hours.

[0081] W4. Termination of reaction and cooling: Add 59.24g of hydroquinone (0.5 parts of total monomer mass), stir for 10min to terminate the reaction, and allow to cool naturally to 30℃;

[0082] W5. Precipitation and purification: Pour 3 times the volume of anhydrous ethanol into the reaction solution, stir at 300 r / min to precipitate the precipitate, and collect it by filtration;

[0083] W6. Washing and drying: Wash three times with anhydrous ethanol (approximately 22L, 20L, and 18L each time), and dry at -0.09MPa and 50℃ for 12 hours to obtain modified acrylic resin. The number-average molecular weight was 6500 and the PDI was 1.31, as determined by GPC.

[0084] Preparation of FISH-specific sealing adhesive:

[0085] X1. Solvent pretreatment: Weigh 28 parts of propylene glycol methyl ether acetate and 12 parts of ethanol, add them to a 500 mL reaction vessel, stir at 25 °C and 300 r / min for 15 min to obtain a mixed solvent;

[0086] X2. Dissolving the base resin: Heat the mixed solvent to 42°C, and add 42 parts of modified acrylic resin in 3 portions (14 parts for the first time, 14 parts for the second time after a 10-minute interval, and 14 parts for the third time after a 10-minute interval). Stir at 500 rpm for 65 minutes until the resin is completely dissolved.

[0087] X3. Mixing of functional components: Cool the resin solution to 32℃, add 6 parts of fluorescent protectant (3.6 parts of UV-9 and 2.4 parts of AP), stir for 20 min, then add 9 parts of hydrogenated rosin glycerol ester, 4.5 parts of HDDA and 1.8 parts of BHT in sequence, and stir at 600 r / min for 95 min.

[0088] X4. Defoaming and mildew prevention treatment: Add 0.9 parts of BYK-066N and 0.9 parts of MIT-10, and stir at 400r / min for 32min;

[0089] X5. Filtration and Degassing: After filtration with 200-mesh filter cloth, the mixture is placed in a vacuum degassing tank at -0.09MPa and 25℃ for 30 minutes to degas and obtain FISH special sealing adhesive.

[0090] Comparative Example 1

[0091] The fluorescent protective agent is missing, and the remaining components and amounts are the same as in Example 1: 40 parts modified acrylic resin, 8 parts thickener, 4 parts crosslinking agent, 1.5 parts stabilizer, 0.8 parts defoamer, 35 parts solvent, and 0.7 parts mildew inhibitor.

[0092] Comparative Example 2

[0093] The tackifier is missing, but the remaining components and amounts are the same as in Example 1: 40 parts modified acrylic resin, 5 parts fluorescent protectant, 4 parts crosslinking agent, 1.5 parts stabilizer, 0.8 parts defoamer, 35 parts solvent, and 0.7 parts mildew inhibitor.

[0094] Comparative Example 3

[0095] The crosslinking agent is missing, and the remaining components and amounts are the same as in Example 1: 40 parts modified acrylic resin, 5 parts fluorescent protectant, 8 parts thickener, 1.5 parts stabilizer, 0.8 parts defoamer, 35 parts solvent, and 0.7 parts mildew inhibitor.

[0096] Comparative Example 4

[0097] The fluorescent protectant is a single 2-hydroxy-4-methoxybenzophenone, UV-9 5 parts, and the remaining components and amounts are the same as in Example 1.

[0098] Comparative Example 5

[0099] The fluorescent protectant is a single ascorbate palmitate, AP 5 parts, and the remaining components and amounts are the same as in Example 1.

[0100] Comparative Example 6

[0101] The tackifier was replaced with ordinary rosin, 8 parts, and the remaining components and amounts were the same as in Example 1.

[0102] Comparative Example 7

[0103] The crosslinking agent was replaced with ethylene glycol diacrylate, 4 parts of EGDA, and the remaining components and amounts were the same as in Example 1.

[0104] Comparative Example 8

[0105] The solvent ratio was changed to propylene glycol methyl ether acetate: ethanol = 5:5, with a total mass of 35 parts. The remaining components and amounts were the same as in Example 1.

[0106] Comparative Example 9

[0107] The amount of stabilizer was 0.5 parts, BHT was 0.5 parts, and the remaining components and amounts were the same as in Example 1.

[0108] Comparative Example 10

[0109] The antifungal agent was replaced with benzalkonium chloride, 0.7 parts, and the remaining components and dosages were the same as in Example 1.

[0110] Comparative Example 11

[0111] The modified acrylic resin was replaced with ordinary methyl methacrylate homopolymer, MMA homopolymer, number average molecular weight 6000, 40 parts, and the remaining components and amounts were the same as in Example 1.

[0112] Performance testing

[0113] The detection method is as follows:

[0114] Fluorescence retention rate: The mounting adhesive was evenly spread on the glass slide (50 μm thick), and the FITC-labeled nucleic acid probe was covered. The initial fluorescence intensity I0 was measured under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 520 nm). The sample was then placed under fluorescence irradiation for 24 h, and the fluorescence intensity I1 was measured again. Fluorescence retention rate = (I1 / I0) × 100%;

[0115] Adhesion test: Using a cross-cut tester, 100 grids of 1mm × 1mm were drawn on the adhesive coating. 3M 610 tape was then applied and quickly peeled off. The extent of coating peeling within the grids was observed, and the results were graded from 1 to 5 (Grade 1: No peeling; Grade 2: Peeling area < 5%; Grade 3: Peeling area 5%-15%; Grade 4: Peeling area 15%-35%; Grade 5: Peeling area > 35%).

[0116] Viscosity: Measured using an NDJ-8S rotational viscometer at 25℃ and 60 r / min.

[0117] Transmittance: The transmittance at a wavelength of 550 nm was measured using a UV-2550 ultraviolet-visible spectrophotometer (with a blank glass slide as a reference).

[0118] Acid and alkali resistance: The sealing adhesive samples were immersed in hydrochloric acid solution with pH=2 and sodium hydroxide solution with pH=12 respectively. After immersion at 25°C for 24 hours, they were taken out and dried until there was no liquid residue on the surface. The adhesion level was determined according to the adhesion test method described above.

[0119] Long-term stability: After sealing with mounting adhesive, the slide was stored in a 4°C refrigerator for 6 months. The fluorescence retention rate after storage was measured, and the difference between the fluorescence retention rate and the initial fluorescence retention rate was calculated (Δ fluorescence retention rate = initial fluorescence retention rate - fluorescence retention rate after storage).

[0120] Autofluorescence intensity: Under a fluorescence microscope (excitation wavelength 488nm, emission wavelength 520nm), the fluorescence intensity of the mounting adhesive coating that does not cover the fluorescent probe is measured. A blank slide is used as a control, and the relative fluorescence intensity value is recorded (the smaller the value, the less interference from autofluorescence).

[0121] Drying speed: At room temperature (25°C, 50% relative humidity), the sealing adhesive was evenly applied to the glass slide (thickness 50μm), and the time it took for the coating to dry completely (not sticky to the touch, and not dripping) was recorded.

[0122] Biocompatibility: Mounting adhesive extract was prepared according to GB / T 16886.12-2017 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials" (extraction ratio 1 g / mL, 37℃ for 24 h); HeLa cells were seeded in 96-well plates (1×10⁻⁶ cells / well). 4 Cells were cultured in wells for 24 hours, and then the extraction medium was added. The cells were cultured for another 24 hours. Cell viability was determined by the CCK-8 assay. Cell viability = (OD value of experimental group / OD value of control group) × 100% (the control group was cell culture medium without extraction medium).

[0123] Resistance to mold: The test was conducted according to GB / T 1741-2020 "Test Method for Resistance to Mold in Coatings". The mounting adhesive sample was placed in a mold incubator (temperature 28℃, relative humidity 95%) and inoculated with a mixed spore suspension of Aspergillus niger and Penicillium sp. After 7 days of incubation, the mold growth was observed and graded from 0 to 4 (Grade 0: no mold growth; Grade 1: mold growth area <10%; Grade 2: mold growth area 10%-30%; Grade 3: mold growth area 30%-60%; Grade 4: mold growth area >60%).

[0124] The test results are shown in Table 1.

[0125] Table 1 Performance test data of Examples 1-3 and Comparative Examples 1-11

[0126]

[0127]

[0128]

[0129]

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A FISH-specific sealing adhesive, characterized in that, It is composed of the following components in parts by weight: 35-45 parts modified acrylic resin, 4-6 parts fluorescent protectant, 6-10 parts tackifier, 3-5 parts crosslinking agent, 1-2 parts stabilizer, 0.5-1 part defoamer, 30-40 parts solvent, and 0.5-1 part mildew inhibitor; The fluorescent protective agent is composed of an ultraviolet absorber and an anti-quenching agent in a mass ratio of 3:

2. The solvent is a mixture of propylene glycol methyl ether acetate and ethanol in a mass ratio of 7:

3. The modified acrylic resin is a copolymer of hydroxyethyl methacrylate and methyl methacrylate, with a molar ratio of 3:

7. The ultraviolet absorber is 2-hydroxy-4-methoxybenzophenone, and the antiquenching agent is ascorbyl palmitate. The thickener is hydrogenated rosin glyceride; The crosslinking agent is 1,6-hexanediol diacrylate; The stabilizer is 2,6-di-tert-butyl-4-methylphenol; The antifungal agent is an isothiazolinone derivative, model number MIT-10.

2. The FISH-specific sealing adhesive according to claim 1, characterized in that, The defoamer is an organosiloxane defoamer, model number BYK-066N.

3. The FISH-specific sealing adhesive according to claim 1, characterized in that, The mass fractions of each component are as follows: 40 parts modified acrylic resin, 5 parts fluorescent protectant, 8 parts tackifier, 4 parts crosslinking agent, 1.5 parts stabilizer, 0.8 parts defoamer, 35 parts solvent, and 0.7 parts mildew inhibitor.

4. A method for preparing a FISH-specific sealing adhesive as described in any one of claims 1-3, characterized in that, The preparation of modified acrylic resin and sealing adhesive includes the following specific steps: S1. Preparation of modified acrylic resin: W1. Weigh out hydroxyethyl methacrylate and methyl methacrylate in a molar ratio of 3:

7. The amount of azobisisobutyronitrile (AIB) initiator is 0.8-1.2 parts of the total mass of the monomers, and the amount of ethyl acetate solvent is 1.5 times the total mass of the monomers. W2. Add hydroxyethyl methacrylate, methyl methacrylate and ethyl acetate to a four-necked flask, stir at 400 r / min and 60℃ for 30 min to form a uniform prepolymer solution; W3. Dissolve azobisisobutyronitrile in a small amount of ethyl acetate and add it dropwise to the prepolymer solution at a rate of 1 mL / min. Heat the solution to 70-75℃ and react at a constant temperature of 400 r / min for 6-8 hours. W4. Add 0.5 parts of hydroquinone by total mass of monomers, stir for 10 min to terminate the reaction, and let it cool naturally to 30℃; W5. Pour the reaction solution into 3 times the volume of anhydrous ethanol, stir at 300 r / min to precipitate, filter and collect the precipitate; W6. The precipitate was washed three times with anhydrous ethanol and dried for 12 hours under vacuum of -0.09 MPa and 50°C to obtain the modified acrylic resin. S2. Preparation of sealing adhesive: X1. Weigh propylene glycol methyl ether acetate and ethanol by weight, add them to the reaction vessel, stir at 25°C and 300 r / min for 15 min, mix evenly to obtain a mixed solvent; X2. Heat the mixed solvent to 40±2℃, and slowly add the modified acrylic resin prepared in step S1 in 3 portions, with an interval of 10 min between each addition. Maintain a stirring speed of 500 r / min and stir for 60±5 min until the resin is completely dissolved to obtain a transparent and homogeneous resin solution. X3. Cool the resin solution to 30±2℃, first add the fluorescent protective agent that has been mixed evenly in a mass ratio of 3:2, stir for 20 min, then add the thickener, crosslinking agent and stabilizer in sequence, adjust the stirring speed to 600 r / min, stir for 90±10 min, so that the components are fully dispersed. X4. Add defoamer and mildew inhibitor, reduce the stirring speed to 400 r / min, and stir for 30 ± 5 min to obtain the mixture; X5. Filter the mixture obtained in step X4 through a 200-mesh filter cloth, then transfer it to a vacuum degassing tank and let it stand for 30 minutes at a vacuum of -0.08 to -0.09 MPa and 25°C to obtain FISH special sealing adhesive.

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