FISH special-purpose sealing sheet glue and preparation method thereof

Abstract

The invention relates to a special mounting adhesive for FISH (fluorescence in situ hybridization) and a preparation method of the special mounting adhesive, and belongs to the technical field of biological materials for fluorescence in situ hybridization detection. The invention aims to solve the problems of fluorescence quenching, insufficient adhesive force and the like of the existing sealing adhesive. The sealing adhesive is composed of modified acrylic resin, a fluorescent protective agent, a tackifier and the like, the fluorescent protective agent is formed by compounding an ultraviolet light absorber and an anti-quenching agent according to the ratio of 3: 2, and the solvent is formed by compounding propylene glycol methyl ether acetate and ethyl alcohol according to the ratio of 7: 3. The preparation method comprises the processes of free radical polymerization preparation of the modified acrylic resin, step-by-step feeding of the sealing adhesive, temperature-controlled stirring, filtering, defoaming and the like. The sealant has the characteristics of excellent fluorescence protection effect, strong adhesive force, acid and alkali resistance, low autofluorescence, moderate drying speed, good long-term stability, good biocompatibility, high transparency and proper viscosity, is suitable for clinical detection and scientific research experiments, is stable in process and is easy for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials for fluorescence in situ hybridization detection, and particularly relates to a special mounting glue for FISH and a preparation method thereof. BACKGROUND

[0002] Fluorescence in situ hybridization (FISH) is a molecular biology technology that combines nucleic acid probes with target nucleic acid sequences in a sample through base complementary pairing, and detects the positioning and quantification of target sequences by using fluorescence signals. With the advantages of high specificity and high sensitivity, FISH has been widely used in chromosomal abnormality detection, gene amplification / loss analysis, pathogen identification and other fields. Mounting glue is a key supporting material for FISH detection, and its core functions include: fixing the cover glass and the glass slide, protecting the fluorescence probe from photo-oxidation and environmental interference, maintaining the stability of the hybridization environment of the sample, ensuring the optical transparency during observation, and avoiding the generation of spontaneous fluorescence interference with the detection signal.

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

[0004] Fluorescence quenching problem is prominent: the existing mounting glue lacks an efficient anti-quenching system, and the fluorescence probe is prone to photo-oxidation under excitation light, which makes it impossible to conduct long-term dynamic observation, especially for low-abundance target sequence detection;

[0005] Insufficient adhesion and environmental stability: the adhesion to the glass carrier (glass slide and cover glass) is weak, and the mounting glue is prone to peeling and warping in subsequent washing, staining or acid-base environment (pH = 2-12 buffer solution commonly used in FISH detection), and is prone to aging and cracking after long-term storage;

[0006] Spontaneous fluorescence interference is obvious: some components of the mounting glue (such as ordinary resin and plasticizer) have inherent spontaneous fluorescence, which increases the background noise of the detection and affects the identification of weak fluorescence signals;

[0007] Dryness speed and convenience are imbalanced: too fast drying is prone to produce bubbles and uneven coating, and too slow drying prolongs the experimental period, so the existing products are difficult to balance the two;

[0008] Poor long-term stability and anti-mildew property: the mounting glue is prone to oxidation and deterioration during storage, and its performance decreases significantly after 3 months of storage at 4℃, and it is prone to mildew in humid environment, resulting in the failure of the mounting glue;

[0009] Poor biocompatibility: some components are toxic to cell samples, which may damage the structure of target nucleic acid and affect the hybridization efficiency and accuracy of the detection results.

[0010] In order to solve the above technical defects, the present application optimizes the component design and preparation process, and develops a FISH special mounting glue with low fluorescence quenching rate, strong adhesion, good environmental stability, low spontaneous fluorescence, moderate drying speed, and good long-term stability, which meets the high-standard requirements of clinical detection and scientific research experiments. SUMMARY

[0011] The present application aims to provide a FISH special mounting glue and a preparation method thereof, which has excellent fluorescence protection effect, strong and stable adhesion, low spontaneous fluorescence, moderate drying speed, good long-term stability, good biocompatibility, high transparency, and appropriate viscosity.

[0012] The object of the present application can be achieved by the following technical solutions:

[0013] A FISH special mounting glue is composed of the following components by mass fraction: modified acrylic resin 35-45 parts, fluorescence protection agent 4-6 parts, tackifier 6-10 parts, crosslinking agent 3-5 parts, stabilizer 1-2 parts, defoaming agent 0.5-1 part, solvent 30-40 parts, and mildew-proof agent 0.5-1 part.

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

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

[0016] As a preferred technical solution of the present application, the modified acrylic resin is a copolymer of hydroxyethyl methacrylate and methyl methacrylate, and the molar ratio of the two is 3:7.

[0017] As a preferred technical solution of the present application, the ultraviolet absorber is 2-hydroxy-4-methoxybenzophenone, and the anti-quenching agent is ascorbic acid palmitate.

[0018] As a preferred technical solution of the present application, the tackifier is hydrogenated rosin glyceride.

[0019] As a preferred technical solution of the present application, the crosslinking agent is 1,6-hexanediol diacrylate.

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

[0021] As a preferred technical solution of the present application, the defoaming agent is an organosiloxane defoaming agent, and the model number is BYK-066N.

[0022] As a preferred technical solution of the present application, the mildew-proof agent is an isothiazolinone derivative, and the model number is MIT-10.

[0023] As a preferred technical solution of the present application, the mass parts of each component are preferably: modified acrylic resin 40 parts, fluorescent protective agent 5 parts, tackifier 8 parts, crosslinking agent 4 parts, stabilizer 1.5 parts, defoaming agent 0.8 parts, solvent 35 parts, and mildew-proof agent 0.7 parts.

[0024] A preparation method of the FISH special mounting glue as described above, comprising preparation of modified acrylic resin and preparation of mounting glue, and the specific steps are as follows:

[0025] S1. Preparation of modified acrylic resin:

[0026] W1. The molar ratio of hydroxyethyl methacrylate to methyl methacrylate is 3:7, the amount of initiator azobisisobutyronitrile is 0.8-1.2 parts of the total mass of monomers, and the amount of solvent ethyl acetate is 1.5 times the total mass of 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, then add it to the prepolymer solution at a rate of 1 mL / min, heat to 70-75℃, and react at 400 r / min for 6-8 hours;

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

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

[0031] W6. Wash the precipitate with anhydrous ethanol 3 times, dry at a vacuum degree of -0.09 MPa and a temperature of 50℃ for 12 hours to obtain the modified acrylic resin;

[0032] S2. Preparation of mounting glue:

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

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

[0035] X3. The resin solution is cooled to 30±2℃, and the fluorescent protective agent is first added, and stirred for 20 min, then the tackifier, crosslinking agent and stabilizer are added in sequence, the stirring speed is adjusted to 600 r / min, and stirred for 90±10 min to make the components fully dispersed;

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

[0037] X5. The above mixture is filtered through a 200-mesh filter cloth, and then transferred to a vacuum degassing tank, and degassed at a vacuum degree of-0.08~-0.09 MPa and 25℃ for 30 min to obtain the FISH special mounting glue.

[0038] The beneficial effects of the present application are:

[0039] (1) The present scheme uses the UV-9 and AP compound fluorescent protection system, cooperates with the BHT stabilizer, and the 24-hour fluorescent intensity retention rate is ≥90%, which can support long-term dynamic observation for more than 24 hours, and meets the detection needs of low-abundance target sequences;

[0040] (2) The modified acrylic resin of the present scheme forms a dense network structure with the HDDA crosslinking agent and hydrogenated rosin glyceride tackifier, and the adhesion reaches the 1st level of the hundred-grade method, and after being soaked in pH=2 hydrochloric acid solution and pH=12 sodium hydroxide solution for 24h, the adhesion still remains the 1st level, effectively solving the problems of film peeling and cracking;

[0041] (3) All components are selected from low spontaneous fluorescence materials, the spontaneous fluorescence intensity is low, which effectively reduces the background noise and improves the recognition accuracy of weak fluorescent signals;

[0042] (4) The BHT stabilizer inhibits oxidation and aging, the MIT-10 mildew-proof agent has high bacteriostatic effect, the fluorescence retention rate changes ≤3% after being stored at 4℃ for 6 months, there is no mildew phenomenon, and the shelf life is extended to more than 12 months;

[0043] (5) The compound solvent of PMA and ethanol optimizes the drying rate, and the sample is completely dried at room temperature for 2-3 hours, which avoids the generation of bubbles caused by too fast drying and ensures the experimental efficiency, and the viscosity is controlled at 500-800 mPa·s, which is convenient for uniform coating and has no flow phenomenon;

[0044] (6) All components meet the biological medical material standard and do not damage the sample cells and target nucleic acid structure, and do not affect the hybridization efficiency;

[0045] (7) High transparency, clear observation of fluorescent signals, and no optical interference;

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

[0047] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects according to the present application are described in detail as follows in combination with examples.

[0048] Example 1

[0049] Preparation of the modified acrylic resin:

[0050] W1. Raw material preparation: 30 mol (3420 g) of hydroxyethyl methacrylate (HEMA) and 70 mol (7350 g) of methyl methacrylate (MMA) were weighed according to a molar ratio of 3:7, the total mass of monomers was 10770 g; the amount of initiator azobisisobutyronitrile (AIBN) was 1.0 part of the total mass of monomers, that is, 107.7 g; the amount of solvent ethyl acetate was 1.5 times the total mass of monomers, that is, 16155 g;

[0051] W2. Prepolymer system building: the above-mentioned HEMA, MMA monomers and ethyl acetate solvent were added to a 50L four-necked flask, the stirrer was started, the stirring speed was set to 400r / min, the temperature was raised to 60℃, and the stirring was continued for 30 min, the system state was observed until the monomers were completely dissolved, and a uniform transparent prepolymer liquid was formed;

[0052] W3. Initiation of polymerization reaction: 107.7g of AIBN was dissolved in 500mL of ethyl acetate, and was slowly added to the prepolymer liquid at a rate of 1mL / min through a constant pressure dropping funnel, after the addition was completed (about 8.3 hours), the reaction temperature was raised to 72℃, the stirring speed was kept at 400r / min, and the temperature was kept constant for 7 hours, during which the system viscosity reached 920mPa·s when the sample was taken for detection at 6 hours, and the reaction was continued for 1 hour and then terminated;

[0053] W4. Reaction termination and temperature reduction: 0.5 parts of hydroquinone (53.85g) of the total mass of monomers was added to the four-necked flask, the polymerization reaction was terminated after stirring for 10 min, and the heating device was turned off to allow the system to naturally cool to 30℃;

[0054] W5. Precipitation and purification: the cooled reaction liquid was slowly poured into 3 times the volume of anhydrous ethanol, a magnetic stirrer was started, and the stirring speed was set to 300r / min, white flocculent precipitate gradually separated out during the stirring process, the stirring was continued for 30 min and then stopped, and the system was allowed to stand for 30 min, then a Buchner funnel was used for suction filtration, and the precipitated product was collected;

[0055] W6. Washing and drying: the precipitated product was washed with anhydrous ethanol, each time using ethanol with a volume of 2 times the mass of the precipitate (about 20 L of ethanol for the first washing, 18 L for the second, and 15 L for the third), a total of 3 times, to remove unreacted monomers and residual solvents; the washed precipitate was transferred to a vacuum drying oven, set to a vacuum of -0.09 MPa and a temperature of 50°C, and dried for 12 hours to obtain a modified acrylic resin (HEMA-MMA copolymer) in powder form, which was detected by GPC to have a number average molecular weight of 6200 and a PDI of 1.35.

[0056] Preparation of a special mounting medium for FISH:

[0057] X1. Solvent pretreatment: 24.5 parts of propylene glycol methyl ether acetate (PMA) and 10.5 parts of ethanol were weighed into a 500 mL stainless steel reaction kettle, set to a temperature of 25°C and a stirring speed of 300 r / min, and continuously stirred for 15 min to obtain a uniform mixed solvent;

[0058] X2. Dissolution of the base resin: the temperature of the reaction kettle was raised to 40°C, and 40 parts of the modified acrylic resin prepared above were added to the mixed solvent in three portions (13 parts for the first portion, 13 parts for the second portion added after 10 min, and 14 parts for the third portion added after another 10 min), while maintaining a stirring speed of 500 r / min, and continuously stirring for 60 min, during which the system was observed every 15 min to ensure complete dissolution of the resin, thereby obtaining a transparent resin solution without particles;

[0059] X3. Mixing of functional components: the resin solution was cooled to 30°C, and 5 parts of a pre-mixed fluorescent protector (3 parts of 2-hydroxy-4-methoxybenzophenone (UV-9) and 2 parts of ascorbyl palmitate (AP)) were added first, and after stirring for 20 min, 8 parts of hydrogenated rosin glyceride (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) were added in sequence, the stirring speed was adjusted to 600 r / min, and the system was continuously stirred for 90 min to ensure that the functional components were fully dispersed, and the system was uniform and transparent;

[0060] X4. Defoaming and mildew-proof treatment: 0.8 parts of a silicone defoamer (BYK-066N) and 0.7 parts of a mildew-proof agent (MIT-10) were added, the stirring speed was reduced to 400 r / min, and the system was stirred for 30 min, during which the bubble situation was observed to ensure that the bubbles were completely eliminated;

[0061] X5. Filtering and defoaming: the above mixed solution is filtered through a 200 mesh nylon filter cloth to remove trace mechanical impurities and incompletely dissolved fine particles, and then the filtered liquid is transferred to a vacuum defoaming tank, the vacuum degree is set to-0.085 MPa, the temperature is 25°C, and the liquid is left to stand for 30 minutes to defoam. Finally, the FISH special mounting medium is obtained, which is uniform, transparent and bubble-free.

[0062] Example 2

[0063] Preparation of modified acrylic resin:

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

[0065] W2. Prepolymer system setup: HEMA, MMA monomers and ethyl acetate solvent are added to a 50 L four-necked flask, the stirrer is started, and stirring is carried out at 400 r / min and 60°C for 30 min until the monomers are completely dissolved to form a uniform prepolymer solution;

[0066] W3. Initiation of polymerization reaction: 87.24 g of AIBN is dissolved in 400 mL of ethyl acetate and added dropwise to the prepolymer solution at a rate of 1 mL / min. After the dropwise addition is completed, the temperature is raised to 70°C, and constant temperature reaction is carried out at 400 r / min for 6.5 hours. The viscosity of the sample taken at 6 hours of reaction is 880 mPa·s, and the reaction is continued for 0.5 hours;

[0067] W4. Reaction termination and cooling: 48.47 g of hydroquinone (0.5 parts of the total mass of monomers) is added, stirring for 10 min to terminate the reaction, and then the temperature is naturally lowered to 30°C;

[0068] W5. Precipitation and purification: the reaction solution is poured into 3 times the volume of anhydrous ethanol, and the precipitate is separated by stirring at 300 r / min. The precipitate is collected by filtration after standing;

[0069] W6. Washing and drying: the precipitate is washed with anhydrous ethanol for 3 times (each time the amount of ethanol is about 15 L, 14 L and 13 L), and then dried in a vacuum drying oven at-0.09 MPa and 50°C for 12 hours to obtain the modified acrylic resin. The number average molecular weight of the modified acrylic resin is 5800 and the PDI is 1.42, as detected by GPC.

[0070] Preparation of FISH special mounting medium:

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

[0072] X2. Dissolution of base resin: heat the mixed solvent to 38°C, add the modified acrylic resin (38 parts) in three portions (13 parts in the first portion, 12 parts in the second portion, and 13 parts in the third portion after an interval of 10 min), and stir at 500 r / min for 55 min 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 the pre-mixed fluorescent protective agent (4 parts of UV-92.4 parts and AP 1.6 parts), stir for 20 min, then add glyceryl hydrogenated rosin (7 parts), HDDA (3.5 parts), and BHT (1.2 parts) in sequence, and stir at 600 r / min for 85 min until the components are fully dispersed;

[0074] X4. Defoaming and mildew-proof treatment: add BYK-066N (0.6 parts) and MIT-10 (0.6 parts), and stir at 400 r / min for 28 min to eliminate air bubbles;

[0075] X5. Filtration and defoaming: filter through a 200-mesh filter cloth, transfer to a vacuum defoaming tank, and stand for defoaming at -0.08 MPa and 25°C for 30 min to obtain the FISH special mounting medium.

[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) according to a molar ratio of 3:7, and the total mass of the monomers is 11847 g; the amount of initiator azobisisobutyronitrile (AIBN) is 1.1 parts of the total mass of the monomers, i.e., 130.32 g; and the amount of solvent ethyl acetate is 1.5 times the total mass of the monomers, i.e., 17770.5 g;

[0079] W2. Construction of pre-polymer system: add the above monomers and solvent into a 50 L four-necked flask, and stir at 400 r / min and 60°C for 30 min to form a uniform pre-polymer solution;

[0080] W3. Initiation of polymerization reaction: dissolve AIBN in 600 mL of ethyl acetate, and add it into the pre-polymer solution at a rate of 1 mL / min; after the addition is completed, heat to 75°C, and react at a constant temperature of 400 r / min for 7.5 hours; when the reaction has proceeded for 7 hours, take a sample for viscosity detection, and continue the reaction for 0.5 hour;

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

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

[0083] W6. Washing and drying: wash with anhydrous ethanol 3 times (each time use about 22 L, 20 L, and 18 L), dry at -0.09 MPa and 50°C for 12 hours to obtain the modified acrylic resin, which has a number average molecular weight of 6500 and a PDI of 1.31 as detected by GPC.

[0084] Preparation of a special mounting glue for FISH:

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

[0086] X2. Dissolution of base resin: heat the mixed solvent to 42°C, add 42 parts of modified acrylic resin in 3 portions (14 parts in the first portion, 14 parts in the second portion after 10 min, and 14 parts in the third portion after another 10 min), and stir at 500 r / min for 65 min until the resin is completely dissolved;

[0087] X3. Mixing of functional components: cool the resin solution to 32°C, add 6 parts of fluorescent protectant (UV-9 3.6 parts and AP 2.4 parts), stir for 20 min, then add 9 parts of hydrogenated rosin glyceride, 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-proof treatment: add 0.9 parts of BYK-066N and 0.9 parts of MIT-10, and stir at 400 r / min for 32 min;

[0089] X5. Filtration and defoaming: filter through 200 mesh filter cloth, then place in a vacuum defoaming tank at -0.09 MPa and 25°C for 30 min of static defoaming to obtain the special mounting glue for FISH.

[0090] Comparative Example 1

[0091] Omit the fluorescent protectant, and the other components and amounts are the same as in Example 1: 40 parts of modified acrylic resin, 8 parts of tackifier, 4 parts of crosslinking agent, 1.5 parts of stabilizer, 0.8 parts of defoaming agent, 35 parts of solvent, and 0.7 parts of mildew-proof agent.

[0092] Comparative Example 2

[0093] Example 1 was repeated except that the crosslinking agent was omitted and the amounts of the other components were adjusted accordingly: modified acrylic resin 40 parts, fluorescent protecting agent 5 parts, tackifier 8 parts, stabilizer 1.5 parts, antifoaming agent 0.8 parts, solvent 35 parts, and mildew preventive 0.7 parts.

[0094] Comparative Example 3

[0095] Example 1 was repeated except that the tackifier was omitted and the amounts of the other components were adjusted accordingly: modified acrylic resin 40 parts, fluorescent protecting agent 5 parts, crosslinking agent 4 parts, stabilizer 1.5 parts, antifoaming agent 0.8 parts, solvent 35 parts, and mildew preventive 0.7 parts.

[0096] Comparative Example 4

[0097] The fluorescent protecting agent was a single compound, UV-9, 5 parts, and the amounts of the other components were adjusted accordingly.

[0098] Comparative Example 5

[0099] The fluorescent protecting agent was a single compound, AP, 5 parts, and the amounts of the other components were adjusted accordingly.

[0100] Comparative Example 6

[0101] The tackifier was replaced by ordinary rosin, 8 parts, and the amounts of the other components were adjusted accordingly.

[0102] Comparative Example 7

[0103] The crosslinking agent was replaced by ethylene glycol diacrylate, EGDA, 4 parts, and the amounts of the other components were adjusted accordingly.

[0104] Comparative Example 8

[0105] The solvent ratio was changed to propylene glycol methyl ether acetate: ethanol = 5:5, total 35 parts, and the amounts of the other components were adjusted accordingly.

[0106] Comparative Example 9

[0107] The amount of stabilizer was 0.5 parts, BHT 0.5 parts, and the amounts of the other components were adjusted accordingly.

[0108] Comparative Example 10

[0109] The mildew preventive was replaced by benzalkonium chloride, 0.7 parts, and the amounts of the other components were adjusted accordingly.

[0110] Comparative Example 11

[0111] The modified acrylic resin was replaced by ordinary methyl methacrylate homopolymer, MMA homopolymer, number average molecular weight 6000, 40 parts, and the amounts of the other components were adjusted accordingly.

[0112] Performance test

[0113] The detection method is as follows:

[0114] Fluorescence retention rate: evenly spread the mounting medium on the slide glass (thickness 50 μm), cover the FITC-labeled nucleic acid probe, and measure the initial fluorescence intensity I0under the fluorescence microscope (excitation wavelength 488 nm, emission wavelength 520 nm); continuously irradiate the sample under the fluorescence irradiation for 24 h, and measure the fluorescence intensity I1again. The fluorescence retention rate = (I1 / I0) x 100%;

[0115] Adhesion: draw 100 grids of 1 mm x 1 mm on the mounting medium coating layer using a cross-cut knife, paste 3M 610 type adhesive tape and quickly peel off, and observe the coating shedding in the grid. The grade is divided into 1-5 levels (1 level: no shedding; 2 level: shedding area < 5%; 3 level: shedding area 5%-15%; 4 level: shedding area 15%-35%; 5 level: shedding area > 35%);

[0116] Viscosity: measured at 25°C and 60 r / min using a NDJ-8S type rotary viscometer;

[0117] Transmittance: measured at 550 nm wavelength using a UV-2550 type ultraviolet-visible spectrophotometer (using a blank slide as a reference);

[0118] Acid and alkali resistance: immerse the mounting medium sample in a hydrochloric acid solution with pH = 2 and a sodium hydroxide solution with pH = 12 respectively, take out after 24 h of immersion at 25°C, and air dry until there is no liquid residue on the surface. Determine the adhesion grade according to the above adhesion detection method;

[0119] Long-term stability: store the mounting medium sealed in a 4°C refrigerator for 6 months, measure the fluorescence retention rate after storage, and calculate the difference from the initial fluorescence retention rate (Δ fluorescence retention rate = initial fluorescence retention rate - fluorescence retention rate after storage);

[0120] Spontaneous fluorescence intensity: measure the fluorescence intensity of the mounting medium coating layer without covering the fluorescent probe under the fluorescence microscope (excitation wavelength 488 nm, emission wavelength 520 nm), using a blank slide as a control, and record the relative fluorescence intensity value (the smaller the value, the smaller the spontaneous fluorescence interference);

[0121] Drying speed: evenly spread the mounting medium on the slide glass (thickness 50 μm) at room temperature (25°C, relative humidity 50%), and record the time for the coating to completely dry (no stickiness and no flow when touched);

[0122] Biocompatibility: according to GB / T 16886.12-2017 "Medical devices-Biological evaluation-Part 12: Sample preparation and reference materials" to prepare the embedding glue immersion extract (immersion ratio 1 g / mL, 37℃ immersion 24h); HeLa cells were inoculated in 96-well plates (1×10 4 The survival rate of cells was determined by CCK-8 method, and the survival rate of cells=(OD value of test group / OD value of control group)×100% (control group was cell culture solution without immersion liquid).

[0123] Mildew resistance: according to GB / T 1741-2020 "Paint film mildew resistance test method", the embedding glue sample was placed in a mold culture box (temperature 28℃, relative humidity 95%), inoculated with mixed spore suspension of Aspergillus niger and Penicillium sp., and observed for mildew after 7 days of culture. The grade was divided into 0-4 levels (0 level: no mildew; 1 level: mildew area <10%; 2 level: mildew area 10%-30%; 3 level: mildew area 30%-60%; 4 level: mildew 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 is only the preferred embodiment of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to make equivalent embodiments with equivalent changes, without departing from the scope of the technical solution of the present application. Any modification, equivalent change and modification of the above embodiments made in accordance with the technical essence of the present application, without departing from the technical solution of the present application, are still within the scope of the technical solution of the present application.

Claims

1. A FISH-specific mounting medium, characterized in that, The modified acrylic resin, the fluorescent protective agent, the tackifier, the crosslinking agent, the stabilizer, the defoaming agent, the solvent and the mildew-proof agent are respectively 40 parts, 5 parts, 8 parts, 4 parts, 1.5 parts, 0.8 parts, 35 parts and 0.7 parts by mass. The fluorescent protective agent is compounded by the ultraviolet absorber and the anti-quenching agent at a mass ratio of 3:

2. The solvent is compounded by propylene glycol methyl ether acetate and ethanol at a mass ratio of 7:

3.

2. The FISH-specific mounting gel according to claim 1, characterized in that, The modified acrylic resin is a copolymer of hydroxyethyl methacrylate and methyl methacrylate, and the molar ratio of the two is 3:

7.

3. The FISH-specific mounting medium of claim 1, wherein, The ultraviolet absorber is 2-hydroxy-4-methoxybenzophenone, and the anti-quenching agent is ascorbic acid palmitate.

4. The FISH-specific mounting medium of claim 1, wherein, The tackifier is hydrogenated rosin glyceride.

5. The FISH-specific mounting medium of claim 1, wherein, The crosslinking agent is 1,6-hexanediol diacrylate.

6. The FISH-specific mounting medium of claim 1, wherein, The stabilizer is 2,6-di-tert-butyl-4-methylphenol.

7. The FISH-specific mounting gel according to claim 1, characterized in that, The defoaming agent is an organosiloxane defoaming agent, and the model number is BYK-066N.

8. The FISH-specific mounting medium of claim 1, wherein, The mildew-proof agent is an isothiazolinone derivative, and the model number is MIT-10.

9. The FISH-specific mounting gel according to claim 1, characterized in that, The mass parts of each component are preferably: the modified acrylic resin is 40 parts, the fluorescent protective agent is 5 parts, the tackifier is 8 parts, the crosslinking agent is 4 parts, the stabilizer is 1.5 parts, the defoaming agent is 0.8 parts, the solvent is 35 parts, and the mildew-proof agent is 0.7 parts.

10. A method of preparing a FISH-specific mounting medium as claimed in any one of claims 1 to 9, characterized in that, The preparation of the modified acrylic resin and the preparation of the mounting medium are included, and the specific steps are as follows: S1. Preparation of the modified acrylic resin: W1. Hydroxyethyl methacrylate and methyl methacrylate are weighed at a molar ratio of 3:7, the initiator azobisisobutyronitrile is used in an amount of 0.8-1.2 parts of the total mass of monomers, and the solvent ethyl acetate is used in an amount of 1.5 times the total mass of monomers; W2. Hydroxyethyl methacrylate, methyl methacrylate and ethyl acetate are added to a four-necked flask, stirred at 400 r / min and 60℃ for 30 min to form a uniform prepolymer solution; W3. The azobisisobutyronitrile is dissolved in a small amount of ethyl acetate and then added dropwise to the prepolymer solution at a rate of 1 mL / min, and the temperature is raised to 70-75℃, and the reaction is carried out at 400 r / min for 6-8 hours; W4. Add 0.5 parts of hydroquinone to the total mass of monomers, stir for 10 min to terminate the reaction, and naturally cool to 30℃; W5. Pour the reaction solution into 3 times the volume of anhydrous ethanol, stir at 300 r / min to precipitate, and collect the precipitate by filtration; W6. Wash the precipitate with anhydrous ethanol 3 times, dry at a vacuum degree of-0.09 MPa and a temperature of 50℃ for 12 hours to obtain the modified acrylic resin; S2. Preparation of the mounting medium: X1. Weigh propylene glycol methyl ether acetate and ethanol by mass parts, add them to a reaction kettle, stir at 25℃ and a stirring speed of 300 r / min for 15 min, mix uniformly, and obtain a mixed solvent; X2. Heat the mixed solvent to 40±2℃, slowly add the modified acrylic resin prepared in step S1 in 3 portions with an interval of 10 min, keep the stirring speed at 500 r / min, and stir for 60±5 min until the resin is completely dissolved to obtain a transparent and uniform resin solution; X3. The resin solution is cooled to 30±2℃, and the fluorescent protective agent is first added, which is previously mixed uniformly at a mass ratio of 3:2, and stirred for 20 min, then the tackifier, crosslinking agent and stabilizer are added in sequence, the stirring speed is adjusted to 600 r / min, and stirred for 90±10 min to make the components fully dispersed; X4. The defoaming agent and mildew-proof agent are added, the stirring speed is reduced to 400 r / min, and stirred for 30±5 min; X5. The above mixture is filtered through a 200-mesh filter cloth, and then transferred to a vacuum degassing tank, and degassed at a vacuum degree of-0.08~-0.09 MPa and 25℃ for 30 min to obtain the FISH special mounting medium.

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