High-adhesion glass slide for indirect immunofluorescence analysis experiment

Abstract

The invention relates to the technical field of glass slides for biomedical detection, and particularly discloses a high-adhesion type glass slide for indirect immunofluorescence analysis experiments. The glass slide comprises a substrate, a transition layer and a functional layer, the transition layer is arranged on the surface of the substrate, is made of metal oxide and is used for improving the bonding stability of the substrate and the functional layer; the functional layer is arranged on the surface of the transition layer and is prepared by compounding modified polyacrylamide containing amino / carboxyl and nano-particles, and the sample adhesion is synergistically enhanced through specific combination of functional groups and a nanoscale surface rough structure. Performance tests show that the glass slide has an adhesion rate of more than or equal to 95% on cell and tissue slices, washing resistance of more than or equal to 5 times and a fluorescence signal variable coefficient of less than or equal to 5%, can effectively solve the problems that the existing glass slide sample is easy to fall off and poor in detection repeatability, remarkably improves the accuracy of indirect immunofluorescence analysis, and is suitable for scenes such as clinical diagnosis and pathological detection.

Description

Technical Field

[0001] This article relates to a highly adhesive glass slide for indirect immunofluorescence analysis experiments. Background Technology

[0002] Indirect immunofluorescence analysis is a commonly used detection technique in the biomedical field. It utilizes the binding of fluorescently labeled secondary antibodies to antigen-primary antibody complexes in the sample, and uses fluorescence signals to achieve antigen localization and quantification. It is widely used in scenarios such as autoantibody detection and tumor marker screening. The surface adhesion properties of the glass slide, as the sample carrier, directly affect the experimental results: insufficient adhesion can cause samples (such as cells and tissue sections) to detach during washing, resulting in a loss of fluorescence signal; non-specific adsorption can increase background fluorescence interference and reduce detection sensitivity.

[0003] In existing technologies, slide surface modification often employs a single silanization treatment (such as 3-aminopropyltriethoxysilane modification) to enhance electrostatic adhesion to the sample by introducing amino groups. However, this type of slide has the following drawbacks:

[0004] Poor adhesion stability: The binding force of single functional groups is weak, and the sample detachment rate exceeds 20% after more than 3 washes;

[0005] Significant nonspecific adsorption: The low surface roughness of the silanized layer makes it easy to adsorb fluorescent secondary antibodies, leading to an increase in background signal intensity;

[0006] Insufficient substrate adhesion: The silanized layer is bonded to the glass substrate only by van der Waals forces, which can easily lead to interlayer delamination during long-term storage.

[0007] Therefore, there is an urgent need to design a glass slide that combines high adhesion stability, low non-specific adsorption, and strong substrate binding to meet the high precision requirements of indirect immunofluorescence analysis. Summary of the Invention

[0008] To address the issues of poor adhesion and strong non-specific adsorption of existing glass slides, a highly adhesive glass slide for indirect immunofluorescence analysis is provided, achieving high sample stability, low background interference, and long-term reliability.

[0009] A highly adhesive glass slide for indirect immunofluorescence analysis comprises, from bottom to top, a substrate, a transition layer, and a functional layer. The substrate is a high-transmittance borosilicate glass with a water contact angle ≤10° after pretreatment. The transition layer, 50-200 nm thick, is disposed on the substrate surface and is made of titanium dioxide or zirconium oxide, forming MO-Si covalent bonds with the substrate via a sol-gel method. The functional layer, 100-300 nm thick, is disposed on the transition layer surface and is composed of a modified polymer, nanoparticles, and a crosslinking agent, in the following mass percentages: modified polymer 80-90%, nanoparticles 10-20%, and crosslinking agent 0.5-1%. The modified polymer is 3-aminopropyltriethoxysilane-modified polyacrylamide with an amino density ≥2 mmol / g. The nanoparticles are silica nanoparticles with a particle size of 10-50 nm. The crosslinking agent is N,N'-methylenebisacrylamide.

[0010] Furthermore, the pretreatment steps for the substrate include: sequentially ultrasonically cleaning with 95% ethanol (300W ultrasonic power, 40kHz frequency) for 15 minutes, ultrasonically cleaning with deionized water for 15 minutes, and drying at 120℃ for 30 minutes. The strong solubility of 95% ethanol can efficiently remove oil, organic residues, and trace impurities from the substrate surface. Combined with ultrasonic vibration at 300W power and 40kHz frequency, it can penetrate deep into the tiny crevices of the substrate surface to achieve thorough cleaning, preventing impurities from affecting the bonding of the subsequent transition layer to the substrate.

[0011] Furthermore, the specific steps for preparing the transition layer using the sol-gel method are as follows: A metal alkoxide is dissolved in anhydrous ethanol at a volume ratio of 1:4, and 5% (v / v) glacial acetic acid is added. The mixture is stirred at 30°C for 1 hour and then allowed to stand for aging for 24 hours to form a sol. The sol is then coated onto the substrate surface using a spin-coating method at a speed of 2500-3500 r / min for 30 seconds, followed by drying at 120°C for 30 minutes, annealing at 500°C for 2 hours, and curing at a heating rate of 5°C / min. The 1:4 volume ratio of metal alkoxide to anhydrous ethanol allows for precise control of the sol concentration, ensuring good fluidity for easy coating while avoiding uneven coating due to excessive concentration.

[0012] Furthermore, polyacrylamide with a molecular weight of 50,000-100,000 Da was dissolved in deionized water to form an 8% (w / w) solution. 3-Aminopropyltriethoxysilane was then added, and the mixture was stirred at 50°C for 30 minutes. Choosing polyacrylamide with a molecular weight of 50,000-100,000 Da ensures that the polymer backbone possesses both good flexibility and structural stability, avoiding the problem of easily breaking functional layers due to excessively small molecular weight, and the problem of excessively large molecular weight leading to excessively high solution viscosity and difficulty in mixing.

[0013] Furthermore, the nanoparticles are silica nanoparticles with a particle size of 15-30 nm, prepared by the following method: tetraethyl orthosilicate is dissolved in anhydrous ethanol at a volume ratio of 1:4, and 2% ammonia solution is added. The mixture is stirred at 25°C for 2 hours to form a sol. After centrifugation at 8000 r / min for 10 minutes and washing three times with deionized water, the sol is dispersed in deionized water to form a suspension with a mass concentration of 15%. The 1:4 volume ratio of tetraethyl orthosilicate to anhydrous ethanol can control the concentration of silica precursor. The use of 2% ammonia solution as an alkaline catalyst promotes the slow hydrolysis of tetraethyl orthosilicate to generate silica. Stirring at 25°C for 2 hours ensures a complete reaction, ultimately forming nanoparticles with a particle size of 15-30 nm.

[0014] Furthermore, the preparation steps of the functional layer are as follows: The modified polymer solution, nanoparticle suspension, and crosslinking agent are mixed and stirred at 50°C for 30 minutes to form a composite solution; this solution is then coated onto the surface of the transition layer using a dip-coating method and cured by irradiation with a 254nm ultraviolet lamp for 30 minutes. Stirring at 50°C for 30 minutes promotes thorough mixing of the modified polymer, nanoparticles, and crosslinking agent, forming a uniform composite solution and ensuring that the amino groups (chemical binding sites) and nanoparticles (physical anchoring points) are uniformly distributed in the solution.

[0015] Furthermore, application scenarios include clinical autoantibody diagnosis, tumor pathology detection, and microbial antigen localization. Suitable sample types include adherent cells, suspension cell smears, 5μm thick frozen sections, and 3μm thick paraffin sections.

[0016] Beneficial effects:

[0017] 1. High adhesion stability: The amino groups of the modified polymer in the functional layer form stable amide bonds (bond energy ≥300kJ / mol) with the carboxyl groups on the sample surface. Combined with the physical anchoring effect of SiO2 nanoparticles, a dual fixation effect of "chemical binding + physical anchoring" is achieved. The adhesion rates to HeLa cells and A549 cells reach 97.2% and 96.8%, respectively. The adhesion rates to 5μm thick frozen sections and 3μm thick paraffin sections reach 96.5% and 95.3%, respectively. It can withstand ≥5 washes with PBS buffer (the sample detachment rate after 5 washes is ≤3%), which is significantly better than existing silanized slides.

[0018] 2. Low background fluorescence interference: The hydrophilic skeleton of modified polyacrylamide (water contact angle ≤25°) can effectively reduce the non-specific adsorption of secondary antibody caused by hydrophobic interactions. At the same time, the high specific surface area of ​​SiO2 nanoparticles can adsorb a small amount of residual free secondary antibody. Under the dual effect, the background fluorescence intensity is reduced by 42.3% (background signal value ≤450AU), and the fluorescence signal variation coefficient is ≤5% (n=10), ensuring the repeatability of the detection results.

[0019] 3. Strong interlayer bonding: The transition layer metal oxide forms MO-Si covalent bonds with the substrate, and the interlayer peel strength is ≥15MPa, which is significantly higher than that of existing silanized glass slides. After long-term storage for 6 months, there is no interlayer peeling or functional layer detachment, and the stability is excellent.

[0020] 4. Wide sample compatibility: No additional sample pretreatment is required. It can adapt to a variety of sample types, including adherent cells (HeLa, A549, HepG2), suspension cells (Jurkat, K562, after centrifugation and smearing), frozen sections (rat liver tissue, human kidney tissue), and paraffin sections (breast cancer tissue, lung cancer tissue), and its compatibility covers commonly used samples in IIFA experiments. Attached Figure Description

[0021] Figure 1 A comparison table of sample adhesion rates for the three embodiments;

[0022] Figure 2 A comparison table of fluorescence signal performance for the three embodiments;

[0023] Figure 3 This is a comparison table of peel strength performance for three embodiments. Detailed Implementation

[0024] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0025] Example 1:

[0026] 1. Substrate pretreatment:

[0027] Take a commercially available borosilicate glass slide (25mm×75mm×1mm, SiO2 content 82%, purchased from Jiangsu Shitai Experimental Equipment Co., Ltd.), place it in an ultrasonic cleaning tank, add 95% ethanol (analytical grade, Sinopharm Group), set the ultrasonic power to 300W and the frequency to 40kHz, and ultrasonically clean for 15 minutes.

[0028] Pour out the ethanol, add deionized water (resistivity ≥18.2MΩ・cm), and clean with the same ultrasonic parameters for 15 minutes;

[0029] After cleaning, place the glass slides in a forced-air drying oven and dry at 120°C for 30 minutes. After removing them, place them in a desiccator to cool to room temperature for later use (the water contact angle on the substrate surface was measured to be 8.5°).

[0030] 2. Preparation of the transition layer (TiO2, 100nm):

[0031] Sol preparation: Add 40 mL of anhydrous ethanol (analytical grade) to a 500 mL three-necked flask, and slowly add 10 mL of tetrabutyl titanate (analytical grade, Aladdin) dropwise under magnetic stirring. After the addition is complete, add 5 mL of glacial acetic acid (analytical grade, Sinopharm Group). Set the stirring speed to 300 r / min and the temperature to 30 °C, and stir at a constant temperature for 1 h. After sealing, let it stand at room temperature for 24 h to obtain a light yellow transparent TiO2 sol.

[0032] Coating and curing: The substrate was fixed on a spin-coater (KW-4A, Institute of Microelectronics, Chinese Academy of Sciences). 0.5 mL of TiO2 sol was dropped onto the center of the substrate. The rotation speed was set to 3000 r / min and the time was 30 s. After coating, the substrate was dried at 120℃ for 30 min and then transferred to a muffle furnace (SX2-4-10, Shanghai Yifeng Electric Furnace Co., Ltd.). The temperature was increased to 500℃ at 5℃ / min and held for 2 h before natural cooling to form a 100 nm thick TiO2 transition layer (measured by a Dektak150 profilometer, error ±5 nm).

[0033] 3. Fabrication of the functional layer (200nm, 20nm SiO2):

[0034] Modified polymer: Add 100 mL of deionized water to a 250 mL beaker, dissolve 8 g of polyacrylamide (molecular weight 80000 Da, Aladdin), add 1.2 g of APTES (Aladdin), stir at 50 °C for 30 min to obtain a modified solution with an amino density of 2.3 mmol / g;

[0035] Nanoparticles: Add 40 mL of anhydrous ethanol and 10 mL of tetraethyl orthosilicate (Sinopharm Group) to a 250 mL beaker, add 2 mL of 25% ammonia water dropwise, stir at 25 °C for 2 h, centrifuge at 8000 r / min for 10 min, wash three times with deionized water, and disperse into 15 mL of 15% suspension (detected by transmission electron microscopy Tecnai G2F20, average particle size 20 nm).

[0036] Composite solution: Mix 85g of modified polymer solution, 15g of nanoparticle suspension, and 0.8g of MBA (Aladdin), and stir at 50℃ for 30min;

[0037] Coating and curing: The coating was applied using a dip coater (ZJ-300, Hangzhou Zhuochi) at a speed of 5 mm / s, followed by irradiation with a 254 nm UV lamp (10 W, 10 cm distance) for 30 minutes to form a 200 nm thick functional layer.

[0038] Example 2

[0039] The only difference between this embodiment and Example 1 is that the transition layer material is ZrO2; all other steps and parameters are exactly the same. The specific transition layer preparation steps are as follows:

[0040] ZrO2 sol preparation: Add 40 mL of anhydrous ethanol to a 500 mL three-necked flask, add 12 mL of zirconium-propoxide (analytical grade, Aladdin) dropwise under magnetic stirring, add 5 mL of glacial acetic acid, set the stirring speed to 300 r / min and the temperature to 30 °C, and stir at a constant temperature for 1.5 h (the reactivity of ZrO2 precursor is lower than that of TiO2, so the stirring time is extended), seal and let stand at room temperature for 24 h to age, and obtain colorless and transparent ZrO2 sol;

[0041] Coating and curing: Same as in Example 1 (3000 r / min rotation speed, 120℃ drying for 30 min, 500℃ annealing for 2 h), forming a 100 nm thick ZrO2 transition layer (measured by a step meter, error ±6 nm).

[0042] Example 3

[0043] The only difference between this embodiment and Example 1 is that the SiO2 nanoparticles have a particle size of 30 nm. All other steps and parameters are exactly the same. The specific nanoparticle preparation steps are as follows:

[0044] Preparation of 30nm SiO2 suspension: Add 40mL of anhydrous ethanol and 10mL of tetraethyl orthosilicate to a 250mL beaker, add 2mL of 25% ammonia water dropwise, adjust the temperature to 30℃ (increasing the temperature promotes the growth of nanoparticles), and stir for 3h (extending the stirring time increases the particle size).

[0045] Separation and dispersion: Centrifuge at 8000 r / min for 15 min (extend the centrifugation time to ensure particle sedimentation), wash 3 times with deionized water, and disperse into 15 mL of 15% suspension (transmission electron microscopy, average particle size 30 nm).

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A highly adhesive glass slide for indirect immunofluorescence analysis experiments, characterized in that, From bottom to top, it includes a base, a transition layer, and a functional layer; The substrate is a high-transmittance borosilicate glass with a water contact angle ≤10° after pretreatment. The transition layer is disposed on the surface of the substrate with a thickness of 50-200nm. It is made of titanium oxide or zirconium oxide and forms MO-Si covalent bonds with the substrate through a sol-gel method. The functional layer is disposed on the surface of the transition layer, with a thickness of 100-300 nm, and is composed of a modified polymer, nanoparticles, and a crosslinking agent, in the following mass percentages: modified polymer 80-90%, nanoparticles 10-20%, and crosslinking agent 0.5-1%; the modified polymer is 3-aminopropyltriethoxysilane-modified polyacrylamide with an amino density ≥2 mmol / g; the nanoparticles are silica nanoparticles with a particle size of 10-50 nm; and the crosslinking agent is N,N'-methylenebisacrylamide.

2. The highly adhesive glass slide for indirect immunofluorescence analysis according to claim 1, characterized in that, The pretreatment steps of the substrate include: ultrasonic cleaning with 95% ethanol (volume concentration) for 15 min, ultrasonic cleaning with deionized water for 15 min, and drying with forced air at 120℃ for 30 min.

3. The highly adhesive glass slide for indirect immunofluorescence analysis according to claim 1, characterized in that, The specific steps for preparing the transition layer by the sol-gel method are as follows: dissolve the metal alkoxide in anhydrous ethanol at a volume ratio of 1:4, add 5% glacial acetic acid, stir at 30°C for 1 hour, and let it stand for aging for 24 hours to form a sol; coat the substrate surface using the spin-coating method at a speed of 2500-3500 r / min for 30 seconds, dry at 120°C for 30 minutes, anneal at 500°C for 2 hours, and cure at a heating rate of 5°C / min.

4. The highly adhesive glass slide for indirect immunofluorescence analysis according to claim 3, characterized in that, Polyacrylamide with a molecular weight of 50,000-100,000 Da was dissolved in deionized water to form a solution with a mass concentration of 8%. 3-aminopropyltriethoxysilane was added and the mixture was stirred at 50°C for 30 minutes.

5. A highly adhesive glass slide for indirect immunofluorescence analysis according to claim 1, characterized in that, The nanoparticles are silica nanoparticles with a particle size of 15-30 nm, and are prepared by the following method: tetraethyl orthosilicate is dissolved in anhydrous ethanol at a volume ratio of 1:4, ammonia water with a volume concentration of 2% is added, and the mixture is stirred at 25°C for 2 hours to form a sol. After centrifugation at 8000 r / min for 10 min and washing with deionized water 3 times, the sol is dispersed in deionized water to form a suspension with a mass concentration of 15%.

6. The highly adhesive glass slide for indirect immunofluorescence analysis according to claim 1, characterized in that, The preparation steps of the functional layer are as follows: the modified polymer solution, nanoparticle suspension and crosslinking agent are mixed and stirred at 50°C for 30 min to form a composite solution; the solution is coated onto the surface of the transition layer by dip coating and cured by irradiation with a 254nm ultraviolet lamp for 30 min.

7. A highly adhesive glass slide for indirect immunofluorescence analysis according to claim 1, characterized in that, Application scenarios include clinical autoantibody diagnosis, tumor pathology testing, and microbial antigen localization. Compatible sample types include adherent cells, suspension cell smears, 5μm thick frozen sections, and 3μm thick paraffin sections.

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