A method for improving the coupling efficiency of an acrylate-based substrate biomolecule

Abstract

This invention relates to the field of medical testing, specifically to a method for improving the biomolecule coupling efficiency on the surface of an acrylate substrate. The acrylate substrate is subjected to plasma treatment; then, it is sequentially soaked in a bridging buffer and an activation buffer. The resulting treated acrylate substrate is used for antibody coupling. The active substance in the bridging buffer is a siloxane containing succinic anhydride; the active substance in the activation buffer includes at least one of EDC, NHS, DCC, DMAP, and HATU. This technical solution can efficiently couple antibodies to an acrylate substrate, further solving the technical problem of the lack of an economical, environmentally friendly, and simple method for coupling biomolecules to the surface of an acrylate substrate. This meets the application needs of using materials containing acrylate substrates, such as optical discs, as bioreaction substrates for qualitative or quantitative analysis, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of medical testing, and more specifically to a method for improving the biomolecule coupling efficiency of acrylate substrates. Background Technology

[0002] Optical discs are a common medium for data storage. Due to their excellent optical properties (reflection and absorption) and smooth surface structure (1-10 nm surface roughness), many studies in recent years have revealed their advantages as a next-generation bioreactor substrate, replacing traditional glass and silicon wafers, and they have been successfully applied in the qualitative and quantitative analysis of substances. The protective layer (surface layer) of optical discs is usually made of UV-curable acrylate, i.e., a UV-curable acrylate substrate.

[0003] In existing technologies, the main methods for using optical discs as a bioreactor substrate for qualitative or quantitative analysis include the following:

[0004] (1) Detection using a standard optical drive: The principle is that the laser head inside the optical drive first uses a high-power or low-power laser to etch the dye layer of the optical disc with the information to be recorded. The dye layer forms pits under the etching of the high-power laser, while the low-power laser has no effect on the dye layer. After continuous etching, a series of pits and bumps appear in the dye layer, representing binary information. When reading the optical disc, due to the different reflectivities of the pits and bumps, the optical drive receives two different light intensity signals, representing binary 0 and 1 respectively. When there are contaminants on the surface of the optical disc, they will interfere with the normal data reading process and generate error correction codes. The value of the error correction code is proportional to the amount of contaminants, thereby achieving qualitative or quantitative detection. For example, Kido et al. constructed a fluorescently labeled protein microarray immunoassay structure on the surface of ordinary CDs and used a standard optical drive to read bio-optical discs for detection. Yu et al. constructed an immunoassay array on the surface of ordinary CDs and BDs, and used gold-labeled antibodies and silver enhancement strategies, combined with a standard optical drive and optical disc quality diagnostic software, to achieve quantitative detection of biomolecules.

[0005] (2) Optical drive modification detection: Alexander et al. modified a regular optical drive into one with two photodetectors to obtain information about DNA on a bioreactor array prepared on the CD surface. Barathur et al. modified a standard optical drive into a laser scanner to detect microfluidic optical discs. Lange et al. used the precise focusing and tracking capabilities of the optical drive's laser head, combined with an optical microscope, to modify it into an optical drive imager for imaging analysis of immune reactions. Although optical drive modification detection schemes can perform qualitative or semi-quantitative detection of biomolecules, they involve the redesign of the internal photoelectric structure of the optical drive and usually require destructive reconstruction of the optical disc, making them impractical.

[0006] The detection methods using bio-optical discs as the reaction substrate described above can perform qualitative or semi-quantitative analysis of loaded biomolecules, but cannot achieve accurate and highly sensitive quantitative detection. One key reason is that the main component of the protective layer on the disc surface is acrylate, which has strong biological inertness, resulting in low antibody conjugation efficiency on the bio-optical disc surface. This directly affects the detection sensitivity and accuracy. Improving the antibody conjugation efficiency on the bio-optical disc surface is one of the keys to improving the detection performance of optical disc detection platforms and is also a research hotspot in this field. Currently, the main methods for modifying the surface of CD or BD discs to improve antibody conjugation efficiency are as follows:

[0007] (1) Ultraviolet treatment: This method mainly utilizes ultraviolet irradiation to harden the surface layer of the optical disc to form an oxide film while introducing hydroxyl, carboxyl, amino groups, etc., and achieves covalent coupling of antibodies through EDC / NHS-mediated amine coupling reaction.

[0008] (2) Spin coating of easily activated SU-8: This method mainly involves fixing the easily activated SU-8 photoresist onto the surface of the optical disc hardening layer using equipment such as a spin coater, constant temperature oven, and ultraviolet lamp.

[0009] (3) Alkaline hydrolysis: This method mainly uses strong alkaline aqueous solutions such as NaOH to perform alkaline hydrolysis on the polyacrylate component in the hardened layer of the optical disc surface to form hydrophilic groups such as carboxylate on its surface, and then achieves covalent coupling of antibodies through EDC / NHS-mediated amine coupling reaction.

[0010] (4) Physical adsorption: This method mainly uses a biological spotting instrument to directly spot biomolecules onto the surface of the optical disc, and uses physical adsorption to adsorb the biomolecules onto the surface of the optical disc.

[0011] However, the above methods for surface modification of optical discs and antibody conjugation still have shortcomings, specifically:

[0012] (1) Ultraviolet treatment: Operators may be exposed to ultraviolet light during the treatment process. The number of active groups generated is small, and only one of the groups, carboxyl or amino, can be linked to the antibody through the subsequent EDC / NHS reaction, resulting in low coupling efficiency.

[0013] (2) Spin coating of easily activated substance SU-8: This method is complicated to operate, the raw materials are expensive, and the process requirements are high. Furthermore, since a layer of SU-8 is spin-coated on the surface of the optical disc, the interference of its refractive index may affect the acquisition process of biological signals.

[0014] (3) The alkaline hydrolysis efficiency is not high, and the strong alkaline solutions such as NaOH may cause burns to the skin and eyes of operators due to improper operation. The strong alkaline waste liquid is harmful to the environment.

[0015] (4) Physical adsorption: Compared with the covalent bonding method mentioned above, physical adsorption has lower fixation and reaction efficiency.

[0016] In summary, there is an urgent need to develop an economical, environmentally friendly, and simple method to significantly improve the antibody conjugation efficiency on the surface of bio-optical discs, in order to meet the application needs of using optical discs as a bioreaction substrate for qualitative or quantitative analysis. Summary of the Invention

[0017] The present invention aims to provide a method for improving the biomolecule coupling efficiency on the surface of acrylate substrates, in order to solve the technical problem that the prior art lacks an economical, environmentally friendly, and simple method for coupling biomolecules and other biomolecules to the surface of acrylate substrates.

[0018] To achieve the above objectives, the present invention adopts the following technical solution:

[0019] A method for improving the biomolecule coupling efficiency on the surface of an acrylate substrate involves plasma treatment of the acrylate substrate; soaking the acrylate substrate in a bridging buffer; soaking the acrylate substrate in an activation buffer; and using the resulting treated acrylate substrate for antibody coupling.

[0020] The active ingredient in the bridging buffer is a siloxane containing succinic anhydride; the active ingredient in the activation buffer includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate.

[0021] Furthermore, the siloxanes containing succinic anhydride include at least one of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, [3-(trimethoxysilyl)propyl]succinic anhydride, and succinic anhydride-terminated polydimethylsiloxanes;

[0022] The concentration of siloxane containing succinic anhydride is 5-30%, and the bridging buffer is prepared using at least one of PBS buffer, morpholine ethanesulfonic acid buffer, and Tris buffer.

[0023] Furthermore, the conditions for plasma treatment are: oxygen pressure 0.05-0.2 bar, power 20-80 W, and treatment time 30-180 s;

[0024] The activation buffer is prepared using morpholine ethanesulfonic acid solution or phosphate buffer; the CD protective layer is soaked in the activation buffer for 5-15 minutes.

[0025] Furthermore, before soaking the optical disc protective layer in the activation buffer solution, the optical disc protective layer is dried at a temperature >120℃; the drying temperature is 120-160℃ and the time is 5-20 minutes.

[0026] Furthermore, the acrylate substrate is derived from the protective layer of the bio-optical disc.

[0027] This technical solution also provides a method for coupling biomolecules to an acrylate substrate, comprising the following sequential steps:

[0028] S1 Washing the acrylate substrate: Wash the acrylate substrate with a mixed solution of methanol and ethanol, and then dry it with nitrogen gas;

[0029] S2 plasma treatment: Acrylic substrate is treated with oxygen plasma;

[0030] S3 Siloxane Bridging: The acrylate substrate is soaked in a bridging buffer; the active substance in the bridging buffer is a siloxane containing succinic anhydride;

[0031] S4 substrate activation: The acrylate substrate is soaked in an activation buffer; the active substance in the activation buffer includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate.

[0032] S5 Antibody / Antigen Conjugation: The antibody / antigen solution is dropped onto an acrylate substrate and incubated to obtain an acrylate substrate conjugated with the antibody.

[0033] Furthermore, in S1, the mixed solution of methanol and ethanol consists of methanol and anhydrous ethanol in a volume ratio of (4-7):(6-3); the washing time is 5 min.

[0034] In S2, the plasma treatment conditions are: oxygen pressure 0.05-0.2 bar, power 20-80 W, and treatment time 30-180 s;

[0035] In S3, the bridging buffer is prepared as follows: Dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione is dissolved in PBS buffer and then sonicated for 10-20 min; the concentration of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione is 5-30%; the soaking time is 12-16 h.

[0036] Alternatively, succinic anhydride-terminated polydimethylsiloxane can be dissolved in PBS buffer and then sonicated for 10-20 minutes to obtain the product; the concentration of succinic anhydride-terminated polydimethylsiloxane is 5-30%; the soaking time is 12-16 hours.

[0037] Alternatively, [3-(trimethoxysilyl)propyl]succinic anhydride can be dissolved in PBS buffer and then sonicated for 10-20 min to obtain the solution; the concentration of [3-(trimethoxysilyl)propyl]succinic anhydride is 5-30%; the soaking time is 12-16 h.

[0038] In S4, the activation buffer is prepared as follows: using 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and making the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 100-150 mmol / L and the final concentration of N-hydroxysuccinimide 25-37.5 mmol / L.

[0039] Alternatively, use a 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and make the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 100-150 mmol / L and the final concentration of 4-dimethylaminopyridine 15-30 mmol / L.

[0040] Alternatively, use a 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and make the final concentration of dicyclohexylcarbodiimide 40-80 mmol / L and the final concentration of 4-dimethylaminopyridine 8-16 mmol / L;

[0041] Alternatively, use 0.01 mol / L phosphate buffer and make the final concentration of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate 40-100 mmol / L;

[0042] Soak the acrylate substrate in the above-mentioned activation buffer for 5-15 minutes;

[0043] In S5, the concentration of antibody in the antibody solution is 50-500 μg / mL; the incubation temperature is 25-37℃, and the incubation time is 3-6 h.

[0044] Furthermore, in S4, the acrylate substrate is first dried at a temperature of 120-160℃ for 5-20 minutes, and then soaked in an activation buffer.

[0045] This technical solution also provides a method for coupling biomolecules to an acrylate substrate to obtain a bio-optical disc coupled with biomolecules.

[0046] This technical solution also provides an application of a bio-disc coupled with biomolecules in the preparation of a bioreaction substrate.

[0047] In summary, the principles and beneficial effects of this invention are as follows:

[0048] This invention discloses a method that can significantly improve the antibody conjugation efficiency on the surface of bio-optical discs, thereby solving the key technical problems of environmentally unfriendly processing methods, high biohazard, low conjugation efficiency, and uneven conjugation in the traditional antibody conjugation process on bio-optical discs. In the method of this invention, the materials and reagents include ordinary optical discs (such as BD, DVD, CD, etc.), washing buffer (such as methanol, ethanol, or a methanol / ethanol mixture in a specific ratio), bridging buffer (such as an aqueous solution of siloxane containing succinic anhydride, such as dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, [3-(trimethoxysilyl)propyl]succinic anhydride, or succinic anhydride-terminated polydimethylsiloxane), activation buffer (such as an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide, as well as a mixture of 4-dimethylaminopyridine, dicyclohexylcarbodiimide, and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate in different ratios), and coating buffer (containing antibody). This invention utilizes siloxanes containing succinic anhydride moiety, such as dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, as bridging agents. The succinic anhydride functional groups of these siloxanes covalently bind to the amino groups of antibodies, achieving highly efficient and uniform antibody conjugation. This method offers high biosafety and environmental friendliness, avoiding the use of substances with significant biohazards, such as ultraviolet light, strong alkalis, and SU-8, in traditional bio-disc surface activation methods. The organosiloxane molecules used in this invention, such as dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, and other siloxanes containing succinic anhydride moiety, have a Si-O backbone that can form stable covalent bonds with any surface containing OH groups. Treating the optical disc surface protective layer with oxygen plasma generates a large number of hydroxyl groups, enabling covalent binding of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, [3-(trimethoxysilyl)propyl]succinic anhydride, and succinic anhydride-terminated polydimethylsiloxanes to the surface of the bio-optical disc. Siloxane molecules with succinic anhydride terminals can achieve dual antibody conjugation. The succinic anhydride functional group of this molecule can directly generate a covalent amide bond with the amino group of the antibody through a ring-opening reaction, effectively conjugating the antibody. Simultaneously, the resulting carboxyl functional group can also conjugate with the antibody molecule. During the bridging process using siloxanes containing succinic anhydride, the inventors also discovered that the concentration of siloxanes has a significant impact on the antibody conjugation effect. Only at an appropriate concentration can a significantly higher conjugation efficiency be obtained than other methods.In addition, after bridging the siloxane and the protective layer of the bio-optical disc, if the disc is dried, it is necessary to use a high-temperature drying process followed by surface activation before subsequent antibody conjugation. Otherwise, the antibody conjugation efficiency will be very low, making it difficult to achieve the subsequent detection objectives. Alternatively, after bridging the siloxane and the protective layer, no drying process is required; functional group activation can be performed directly. This method ensures a higher antibody conjugation rate compared to drying. Therefore, the method for improving antibody conjugation efficiency on the surface of bio-optical discs created in this invention is highly efficient, economical, environmentally friendly, and simple. It can significantly improve the antibody conjugation efficiency on the surface of bio-optical discs, providing an effective antibody conjugation strategy for bio-optical disc-based biological detection, and has great potential for widespread application.

[0049] In summary, this technical solution only requires simple plasma treatment, the introduction of a bridging agent, and an activation step to prepare a bio-optical disc reaction substrate with high surface antibody binding capacity. Testing shows that the bio-optical disc reaction substrate prepared by this method exhibits significantly improved fluorescent antibody conjugation efficiency and uniform conjugation signal, solving the key technical problems of traditional bio-optical disc surface antibody conjugation methods, such as environmentally unfriendly processing, high biohazard, low conjugation efficiency, and uneven conjugation. Attached Figure Description

[0050] Figure 1 The results of droplet contact angle detection for different treatment groups in Example 1 are shown (A: oxygen plasma treatment; B: alkaline hydrolysis; C: untreated).

[0051] Figure 2 Typical fluorescence images of the antibody-coupled effects of different treatment groups in Example 1 are shown below (A: Plasma treatment followed by addition of bridging agent dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione (concentration 10%); B: Alkaline hydrolysis method; C: Physical adsorption in the untreated group).

[0052] Figure 3 The quantitative results of average fluorescence intensity for different treatment groups in Example 1 are shown (A: oxygen plasma treatment; B: alkaline hydrolysis method; C: physical adsorption of untreated group).

[0053] Figure 4 This is a typical fluorescence image showing the effect of different concentrations of bridging buffer on the protein-antibody conjugation efficiency in Example 2.

[0054] Figure 5 The results show the quantitative results of the average fluorescence intensity corresponding to different concentrations of bridging buffer in Example 2.

[0055] Figure 6 This is a typical fluorescence image showing the effect of different drying temperatures on the protein-antibody conjugation efficiency in Example 3.

[0056] Figure 7 The quantitative results of the average fluorescence intensity corresponding to different drying temperatures in Example 3 are shown.

[0057] Figure 8 Typical fluorescence images and quantitative results of average fluorescence intensity of the optical discs containing conjugated antibodies obtained by the hydrolysis-reconstruction method and the high-temperature dehydration method in Example 4.

[0058] Figure 9 Typical fluorescence images and average fluorescence intensity quantification results of optical discs containing conjugated antibodies using different bridging buffers and activation buffers, as shown in Example 5. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0060] Example 1:

[0061] This technical solution involves conjugating antibodies to the surface of conventional optical discs, including digital optical discs such as Blu-ray, DVD, and CD. Existing optical discs mainly consist of the following stacked parts:

[0062] Substrate: It is the physical carrier of optical discs. It is usually made of polycarbonate (PC) and has the characteristics of good impact toughness, wide operating temperature range, good dimensional stability, weather resistance, and non-toxicity. Its main function is to provide mechanical support and protect the bottom surface of the optical disc.

[0063] Recording layer: The structure containing the recorded data. In CDs, the data layer is typically made of organic dye material, which is ablated using lasers to create tiny pits and planes during recording. In DVDs and Blu-ray discs, the data layer is formed by a highly precise uneven structure or a structure followed by a higher density blue-violet laser.

[0064] Reflective layer: The area used to reflect the laser beam of the optical drive, usually made of metal (such as aluminum or silver) or semiconductor material (such as cadmium selenide).

[0065] Protective layer: Usually made of acrylate, its main function is to protect the data layer from scratches or contamination, while preventing oxidation and premature degradation.

[0066] This technical solution mainly involves treating the protective layer of optical discs. The general process includes washing the protective layer (removing surface contaminants), oxygen plasma treatment (to generate abundant hydroxyl groups in the protective layer), siloxane molecule bridging (bridging the Si-O backbone of siloxane molecules with hydroxyl groups), high-temperature drying and activation, and antibody conjugation. After activation, the optical disc forms a bio-optical disc reaction substrate with high surface antibody binding capacity, allowing for more effective antibody conjugation. The specific process is as follows:

[0067] (1) Washing the protective layer on the surface of the optical disc

[0068] Prepare 10 mL of washing solution with a methanol:anhydrous ethanol ratio of 7 mL:3 mL. Place the optical disc in the washing solution and wash thoroughly for 5 minutes to remove surface contaminants that could affect the binding affinity of proteins and antibodies. Immediately dry the washed optical disc with nitrogen gas.

[0069] (2) Plasma treatment of optical disc surface

[0070] Processing Group 1: Place the optical disc obtained in the above steps into an oxygen plasma treatment device. Parameter settings: oxygen pressure 0.1 bar, power 50 W, cleaning time 60 s.

[0071] Treatment Group 2: Take an identical CD and use the alkaline hydrolysis method as a control experiment. Prepare 100 mL of 1 mol / L NaOH solution, place the CD in the solution, incubate in a constant temperature oven at 55℃ for 90 min, then take it out, rinse it thoroughly with ultrapure water, and blow it dry with nitrogen gas.

[0072] Processing Group 3: Take an identical CD and do not perform any surface treatment.

[0073] A water droplet contact angle experiment was conducted on three groups of optical discs with different treatments. For example... Figure 1 As shown, the water droplet contact angle of the untreated optical disc is 82.2° due to its hydrophobic surface; the water droplet contact angle of the disc after alkaline hydrolysis is 31.9°; and the water droplet contact angle of the disc after oxygen plasma treatment is 11°. These results indicate that the density of hydrophilic groups generated on the optical disc surface is highest after oxygen plasma treatment, which is beneficial for subsequent antibody conjugation steps.

[0074] (3) Bridging between the Si-O framework of siloxane molecules and the surface of optical discs

[0075] Prepare 1 mL of bridging buffer: Add 100 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione (bridging agent, CAS: 93642-68-3) to 900 μL of 1×PBS buffer to prepare a 10% bridging buffer. Sonicate the bridging buffer in an ultrasonicator for 10 min until the solution is clear and homogeneous. Place the resulting solution on the surface of a surface-activated optical disc (treatment group 1) and incubate at 4℃ for 12-16 h, then thoroughly rinse with ultrapure water.

[0076] (4) High-temperature drying and functional group activation

[0077] After cleaning, the optical discs (treatment group 1) were placed in a drying oven and heated at 120°C for 10 min. An activation buffer containing 150 mmol / L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 37.5 mmol / L NHS (N-hydroxysuccinimide) was prepared in 10 mL of 0.1 mol / L morpholine ethanesulfonic acid solution. The dried optical discs (treatment group 1) were immediately placed in the activation buffer and activated at room temperature for 15 min, followed by thorough washing with ultrapure water.

[0078] The optical discs obtained from treatment group 2 in step (2) were placed in an activation buffer and activated at room temperature for 15 minutes before being thoroughly washed with ultrapure water.

[0079] (5) Antibody conjugate

[0080] Immediately add 50 μL of fluorescein isothiocyanate-labeled antibody (250 μg / mL) to the surface of the optical discs in treatment groups 1 and 2 obtained in step (4) above, and incubate at 37°C in the dark for 3 hours. Add the same concentration and volume of the same antibody to the surface of the optical disc obtained in treatment group 3, and incubate at 37°C in the dark for 3 hours. The antibody used in this scheme is specifically: fluorescein isothiocyanate (FITC)-functionalized anti-C-reactive protein antibody. In fact, this method can be used to couple any protein (containing amino groups) onto the optical disc. The above is all illustrated using antibodies as an example. Antigens and antibodies are essentially proteins, so this method can also be used to couple antigens and other protein substances onto the optical disc. After thoroughly washing with ultrapure water, observe the experimental results using a fluorescence confocal microscope. Figure 2 As shown, due to the introduction of the linker, the antibody conjugation efficiency and uniformity in treatment group 1 were significantly higher than those in treatment group 2 (alkaline hydrolysis method) and treatment group 3 (physical adsorption method). The quantitative fluorescence image plotted based on the average fluorescence intensity is shown below. Figure 3 As shown. Figure 3 The average fluorescence intensity was obtained as follows: When observing the experimental results using a fluorescence confocal microscope, three discontinuous fields of view were randomly selected for each sample, and the total fluorescence intensity (green pixels) in each field of view was extracted using ImageJ software. This total intensity was then divided by the area of ​​a single field of view to calculate the average fluorescence intensity value. t-test statistical analysis showed significant differences between group A and groups B and C, respectively. The results demonstrate that the surface modification method for the bio-disc proposed in this invention can significantly improve the antibody conjugation efficiency on the bio-disc surface, thereby effectively enhancing the analytical performance of the bio-disc.

[0081] Example 2

[0082] Prepare a 5% bridging buffer by dissolving 50 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione in 950 μL of 1×PBS buffer; prepare a 10% bridging buffer by dissolving 100 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione in 900 μL of 1×PBS buffer; prepare a 15% bridging buffer by dissolving 150 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione in 850 μL of 1×PBS buffer; and prepare a 15% bridging buffer by dissolving 200 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione in 800 μL of 1×PBS buffer. Prepare a 20% bridging buffer by adding 300 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione to 700 μL of 1×PBS buffer to prepare a 30% bridging buffer.

[0083] The remaining operating steps are the same as in Example 1, except that this example investigated the solute concentration of the dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione bridging buffer. Experimental results are as follows: Figure 4 The fluorescence images show that the bio-disc treated with 10% bridging buffer exhibits the best antibody conjugation efficiency. The antibody conjugation effects of low-concentration (5%) and high-concentration (15%, 20%, 30%) bridging buffers are all inferior to those of the 10% bridging buffer. The analysis indicates that the low-concentration (5%) bridging buffer itself is insufficient to provide enough antibody conjugation sites, while the high-concentration (15%, 20%, 30%) bridging buffers are also not ideal. The inventors believe this may be due to the self-crosslinking of the high-concentration bridging agent in aqueous solution, which affects the antibody conjugation efficiency. The antibody conjugation efficiency is highly sensitive to the solute concentration in the bridging buffer, a fact the inventors had not anticipated before the experiment.

[0084] The quantitative fluorescence image plotted based on the average fluorescence intensity is as follows: Figure 5 As shown (statistical methods are the same) Figure 3 Significant differences were found between the 10% group and other concentration groups (p < 0.0001, t-test). Therefore, a 10% bridging buffer concentration was determined to be the optimal concentration for protein antibody conjugation. The fluorescence intensity produced by the 10% bridging buffer was more than 2.5 times that of other concentrations, and even slight increases or decreases in the 10% concentration resulted in a precipitous drop in detection performance (fluorescence intensity), which was unexpected by the inventors. The selection of the 10% bridging buffer concentration exceeded expectations in terms of quantity (detection of fluorescence intensity values), achieving unexpected technical effects.

[0085] Example 3

[0086] This embodiment is basically the same as Embodiment 1, except that the high-temperature drying in step (4) is set to 60, 80, 100, 120, 140, and 160°C. The drying process lasts for 10 minutes. Under these conditions, no liquid residue is visible on the surface of the optical disc, indicating that the moisture has been sufficiently dried. After antibody conjugation, the experimental results are observed using a fluorescence confocal microscope. Figure 6 As shown in the fluorescence images, the antibody conjugation efficiency gradually increases with increasing dehydration temperature. The highest antibody conjugation efficiency is observed at temperatures of 120℃ and above, indicating that this temperature treatment provides the highest protein-antibody binding affinity. The quantitative fluorescence image plotted based on the average fluorescence intensity is shown below. Figure 7 As shown (statistical methods are the same) Figure 3 Significant differences were found between the 120℃ group and the 100℃, 80℃, and 60℃ groups (p < 0.01, t-test). When the drying temperature reached 120℃ or higher, the fluorescence intensity of the conjugated antibody was significantly improved, indicating that the drying temperature is crucial for antibody conjugation efficiency and is a key technical factor affecting the effectiveness of the protocol. This finding was obtained independently by the inventors.

[0087] In addition, the inventors also tried non-high-temperature drying methods, such as drying with conventional nitrogen gas followed by thorough drying at room temperature, and then performing antibody conjugation. The results showed that the antibody conjugation efficiency of the activated optical discs prepared in this way was lower than that obtained after drying at 60°C or higher (reflected in the average fluorescence intensity and fluorescence microscopy images, with the average fluorescence intensity value lower than the 60°C group). This demonstrates that drying the optical discs after bridging buffer treatment (to avoid hydrolysis of the succinic anhydride groups) requires high-temperature drying (120°C being the optimal drying temperature). Otherwise, it is difficult to effectively guarantee the antibody conjugation effect of the activated optical discs. This further illustrates the criticality of the high-temperature drying step, which is a key point in ensuring the technical implementation of bridging the optical disc protective layer and antibody protein using siloxane containing the succinic anhydride moiety.

[0088] Example 4

[0089] Activated optical discs obtained according to the plasma treatment method in Example 1 were used for subsequent experiments. Bridging buffer was added to the surface of the bio-discs and labeled as treatment group 1 and treatment group 2. After being placed in a 4°C refrigerator for 12 hours, they were thoroughly washed with ultrapure water. The bio-discs of treatment group 1 were placed in a drying oven and heated at 120°C for 10 minutes. Immediately after high-temperature dehydration, the bio-discs of treatment group 1 were placed in a 0.1 mol / L morpholine ethanesulfonic acid solution containing 150 mmol / L EDC and 37.5 mmol / L NHS, incubated at room temperature for 15 minutes, and then thoroughly washed with ultrapure water. The bio-discs of treatment group 2 were directly placed in a 0.1 mol / L morpholine ethanesulfonic acid solution containing 150 mmol / L EDC and 37.5 mmol / L NHS, incubated at room temperature for 15 minutes, and then thoroughly washed with ultrapure water.

[0090] Immediately, 50 μL of fluorescein isothiocyanate-labeled antibody (250 μg / mL) was added dropwise to the surfaces of bio-disc discs in treatment groups 1 and 2, and incubated at 37°C in the dark for 3 hours. After thorough washing with ultrapure water, the experimental results were observed using a fluorescence confocal microscope. See [link to experimental results]. Figure 8 (The method for statistical analysis of fluorescence intensity is the same as...) Figure 3 Statistical analysis using a t-test showed a significant difference between the hydrolysis-reconstruction group and the high-temperature dehydration group. Fluorescence images and quantitative images of average fluorescence intensity showed that the bio-discs in the hydrolysis-reconstruction group (without high-temperature treatment) exhibited higher antibody conjugation efficiency. "Hydrolysis-reconstruction" refers to treatment group 2, and "high-temperature dehydration" refers to treatment group 1 (the method used in Example 1). The average fluorescence intensity of the former was nearly three times that of the latter, demonstrating a significant improvement of nearly 300%, exceeding the inventors' expectations. In the prior art, it is generally believed that the succinic anhydride group in dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione is highly susceptible to hydrolysis, thus affecting antibody conjugation. Therefore, those skilled in the art tend to use a drying process on the discs treated with bridging buffer, particularly by reactivating dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione through high-temperature dehydration, to improve antibody conjugation efficiency. However, the actual situation was not as expected. The inventors adopted a step of not performing any form of drying treatment on the optical discs treated with the bridging buffer (e.g., not performing high-temperature drying treatment as in Example 3), and directly added the activation solution to the optical discs after the bridging buffer treatment. This overcame the bias of the prior art and the antibody conjugation efficiency of the final product obtained was higher than that expected by those skilled in the art.

[0091] Therefore, for the protective layer of optical discs treated with bridging buffer (after incubation in bridging buffer at 4°C and thorough washing with ultrapure water), a high-temperature drying method followed by activation (EDC+NHS) can improve antibody conjugation efficiency compared to low-temperature (room temperature) drying. However, if the protective layer of the optical disc treated with bridging buffer is not subjected to any form of high-temperature drying and is directly activated, it significantly improves the subsequent antibody conjugation efficiency compared to any form of high-temperature drying, achieving unexpected technical results.

[0092] Example 5

[0093] (1) Washing the protective layer on the surface of the optical disc

[0094] Prepare 10 mL of washing solution with a methanol:anhydrous ethanol ratio of 7 mL:3 mL. Place the optical disc in the washing solution and wash thoroughly for 5 minutes to remove surface contaminants that could affect the binding affinity of proteins and antibodies. Immediately dry the washed optical disc with nitrogen gas.

[0095] (2) Plasma treatment of optical disc surface

[0096] The optical disc obtained in the above steps was placed in an oxygen plasma treatment device with the following parameters: oxygen pressure 0.1 bar, power 50 W, and cleaning time 60 s.

[0097] (3) Configure different bridging buffers

[0098] Preparation of bridging buffer 1: 100 μL of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione (bridging agent, CAS: 93642-68-3) was added to 900 μL of 1×PBS buffer to prepare 10% bridging buffer 1. The bridging buffer was ultrasonically mixed for 10 min until the solution was clear and homogeneous. The resulting solution was placed on the surface of a surface-activated optical disc (treatment group 1) and incubated at 4℃ for 12-16 h, followed by thorough rinsing with ultrapure water.

[0099] Preparation of bridging buffer 2: 100 μL of succinic anhydride-terminated polydimethylsiloxane (bridging agent, CAS: 161205-23-8) was added to 900 μL of 1×PBS buffer to prepare 10% bridging buffer 2. The bridging buffer was ultrasonically mixed for 10 min until the solution was clear and homogeneous. The resulting solution was placed on the surface of the surface-activated optical disc (treatment group 2) and incubated at 4℃ for 12-16 h, followed by thorough rinsing with ultrapure water.

[0100] Preparation of bridging buffer 3: 100 μL of [3-(trimethoxysilyl)propyl]succinic anhydride (bridging agent, CAS: 156088-53-8) was added to 900 μL of 1×PBS buffer to prepare 10% bridging buffer 3. The above bridging buffer was ultrasonically mixed for 10 min until the solution was clear and homogeneous. The resulting solution was placed on the surface of the surface-activated optical disc (treatment group 3) and incubated at 4℃ for 12-16 h, followed by thorough rinsing with ultrapure water.

[0101] (4) Prepare different activation buffers

[0102] Preparation of Activation Buffer 1: Prepare Activation Buffer 1 containing 150 mmol / L EDC and 30 mmol / L 4-dimethylaminopyridine (DMAP, CAS: 1122-58-3) in 10 mL of 0.1 mol / L morpholine ethanesulfonic acid solution. Immediately place the optical discs treated with bridging buffer 1 (treatment group 1) into Activation Buffer 1, activate at room temperature for 30 min, and then thoroughly wash with ultrapure water.

[0103] Prepare activation buffer 2: Prepare activation buffer 2 containing 80 mmol / L dicyclohexylcarbodiimide (DCC, CAS: 538-75-0) and 16 mmol / L 4-dimethylaminopyridine (DMAP) in 10 mL of 0.1 mol / L morpholine ethanesulfonic acid solution. Immediately place the optical discs treated with bridging buffer 2 (treatment group 2) into activation buffer 2, activate at room temperature for 30 min, and then thoroughly wash with ultrapure water.

[0104] Preparation of Activation Buffer 3: Prepare Activation Buffer 3 containing 100 mmol / L O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU, CAS: 148893-10-1) in 10 mL of 0.01 mol / L phosphate buffer. Immediately place the CD (treatment group 3) treated with bridging buffer 3 into activation buffer 3, activate at room temperature for 30 min, and then thoroughly wash with ultrapure water.

[0105] (5) Antibody conjugate

[0106] Immediately add 50 μL of fluorescein isothiocyanate-labeled antibody (250 μg / mL) to the surfaces of the optical discs in treatment groups 1, 2, and 3 obtained in step (4) above, and incubate at 37°C in the dark for 3 hours. After thorough washing with ultrapure water, observe the experimental results using a fluorescence confocal microscope. Figure 9 As shown, Figures A, B, and C display typical fluorescence images of the bio-disc surfaces of treatment groups 1, 2, and 3, respectively, obtained under a fluorescence confocal microscope (the fluorescence intensity statistical method is the same as that used in the previous figures). Figure 3As can be seen from the fluorescence images and quantitative images of average fluorescence intensity, the different bridging buffers and activation buffer systems used can significantly improve the antibody conjugation efficiency on the surface of the bio-optical disc. Therefore, using siloxanes containing succinic anhydride as bridging buffers, and using EDC, NHS, DCC, DMAP, and HATU to activate the optical disc surface, can all achieve the goal of improving the efficiency of protein conjugation (such as antibodies and antigens) on the optical disc surface.

[0107] The inventors also attempted the operation method of Example 1 in this embodiment, namely, after treating the CD with bridging buffer, high-temperature drying (heating at 120°C for 10 min) was performed followed by functional group activation (activation buffer treatment). Following the above operation method (the bridging buffer and activation buffer still used the type of this embodiment), the fluorescence intensity of treatment groups 1, 2, and 3 in this embodiment compared to... Figure 9 All corresponding groups showed a significant decrease. These experimental results further demonstrate that, to further improve antibody conjugation efficiency using this treatment method, activation buffer can be added directly after bridging buffer treatment. In other words, the hydrolysis-reconstruction method is superior to the high-temperature dehydration method.

[0108] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for improving the biomolecule coupling efficiency on the surface of an acrylate substrate, characterized in that: The acrylate substrate was subjected to oxygen plasma treatment; then the acrylate substrate was soaked in bridging buffer; the acrylate substrate soaked in bridging buffer was washed with ultrapure water; then, without drying, the washed acrylate substrate was directly added to activation buffer; the acrylate substrate was soaked in activation buffer, and the resulting treated acrylate substrate was used for antibody conjugation. The active ingredient in the bridging buffer is a siloxane containing succinic anhydride; the active ingredient in the activation buffer is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, or a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, or a combination of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate; Siloxanes containing succinic anhydride include at least one of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, [3-(trimethoxysilyl)propyl]succinic anhydride, and succinic anhydride-terminated polydimethylsiloxanes. The concentration of siloxane containing succinic anhydride is 10%, and the bridging buffer is prepared using at least one of PBS buffer, morpholine ethanesulfonic acid buffer, and Tris buffer.

2. The method for improving the biomolecule coupling efficiency on the surface of an acrylate substrate according to claim 1, characterized in that: The conditions for plasma treatment are: oxygen pressure 0.05-0.2 bar, power 20-80 W, and treatment time 30-180 s; The activation buffer is prepared using morpholine ethanesulfonic acid solution or phosphate buffer; the CD protective layer is soaked in the activation buffer for 5-15 minutes.

3. The method for improving the biomolecule coupling efficiency on the surface of an acrylate substrate according to claim 2, characterized in that: The acrylate substrate is derived from the protective layer of the bio-optical disc.

4. A method for coupling biomolecules to an acrylate substrate, characterized in that: The following steps are performed sequentially: S1 Washing the acrylate substrate: Wash the acrylate substrate with a mixed solution of methanol and ethanol, and then dry it with nitrogen gas; S2 plasma treatment: Acrylic substrate is treated with oxygen plasma; S3 Siloxane Bridging: The acrylate substrate is soaked in a bridging buffer; the active substance in the bridging buffer is a siloxane containing succinic anhydride; Siloxanes containing succinic anhydride include at least one of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione, [3-(trimethoxysilyl)propyl]succinic anhydride, and succinic anhydride-terminated polydimethylsiloxanes. The concentration of siloxane containing succinic anhydride is 10%, and the bridging buffer is prepared using at least one of PBS buffer, morpholine ethanesulfonic acid buffer, and Tris buffer. S4 substrate activation: The acrylate substrate soaked in bridging buffer is washed with ultrapure water; then, without drying, the washed acrylate substrate is directly added to the activation buffer; the acrylate substrate is soaked in the activation buffer; the active substance in the activation buffer is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, or a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, or a combination of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, or O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate; S5 Antibody / Antigen Conjugation: The antibody / antigen solution is dropped onto an acrylate substrate and incubated to obtain an acrylate substrate conjugated with the antibody.

5. The method for coupling biomolecules to an acrylate substrate according to claim 4, characterized in that: In S1, the mixed solution of methanol and ethanol consists of methanol and anhydrous ethanol in a volume ratio of 7:3; the washing time is 5 min. In S2, the plasma treatment conditions are: oxygen pressure 0.05-0.2 bar, power 20-80 W, and treatment time 30-180 s; In S3, the bridging buffer is prepared as follows: dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione is dissolved in PBS buffer and then sonicated for 10-20 min; the concentration of dihydro-3-[3-(triethoxysilyl)propyl]furan-2,5-dione is 10%; the soaking time is 12-16 h; Alternatively, succinic anhydride-terminated polydimethylsiloxane can be dissolved in PBS buffer and then sonicated for 10-20 minutes to obtain the product; the concentration of succinic anhydride-terminated polydimethylsiloxane is 10%; the soaking time is 12-16 hours. Alternatively, [3-(trimethoxysilyl)propyl]succinic anhydride can be dissolved in PBS buffer and then sonicated for 10-20 min to obtain the solution; the concentration of [3-(trimethoxysilyl)propyl]succinic anhydride is 10%; the soaking time is 12-16 h. In S4, the activation buffer is prepared as follows: using 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and making the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 100-150 mmol / L and the final concentration of N-hydroxysuccinimide 25-37.5 mmol / L; Alternatively, use a 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and make the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride 100-150 mmol / L and the final concentration of 4-dimethylaminopyridine 15-30 mmol / L. Alternatively, use a 0.05-0.1 mol / L morpholine ethanesulfonic acid solution, and make the final concentration of dicyclohexylcarbodiimide 40-80 mmol / L and the final concentration of 4-dimethylaminopyridine 8-16 mmol / L; Alternatively, use 0.01 mol / L phosphate buffer and make the final concentration of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate 40-100 mmol / L; Soak the acrylate substrate in the above-mentioned activation buffer for 5-15 minutes; In S5, the concentration of antibody in the antibody solution is 50-500 μg / mL; the incubation temperature is 25-37℃, and the incubation time is 3-6 h.

6. A bio-optical disc coupled with biomolecules, obtained by a method for coupling biomolecules to an acrylate substrate according to any one of claims 4-5.

7. The application of the bio-disc coupled with biomolecules as described in claim 6 in the preparation of bioreaction substrates.

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