Synthetic bio-derived chemical signal amplification system and high-sensitivity antigen-antibody kit

Abstract

The invention relates to the field of biological detection, in particular to a synthetic bio-derived chemical signal amplification system and a high-sensitivity antigen-antibody kit. The invention discloses a synthetic bio-derived chemical signal amplification system and a high-sensitivity antigen-antibody kit. The kit comprises carboxylated silicon dioxide fluorescent microsphere dispersion liquid, an antibody, an activating agent, a combined stabilizer, a sealing agent and a buffer solution, the carboxylated silicon dioxide fluorescent microsphere dispersion liquid is obtained after silicon dioxide fluorescent microspheres are subjected to surface modification by a carboxylated polyether silane coupling agent; and the antibody is specifically and covalently bound with the carboxylated silicon dioxide fluorescent microspheres. According to the present invention, the efficient amplification of the detection signal is achieved, the detection lower limit is reduced, the detection capability on the low-concentration antigen is effectively improved, the stable pg-level detection effect can be achieved, and the obtained kit has characteristics of high sensitivity, high precision and high stability, and can be used in the wide antigen-antibody detection diagnosis item.

Description

Technical Field

[0001] This application relates to the field of biological detection, specifically to synthetic biologically derived chemical signal amplification systems and highly sensitive antigen-antibody kits. Background Technology

[0002] Lateral flow immunoassay is an immunoassay technique that relies on the interaction between biomolecules. Known antigens or antibodies are pre-conjugated onto a carrier as a capture line. The sample to be tested flows through the capture line under capillary action of the carrier. If the sample contains the corresponding antibody or antigen, it will specifically react with the antigen or antibody on the carrier and be adsorbed onto the carrier. The presence or concentration of the analyte is determined by color development or luminescence of a labeled substance. This technique achieves macroscopic visualization or microscopic detection of antigen-antibody reactions through signal amplification. Due to its simplicity, speed, and low cost, it has been widely used in medical testing, environmental monitoring, food testing, and animal disease control.

[0003] The antibodies used in lateral immunochromatographic medical diagnostics are mostly recombinant monoclonal antibodies, such as murine recombinant monoclonal antibodies and human-mouse chimeric monoclonal antibodies. Their application in medical diagnostic test kits offers advantages such as high specificity, high batch-to-batch consistency, scalable production, and stable and controllable performance, significantly improving diagnostic sensitivity, accuracy, and clinical applicability. Recombinant monoclonal antibodies are monoclonal antibodies produced through synthetic biology techniques, where artificially designed antibody genes are efficiently expressed in vitro. The core technology involves constructing antibody genes using DNA recombination technology, followed by large-scale production using host cells (such as bacteria, yeast, or mammalian cells). Compared to traditional hybridoma techniques and conventional genetic engineering methods, the core advantages of using synthetic biology to prepare recombinant monoclonal antibodies lie in rational design, efficient construction, precise regulation, and performance customization, enabling optimization of the entire antibody research and production chain from the gene level to the cell factory level.

[0004] Fluorescent immunochromatography is a novel detection technique that combines immunofluorescence and immunochromatography. This technique utilizes fluorescent microspheres that bind to antibodies to form fluorescently labeled antibodies. Using a nitrocellulose membrane as a carrier, the sample moves along the chromatography strip via capillary action. After the target analyte in the sample binds to the fluorescently labeled antibody, it is captured by the immobilized antibody at the detection line, forming a complex. Irradiation with excitation light produces a fluorescent signal. This technique achieves qualitative or quantitative detection through the specific reaction between antigen and antibody, and its sensitivity is higher than that of traditional colloidal gold chromatography.

[0005] However, in actual testing, since the content of specific biomarkers in the sample, namely antibodies or antigens, is usually in the range of picograms / mL to nanograms / mL, most immunofluorescence detection reagents prepared by traditional methods have problems such as non-specific aggregation of fluorescent microspheres, non-specific adsorption and binding of antibodies, poor stability of physical adsorption antibody binding, and easy impact on antibody biological activity, making it difficult to achieve the required high sensitivity and high accuracy.

[0006] Therefore, it is necessary to develop a synthetic bio-derived chemical signal amplification system to achieve efficient amplification of detection signals, lower the detection limit, and improve detection accuracy while achieving high sensitivity. This would result in antigen-antibody kits with high sensitivity and high accuracy, optimize the detection process, and provide patients with more convenient and high-quality medical diagnostic services. Summary of the Invention

[0007] To achieve efficient amplification of detection signals, lower the detection limit, and improve detection accuracy while achieving high sensitivity, this application provides a synthetic biologically derived chemical signal amplification system and a highly sensitive antigen-antibody kit.

[0008] In a first aspect, this application provides a synthetic biology-derived chemical signal amplification system, employing the following technical solution: A synthetic bio-derived chemical signal amplification system includes a dispersion of carboxylated silica fluorescent microspheres, an antibody, an activator, a combined stabilizer, a blocking agent, and a buffer solution; The carboxylated silica fluorescent microsphere dispersion is obtained by surface modification of silica fluorescent microspheres with a carboxylated polyether silane coupling agent; the chemical formula of the carboxylated polyether silane coupling agent is (RO)3Si-(CH2)3-O-(C2H4O). n -(C3H6O) m -COOH; where R is an alkyl group, n is an integer in the range of 5 to 20, and m is an integer in the range of 0 to 5; The antibody specifically covalently binds to carboxylated silica fluorescent microspheres.

[0009] By adopting the above technical solution, silica fluorescent microspheres are used as signal carriers. Compared with traditional polystyrene fluorescent microspheres, they have better chemical stability, and the surface of silica fluorescent microspheres is rich in active groups, making them easier to modify. Simultaneously, the fluorescent material is less affected by the environment, resulting in a more stable fluorescence signal. Furthermore, during antibody labeling, silica fluorescent microspheres exhibit superior dispersion stability and antibody binding ability compared to traditional polystyrene fluorescent microspheres. They also show better uniformity of migration on the chromatography membrane, effectively reducing band tailing and false positives, making them more suitable for detection scenarios requiring high sensitivity, high precision, and high stability.

[0010] Secondly, this application uses a carboxylated polyether silane coupling agent to modify the surface of silica fluorescent microspheres, introducing carboxyl groups that can specifically covalently react with antibodies on the surface of the silica fluorescent microspheres. After the action of an activator, these carboxyl groups can undergo an amidation reaction with the amino groups of the antibody to form stable amide bonds, thereby achieving specific covalent binding between the antibody and the silica fluorescent microspheres. This can effectively improve the binding stability between the silica fluorescent microspheres and the antibody, which is of positive significance for achieving high sensitivity and high accuracy in detection.

[0011] Meanwhile, the carboxylated polyether silane coupling agent used has a siloxane group at its hydrophilic end, which can react with the hydroxyl groups on the surface of silica fluorescent microspheres to form Si-O-Si covalent bonds, and the binding strength is much stronger than physical adsorption; the carboxyl group at its hydrophobic end can serve as a specific binding site for antibodies after activation, and the antibody binding stability is good after coupling; it also has excellent biocompatibility and has little impact on antibody activity.

[0012] Furthermore, in traditional antibody labeling processes, nonionic surfactants are typically added to optimize the dispersibility of fluorescent microspheres and reduce their aggregation. This also helps reduce nonspecific antibody adsorption, improving labeling efficiency and effectiveness, and can inhibit false positives in subsequent detections. Additionally, it effectively reduces antibody hydrophobic denaturation, thus protecting antibody activity. However, in practice, environmental factors such as temperature, pH, and surfactant concentration can cause the addition of nonionic surfactants to lead to antibody detachment from the fluorescent microspheres. Moreover, the improvement in labeling efficiency is easily affected by environmental fluctuations, potentially negatively impacting the sensitivity and accuracy of subsequent detections.

[0013] Based on the above problems, the carboxylated polyether silane coupling agent used in this application, on the basis of achieving functionalized modification of silica microspheres, also has the structural characteristics of a nonionic surfactant, and its structure contains -(C2H4O). n - The chain segments can provide a steric hindrance effect. By controlling the repeating unit value n, a suitable chain segment length can be obtained, allowing it to exert an appropriate steric hindrance effect. The dispersibility of the modified silica fluorescent microspheres in aqueous phase or buffer solution is further improved, and long-term dispersion stability can be maintained under the influence of the steric hindrance effect. -(C3H6O) m - The chain segment is a hydrophobic group that connects to the carboxyl group. By controlling the repeating unit value m, a suitable number of antibody binding sites can be obtained, thereby improving the antibody binding efficiency.

[0014] Therefore, this application can effectively overcome the adverse effects caused by the addition of nonionic surfactants while maintaining their positive effects. The resulting synthetic bio-derived chemical signal amplification system can effectively amplify the detection signal and reduce the detection limit. When applied to detection kits, it can improve detection accuracy while achieving high sensitivity, providing patients with more convenient and high-quality medical diagnostic services.

[0015] Preferably, in the chemical formula of the carboxylated polyether silane coupling agent, R is ethyl, n is an integer in the range of 8 to 15, and m is an integer in the range of 1 to 3.

[0016] By adopting the above technical solution, the chemical formula of the carboxylated polyether silane coupling agent is further optimized so that R is ethyl, and a suitable hydrolysis rate is controlled, resulting in better controllability of the surface-modified silica fluorescent microspheres. Simultaneously, n is further optimized to be an integer in the range of 8 to 15 to control a suitable steric hindrance effect, thereby further reducing the problem of insufficient dispersibility caused by insufficient steric hindrance, which hinders the coupling reaction between the hydrophobic carboxyl group and the antibody; and further reducing the problem of insufficient dispersibility caused by insufficient steric hindrance. Furthermore, m is further optimized to be an integer in the range of 1 to 3 to further balance the affinity / reluctance level, ensuring the number of antibody binding sites while further reducing the problem of aggregation caused by excessive hydrophobicity, thus improving the antibody binding efficiency.

[0017] Preferably, the surface modification method for the silica fluorescent microspheres includes the following steps: S1. Prepare a silica fluorescent microsphere dispersion. After dilution with deionized water, the dispersion is subjected to ultrasonication, centrifugation, and resuspension in sequence to obtain the pretreated silica fluorescent microsphere dispersion. S2. Dissolve the carboxylated polyether silane coupling agent in anhydrous ethanol aqueous solution, adjust the pH to 4-5, and add the pretreated silica fluorescent microsphere dispersion under mixing and stirring. After the reaction is completed, the dispersion is obtained by centrifugation, washing and resuspension.

[0018] By adopting the above technical solution, the silica fluorescent microspheres are first activated and pretreated to optimize the subsequent surface modification effect; in step S2, the weak acid environment of pH 4-5 helps to catalyze the hydrolysis of silane, resulting in a better subsequent surface modification effect.

[0019] Preferably, in step S2, after adjusting the pH, the mixture is stirred for 5-10 minutes, and then the pretreated silica fluorescent microsphere dispersion is added while maintaining the stirring state.

[0020] By adopting the above technical solution, the mixture is first stirred for 5-10 minutes to induce partial hydrolysis of the carboxylated polyether silane coupling agent. Then, the pretreated silica fluorescent microsphere dispersion is added. At this time, the partially hydrolyzed carboxylated polyether silane coupling agent reacts with the silica fluorescent microspheres for modification. Meanwhile, the temporarily unhydrolyzed carboxylated polyether silane coupling agent can play a good dispersing role, allowing the silica fluorescent microspheres to undergo surface modification in a relatively stable dispersion system. This achieves a relatively stable and continuous reaction system of hydrolysis, dispersion, and modification, with good surface modification uniformity. This further optimizes the subsequent signal amplification system and has positive significance for achieving high sensitivity and high accuracy detection.

[0021] Preferably, in step S2, the mass ratio of carboxylated polyether silane coupling agent to silica fluorescent microspheres is 1:(12-16), the reaction time is 12-24 h, and the reaction temperature is 25-35 °C.

[0022] By adopting the above technical solution, the mass ratio of carboxylated polyether silane coupling agent to silica fluorescent microspheres, reaction time, and reaction temperature are further optimized, thereby further optimizing the reaction rate and surface modification effect.

[0023] Preferably, the activator is N-hydroxysuccinimide ester, which reacts with the carboxyl groups modified on the silica fluorescent microspheres to form an intermediate activated ester.

[0024] By adopting the above technical solution, using N-hydroxysuccinimide ester as an activator, the carboxyl groups modified on the silica fluorescent microspheres form an intermediate activated ester through an activation reaction, which can react with the antibody amino group to generate a stable amide bond. Compared with the traditional method of using EDC to conjugate antibodies, it is more stable and has a better effect on specifically binding antibodies.

[0025] Preferably, the activation reaction includes the following steps: A1. Prepare aqueous solutions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester, respectively; A2. Mix and stir the carboxylated silica fluorescent microsphere dispersion, the aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the aqueous solution of N-hydroxysuccinimide ester. After reacting for 20-30 min, the mixture is washed and resuspended sequentially to obtain the activated silica fluorescent microsphere dispersion. A3. Add 0.1-0.5 wt% of combined stabilizer to the activated silica fluorescent microsphere dispersion, mix and stir to obtain silica fluorescent microsphere composite solution; The combination stabilizers include calcium phosphate, calcium citrate, and gentamicin.

[0026] In traditional methods of antibody conjugation using EDC (electrode precipitate), the activation of fluorescent microspheres is prone to hydrolysis, requiring immediate washing before antibody conjugation, which presents significant practical limitations. While the intermediate activated ester used in this application is more stable than EDC, it may still be subject to some degree of hydrolysis.

[0027] Therefore, by adopting the above technical solution, in step A3, an appropriate amount of combined stabilizer is further added to the activated silica fluorescent microsphere dispersion, wherein gentamicin has a good antibacterial effect and effectively inhibits the excessive proliferation and interference of microorganisms that may exist in the system.

[0028] Secondly, the small amount of free calcium ions in calcium phosphate and calcium citrate can bind to specific amino acid residues in antibody molecules, helping to maintain the spatial conformation of the antibody and ensuring its specificity in binding to the antigen. Simultaneously, calcium phosphate adsorbs positively onto the surface of N-hydroxysuccinimide-activated silica fluorescent microspheres, while antibodies are mostly negatively charged in the system. Therefore, under the influence of the charge effect, negatively charged antibody molecules tend to approach the intermediate activated ester on the surface of the silica fluorescent microspheres, which helps to increase the collision probability and binding efficiency of the amidation reaction, meaning that the subsequent specific covalent binding of antibodies by the silica fluorescent microspheres is more effective. Furthermore, an appropriate amount of phosphate can optimize the ionic strength of the buffer solution, reduce the non-specific repulsion between the silica fluorescent microspheres and the antibody, and help inhibit the hydrolysis of the intermediate activated ester. In addition, calcium phosphate can enhance fluorescence; its crystal structure helps reduce the self-quenching of fluorescent molecules, which is of positive significance for signal amplification.

[0029] Furthermore, the soluble complex formed by calcium citrate in the system can bind to the amino groups of the antibody, helping to anchor the antibody near the intermediate activated ester on the surface of the silica fluorescent microspheres. This promotes amide bond formation, further improving antibody binding. Because a small amount of free citrate can complex with any trace amounts of unbound free Ca2+, this process also helps to further improve antibody binding. 2+ This effectively reduces the damage to the intermediate activated ester and indirectly inhibits its hydrolysis, as well as other potentially interfering metal ions. Furthermore, since the silica fluorescent microspheres in this application use rare earth ions as fluorescent materials, and calcium citrate has a certain enrichment effect on rare earth ions in the system, it effectively enhances the fluorescence signal and luminescence efficiency.

[0030] In summary, this application, by adding a combination of stabilizers to the activated silica fluorescent microsphere dispersion, effectively inhibits the hydrolysis of intermediate activated esters and further optimizes the signal amplification, antibody binding, stability, and storage stability of the system. This effectively solves the problem of poor storage stability and significant limitations in practical operation of activated fluorescent microspheres; simultaneously, it reduces non-specific reactions in subsequent detection, resulting in a significant improvement in both detection sensitivity and accuracy.

[0031] Preferably, the blocking agent includes one of bovine serum albumin, ovalbumin, and casein, and the buffer includes at least one of acetate buffer, phosphate buffer, borate buffer, and MES buffer.

[0032] Preferably, the method for preparing the signal amplification system includes the following steps: The antibody was dissolved and diluted with buffer solution to obtain an antibody solution. The antibody solution was added to a silica fluorescent microsphere composite solution and stirred at room temperature for 1-2 hours. A blocking agent was added and stirring was continued for 0.5-1 hour. The solution was then centrifuged, washed, and resuspended to obtain a signal amplification system.

[0033] Secondly, this application provides a highly sensitive antigen-antibody kit, which adopts the following technical solution: A highly sensitive antigen-antibody kit includes a synthetic bio-derived chemical signal amplification system, a sample pad, a conjugate pad, an NC membrane, absorbent paper, and a cartridge. The sample pad has a sample application well. The NC membrane has a detection line and a control line. The detection line (T line) can be coated with a mouse recombinant monoclonal antibody, and the control line (C line) can be coated with an IgG polyclonal antibody. The synthetic bio-derived chemical signal amplification system is sprayed onto the conjugate pad, and the sample pad, conjugate pad, NC membrane, and absorbent paper are sequentially attached. The sample pad is then cut into test strips of appropriate width, and the test strips are inserted into the cartridge to obtain a highly sensitive antigen-antibody kit.

[0034] By employing the above technical solution, during the testing process, the sample is diluted, and a certain amount is added to the sample well. After reacting for 5–10 minutes, the test line (T line) and control line (C line) are observed. If two clear red bands are visible to the naked eye at both the test line (T line) and control line (C line), the sample is positive. If a red band appears at the control line (C line), but no band appears at the test line (T line), or it is impossible to determine whether a band appears to the naked eye, the sample is negative. If no red bands appear at either the test line (T line) or control line (C line), or if a red band appears only at the test line (T line), it is an incorrect result and retesting is required.

[0035] The above testing procedures are relatively simple and can be well applied to patient self-examination or diagnostic examinations in rural medical institutions. They have excellent practical value and provide patients with more convenient and high-quality medical diagnostic services.

[0036] In summary, this application has the following beneficial effects: 1. This application uses a carboxylated polyether silane coupling agent to modify the surface of silica fluorescent microspheres, which can achieve specific covalent binding between the antibody and the silica fluorescent microspheres, effectively improving the binding stability between the silica fluorescent microspheres and the antibody; moreover, the carboxylated polyether silane coupling agent has excellent biocompatibility and has little impact on the activity of the antibody, effectively improving the detection sensitivity and accuracy.

[0037] 2. The carboxylated polyether silane coupling agent used in this application, on the basis of functionalizing silica microspheres, also has the structural characteristics of nonionic surfactants. While maintaining the positive effect of nonionic surfactants, it effectively overcomes the adverse effects caused by their addition. The resulting synthetic bio-derived chemical signal amplification system can effectively achieve efficient amplification of detection signals and reduce the detection limit. When applied to detection kits, it can improve detection accuracy while achieving high sensitivity.

[0038] 4. In the process of modifying silica fluorescent microspheres on the surface of carboxylated polyether silane coupling agent, this application can carry out surface modification in a relatively stable dispersion system, realize a relatively stable and continuous hydrolysis, dispersion and modification reaction system, and have good surface modification uniformity, thereby further optimizing the subsequent signal amplification system, which is of positive significance for achieving high sensitivity and high accuracy detection.

[0039] 5. This application, by adding a combined stabilizer to the activated silica fluorescent microsphere dispersion, further optimizes the signal amplification, antibody binding effect, stability, and storage stability of the system while effectively inhibiting the hydrolysis of intermediate activated esters. This effectively solves the problem of poor storage stability of activated fluorescent microspheres and significant limitations in practical operation; at the same time, it reduces non-specific reactions in subsequent detection, resulting in a significant improvement in both detection sensitivity and accuracy. Detailed Implementation

[0040] In this embodiment, the choice of antibody is not specifically limited and can be adjusted according to the actual detection needs. The following embodiments use murine recombinant monoclonal antibody and IgG polyclonal antibody as examples. The murine recombinant monoclonal antibody can be murine anti-human KIM-1 recombinant monoclonal antibody, murine anti-human TIMP-2 recombinant monoclonal antibody, murine anti-human PCT recombinant monoclonal antibody, etc., and the IgG polyclonal antibody is goat anti-rabbit IgG polyclonal antibody. In this embodiment, the fluorescent material of the silica fluorescent microspheres is rare earth ions, which can be Eu. 3+ 、Tb 3+ Ce 3+ 、Sm 3+ In this embodiment, a particle size of 300 nm or less is preferred. 3+ Examples of silica fluorescent microspheres as fluorescent materials are provided. In this embodiment, the chemical formula of the carboxylated polyether silane coupling agent is as follows: (RO)3Si-(CH2)3-O-(C2H4O) n -(C3H6O) m -COOH; wherein R is an alkyl group, n is an integer in the range of 5 to 20, and m is an integer in the range of 0 to 5; more preferably R is an ethyl group, n is an integer in the range of 8 to 15, and m is an integer in the range of 1 to 3; In this embodiment, the activator is N-hydroxysuccinimide ester; In this embodiment, the sealing agent includes one of bovine serum albumin, ovalbumin, and casein. Bovine serum albumin is preferred as the sealing agent in this embodiment. In this embodiment, the buffer solution includes at least one of acetate buffer, phosphate buffer, borate buffer, and MES buffer. In this embodiment, phosphate buffer and MES buffer are used as examples. The phosphate buffer has a specification of 0.01 mol / L and a pH of 7.4; the MES buffer has a specification of 0.5M and a pH of 6 to 6.5. In this embodiment, EDC refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; In this embodiment, the volume ratio of anhydrous ethanol to deionized water in the anhydrous ethanol aqueous solution is 9:1, and the anhydrous ethanol is of analytical grade. The reconstitution solution in this embodiment consists of: phosphate buffer solution with a strength of 0.01 mol / L and pH 7.4; and 0.2 wt% bovine serum albumin. The present application will be further described in detail below with reference to embodiments, application examples, comparative examples and comparative application examples. The raw materials involved in the present application can all be obtained commercially.

[0041] Example 1 A synthetic bio-derived chemical signal amplification system comprises a carboxylated silica fluorescent microsphere dispersion, a mouse anti-human KIM-1 recombinant monoclonal antibody, a goat anti-rabbit IgG polyclonal antibody, N-hydroxysuccinimide ester, a combined stabilizer, bovine serum albumin, and MES buffer. The carboxylated silica fluorescent microsphere dispersion is obtained by surface modification of silica fluorescent microspheres with a carboxylated polyether silane coupling agent. The chemical formula of the carboxylated polyether silane coupling agent is (RO)3Si-(CH2)3-O-(C2H4O).n -(C3H6O) m -COOH; where R is ethyl, n is 10, and m is 2; the combined stabilizers include calcium phosphate, calcium citrate, and gentamicin, with a mass ratio of 2:1:1; The preparation method of the above signal amplification system includes the following steps: 1. Preparation of carboxylated silica fluorescent microsphere dispersion S1. Take 10 mL of 10 wt% silica fluorescent microsphere dispersion, dilute with deionized water to 1% solid content, sonicate for 30 min, centrifuge at 3000 rpm for 10 min, discard the supernatant, and resuspend with anhydrous ethanol to 1% solid content to obtain the pretreated silica fluorescent microsphere dispersion. S2. Weigh 20 mg of carboxylated polyether silane coupling agent and dissolve it in 10 mL of anhydrous ethanol aqueous solution. Adjust the pH to 4-5 and mix and stir at 300 rpm for 10 min at room temperature. Then, add 30 mL of pretreated silica fluorescent microsphere dispersion while mixing and stirring. After reacting at 30 °C for 12 h, centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend the precipitate with anhydrous ethanol, sonicate for 5 min, wash twice with phosphate buffer to remove residual ethanol, and finally resuspend with phosphate buffer to a solid content of 1% to obtain carboxylated silica fluorescent microsphere dispersion. 2. Preparation of silica fluorescent microsphere composite solution A1. Prepare an aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration of 20 mg / ml, and prepare an aqueous solution of N-hydroxysuccinimide at a concentration of 50 mg / ml. A2. Mix and stir 20 ml of carboxylated silica fluorescent microsphere dispersion, 2 ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide aqueous solution and 2 ml of N-hydroxysuccinimide aqueous solution at 100 rpm. After reacting at room temperature for 30 min, wash twice with MES buffer and resuspend in deionized water to a solid content of 1% to obtain activated silica fluorescent microsphere dispersion. A3. Add 0.2wt% of combined stabilizer to the activated silica fluorescent microsphere dispersion, mix and stir at 600rpm for 30min to obtain silica fluorescent microsphere composite solution; 3. Preparation of the signal amplification system Mouse anti-human KIM-1 recombinant monoclonal antibody and goat anti-rabbit IgG polyclonal antibody were dissolved and diluted separately with phosphate buffer to obtain 5 mg / ml mouse anti-human KIM-1 recombinant monoclonal antibody solution and 5 mg / ml goat anti-rabbit IgG polyclonal antibody solution. 1 ml of mouse anti-human KIM-1 recombinant monoclonal antibody solution was added to 5 ml of silica fluorescent microsphere composite solution and stirred at room temperature for 1 h. Bovine serum albumin was added and stirring continued for 1 h, bringing the final concentration of bovine serum albumin to 1 wt%. The mixture was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in phosphate buffer, sonicated for 5 min, washed twice with phosphate buffer, and resuspended in 50 ml of reconstitution solution to a solid content of 1%, yielding a silica fluorescent microsphere dispersion labeled with mouse anti-human KIM-1 recombinant monoclonal antibody. The same procedure was repeated for the goat anti-rabbit IgG polyclonal antibody solution to obtain a silica fluorescent microsphere dispersion labeled with goat anti-rabbit IgG polyclonal antibody. The two solutions were then mixed at a volume ratio of 1:1 to obtain the signal amplification system.

[0042] Example 2 The difference between this embodiment and Embodiment 1 is that the mouse-derived recombinant monoclonal antibody is a mouse anti-human TIMP-2 recombinant monoclonal antibody.

[0043] Example 3 The difference between this embodiment and Embodiment 1 is that the mouse-derived recombinant monoclonal antibody is a mouse anti-human PCT recombinant monoclonal antibody.

[0044] Example 4 The difference between this embodiment and Embodiment 1 is that n in the chemical formula of the carboxylated polyether silane coupling agent is 8.

[0045] Example 5 The difference between this embodiment and Embodiment 1 is that n in the chemical formula of the carboxylated polyether silane coupling agent is 15.

[0046] Example 6 The difference between this embodiment and Embodiment 1 is that m in the chemical formula of the carboxylated polyether silane coupling agent is 1.

[0047] Example 7 The difference between this embodiment and Embodiment 1 is that m in the chemical formula of the carboxylated polyether silane coupling agent is 3.

[0048] Example 8 The difference between this embodiment and Embodiment 1 is that the combined stabilizer includes calcium phosphate and gentamicin, with a mass ratio of 2:1.

[0049] Example 9 The difference between this embodiment and Embodiment 1 is that the combined stabilizer includes calcium citrate and gentamicin, with a mass ratio of 2:1.

[0050] Example 10 The difference between this embodiment and Embodiment 1 is that the combined stabilizer is gentamicin.

[0051] Example 11 The difference between this embodiment and Example 1 is that in step S2, 20 mg of carboxylated polyether silane coupling agent is weighed and dissolved in 10 mL of anhydrous ethanol aqueous solution, the pH is adjusted to 4-5, and the mixture is stirred at 300 rpm for 30 min at room temperature. Then, 30 mL of pretreated silica fluorescent microsphere dispersion is added while stirring. After reacting at 30°C for 12 h, the mixture is centrifuged at 4000 rpm for 10 min, the supernatant is discarded, the precipitate is resuspended in anhydrous ethanol, sonicated for 5 min, and then washed twice with phosphate buffer to remove residual ethanol. Finally, the mixture is resuspended in phosphate buffer until the solid content is 1%, thus obtaining the carboxylated silica fluorescent microsphere dispersion.

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that commercially available carboxyl-functionalized silica fluorescent microspheres are used instead of the carboxylated silica fluorescent microspheres modified with carboxylated polyether silane coupling agent.

[0053] Comparative Example 2 The difference between this comparative example and Example 1 is that commercially available carboxylated functionalized silica fluorescent microspheres are used instead of the carboxylated silica fluorescent microspheres modified with carboxylated polyether silane coupling agent; and in step A2, 0.1 wt% of carboxylated polyether silane coupling agent is added to the dispersion of carboxylated silica fluorescent microspheres.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that commercially available carboxylated functionalized silica fluorescent microspheres are used instead of the carboxylated silica fluorescent microspheres modified with carboxylated polyether silane coupling agent; and 0.1 wt% of carboxylated polyether silane coupling agent is added to the complex solution.

[0055] Comparative Example 4 The difference between this comparative example and Example 1 is that commercially available carboxylated functionalized silica fluorescent microspheres are used instead of the carboxylated silica fluorescent microspheres modified with carboxylated polyether silane coupling agent; and in step A2, 0.1 wt% of Tween 20 is added to the dispersion of carboxylated silica fluorescent microspheres.

[0056] Comparative Example 5 The difference between this comparative example and Example 1 is that no combined stabilizer is added.

[0057] Application Example 1 A highly sensitive antigen-antibody kit includes the signal amplification system obtained in Example 1, a sample pad, a conjugate pad, an NC membrane, absorbent paper, and a cartridge. The sample pad has sample application wells, and the NC membrane is marked with a detection line and a control line. The detection line (T line) is coated with mouse anti-human KIM-1 recombinant monoclonal antibody, and the control line (C line) is coated with goat anti-rabbit IgG polyclonal antibody. The preparation method of the above-mentioned highly sensitive antigen-antibody kit is as follows: 1) The signal amplification system obtained in step 1 was uniformly sprayed onto the bonding pad at a spraying rate of 5 μL / cm, dried at 35°C, and then packaged for later use. 2) Fix the NC membrane onto the cartridge. Equilibrate the NC membrane temperature and humidity at 35℃ beforehand. The distance between the detection line (T line) and the control line (C line) is 5mm, and the distances between the detection line (T line) and the conjugate pad end, and between the control line (C line) and the absorbent paper end are 10mm each. The detection line (T line) contains mouse anti-human KIM-1 recombinant monoclonal antibody, and the control line (C line) contains goat anti-rabbit IgG polyclonal antibody. Dilute each antibody to 1mg / ml with phosphate buffer. Using a contact spotting head, the streaking volume is set to 0.5μL / cm. Streak parallel lines onto the NC membrane to form the detection line (T line) and the control line (C line). After the operation, place the sheet at 35℃ to dry for 12 hours. After drying, store it in an aluminum foil bag away from light for later use. 3) Paste the sample pad, conjugate pad, NC membrane, and absorbent paper in sequence, cut them into test strips of appropriate width, insert the test strips into the cartridge, and obtain the high-sensitivity antigen-antibody kit.

[0058] Application Example 2 The difference between this application example and application example 1 is that the signal amplification system in step 1) was prepared in example 2, and in step 2), the detection line (T line) is a mouse anti-human TIMP-2 recombinant monoclonal antibody.

[0059] Application Example 3 The difference between this application example and application example 1 is that the signal amplification system in step 1) was prepared in example 3, and in step 2), the detection line (T line) contains mouse anti-human PCT recombinant monoclonal antibody.

[0060] The difference between Application Examples 4-11 and Application Example 1 is that the signal amplification system in step 1) is prepared according to Examples 4-11 respectively.

[0061] The difference between Comparative Examples 1-5 and Comparative Example 1 is that the signal amplification system in step 1) is prepared according to Comparative Examples 1-5 respectively.

[0062] Performance testing methods The antigen-antibody kits obtained in Application Examples 1-11 and Comparative Application Examples 1-5 were used as detection tools. The corresponding biomarkers (KIM-1 in Application Examples 1 and 4-11, TIMP-2 in Application Example 2, and PCT in Application Example 3) were used as detection samples, and detection was performed at gradient concentrations of 0 ng / ml, 0.1 ng / ml, 1 ng / ml, 10 ng / ml, and 100 ng / ml. During the testing process, after diluting the sample, a certain amount is added to the sample well. After reacting for 10 minutes, the test line (T line) and control line (C line) are observed. If two clear red bands are visible to the naked eye at both the test line (T line) and control line (C line), the sample is positive. If a red band appears at the control line (C line), but no band appears at the test line (T line) or it is impossible to determine whether a band appears to the naked eye, the sample is negative. If no red bands appear at either the test line (T line) or control line (C line), or if a red band appears only at the test line (T line), it is an incorrect result and needs to be retested. The detection sensitivity of the antigen-antibody kit should be analyzed based on the test results. Each concentration of biomarker was tested 10 times, for a total of 50 tests. If the 50 results were consistent, the accuracy was recorded as 100%. If there were inconsistencies, the corresponding accuracy was calculated. In addition, following the above testing steps, each test group was tested for IL-18 biomarker at a concentration of 100 ng / ml, and the positive results were recorded. The detection accuracy of the antigen-antibody kit was analyzed based on the test results. The antigen-antibody kits obtained in Application Examples 1-11 and Comparative Application Examples 1-5 were placed in an accelerated aging environment at 45°C. After accelerated aging for 7, 14, 30, and 60 days, the biomarkers at a concentration of 1 ng / ml were detected according to the above steps, and the accelerated aging stability of the antigen-antibody kits was analyzed based on the detection results.

[0063] Table 1 Sensitivity Detection Results Note: In the table, "-" represents negative and "+" represents positive. The number of "+" indicates the intensity of the color as observed by the naked eye, and the intensity of the color is positively correlated with the strength of the positive result.

[0064] Based on the above test results, the kits in Application Examples 1 to 11 can all detect biomarkers at a concentration of 0.1 ng / ml with high sensitivity, which fully verifies the ability of this application to achieve efficient amplification of the detection signal and reduce the detection limit. The obtained kits have high sensitivity and can achieve pg-level detection effect. Specifically, based on the test results of Application Examples 1 to 3, the kits obtained by this application can be well applied to the detection of different biomarkers and can be used in a wide range of antigen and antibody detection and diagnostic projects.

[0065] Specifically, considering the detection results of Application Example 1 and Application Examples 4-7, the kits in Application Examples 4-7 showed a certain degree of decrease in positive detection capability compared to Application Example 1 in the detection of multiple biomarker concentrations. This may be due to changes in the molecular structure of the carboxylated polyether silane coupling agent, specifically caused by changes in steric hindrance effect and changes in antibody-specific binding sites. This further verifies the influence of the molecular structure of the carboxylated polyether silane coupling agent on the detection capability of the kit.

[0066] Specifically, considering the detection results of Application Examples 1 and 8-10, the kits used in Application Examples 8-10 also showed a certain degree of decrease in positive detection capability. This may be due to the absence of calcium phosphate and / or calcium citrate components in the combined stabilizer. Further verification confirmed the positive effects of both on inhibiting intermediate activated ester hydrolysis, enhancing signal, optimizing antibody binding and stability, etc., which can, to some extent, improve the problem of significant limitations in practical operation, and the detection sensitivity of the kit has been significantly further optimized.

[0067] Specifically, comparing the detection results of Application Example 1 and Comparative Application Examples 1-5, none of the kits in Comparative Application Examples 1-4 could detect the biomarker at 0.1 ng / ml, and their positive detection ability at concentrations greater than or equal to 1 ng / ml was significantly lower than that of Application Example 1. This further verifies the positive effect of surface modification of silica fluorescent microspheres with carboxylated polyether silane coupling agents. In Comparative Application Example 5, without the addition of the combined stabilizer, although pg-level detection was still achieved, the detection ability was significantly lower than that of Application Example 1, further verifying the positive effect of the combined stabilizer.

[0068] Table 2 Accuracy Test Results Based on the above test results, the kits in Examples 1 to 11 can all achieve a detection accuracy of 100%, and no false positives were found for non-specific biomarkers, which fully demonstrates that the kits in this application have high accuracy and strong reliability in actual detection and diagnosis.

[0069] False positives were observed in all of the comparative application examples 1-4, and non-specific detections were also observed in comparative application example 3, further verifying the positive effect of surface modification of silica fluorescent microspheres with carboxylated polyether silane coupling agent.

[0070] Table 3. Accelerated Aging Stability Test Results Note: In the table, "-" represents negative and "+" represents positive. The number of "+" indicates the intensity of the color as observed by the naked eye, and the intensity of the color is positively correlated with the strength of the positive result.

[0071] Based on the above test results, after accelerated aging for 7, 14, 30 and 60 days, the kits in Examples 1 to 11 still exhibited excellent positive detection capabilities and maintained good detection sensitivity. This fully demonstrates that the kits in this application have good storage stability, which largely overcomes the limitations of kit application and promotion, and can provide patients with more convenient and high-quality medical diagnostic services.

[0072] In contrast, the kits in Examples 1-5 lost their positive detection capability after accelerated aging for 7 or 14 days, further verifying the positive effects of surface modification of silica fluorescent microspheres with carboxylated polyether silane coupling agent and combined stabilizer.

[0073] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A synthetic bio-derived chemical signal amplification system, characterized in that, Includes carboxylated silica fluorescent microsphere dispersion, antibody, activator, combination stabilizer, blocking agent, and buffer solution; The carboxylated silica fluorescent microsphere dispersion is obtained by surface modification of silica fluorescent microspheres with a carboxylated polyether silane coupling agent; the chemical formula of the carboxylated polyether silane coupling agent is (RO)3Si-(CH2)3-O-(C2H4O). n -(C3H6O) m -COOH; where R is an alkyl group, n is an integer in the range of 5 to 20, and m is an integer in the range of 0 to 5; The antibody specifically covalently binds to carboxylated silica fluorescent microspheres.

2. The synthetic biology-derived chemical signal amplification system according to claim 1, characterized in that, In the chemical formula of the carboxylated polyether silane coupling agent, R is ethyl, n is an integer in the range of 8 to 15, and m is an integer in the range of 1 to 3.

3. The synthetic biology-derived chemical signal amplification system according to claim 1, characterized in that, The surface modification method for the silica fluorescent microspheres includes the following steps: S1. Prepare a silica fluorescent microsphere dispersion. After dilution with deionized water, the dispersion is subjected to ultrasonication, centrifugation, and resuspension in sequence to obtain the pretreated silica fluorescent microsphere dispersion. S2. Dissolve the carboxylated polyether silane coupling agent in anhydrous ethanol aqueous solution, adjust the pH to 4-5, and add the pretreated silica fluorescent microsphere dispersion under mixing and stirring. After the reaction is completed, the dispersion is obtained by centrifugation, washing and resuspension.

4. The synthetic biology-derived chemical signal amplification system according to claim 3, characterized in that, In step S2, after adjusting the pH, mix and stir for 5-10 minutes, then add the pretreated silica fluorescent microsphere dispersion while maintaining the stirring state.

5. The synthetic biology-derived chemical signal amplification system according to claim 3, characterized in that, In step S2, the mass ratio of carboxylated polyether silane coupling agent to silica fluorescent microspheres is 1:(12-16), the reaction time is 12-24 h, and the reaction temperature is 25-35 °C.

6. The synthetic biology-derived chemical signal amplification system according to claim 1, characterized in that, The activator is N-hydroxysuccinimide ester, which reacts with the carboxyl groups modified on the silica fluorescent microspheres to form an intermediate activated ester.

7. The synthetic biology-derived chemical signal amplification system according to claim 6, characterized in that, The activation reaction includes the following steps: A1. Prepare aqueous solutions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide ester, respectively; A2. Mix and stir the carboxylated silica fluorescent microsphere dispersion, the aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the aqueous solution of N-hydroxysuccinimide ester. After reacting for 20-30 min, the mixture is washed and resuspended sequentially to obtain the activated silica fluorescent microsphere dispersion. A3. Add 0.1-0.5 wt% of combined stabilizer to the activated silica fluorescent microsphere dispersion, mix and stir to obtain silica fluorescent microsphere composite solution; The combination stabilizers include calcium phosphate, calcium citrate, and gentamicin.

8. The synthetic biology-derived chemical signal amplification system according to claim 1, characterized in that, The blocking agent includes one of bovine serum albumin, ovalbumin, and casein, and the buffer includes at least one of acetate buffer, phosphate buffer, borate buffer, and MES buffer.

9. The synthetic bio-derived chemical signal amplification system according to any one of claims 1 to 8, characterized in that, The method for preparing the signal amplification system includes the following steps: The antibody was dissolved and diluted with buffer solution to obtain an antibody solution. The antibody solution was added to a silica fluorescent microsphere composite solution and stirred at room temperature for 1-2 hours. A blocking agent was added and stirring was continued for 0.5-1 hour. The solution was then centrifuged, washed, and resuspended to obtain a signal amplification system.

10. A highly sensitive antigen-antibody kit, characterized in that, Includes the synthetic bio-derived chemical signal amplification system as described in any one of claims 1 to 9.

Images