Preparation method of double-signal intelligent immunosensor and application of double-signal intelligent immunosensor in silk fibroin detection

Abstract

The invention relates to the field of immunosensors, and discloses a preparation method of a double-signal intelligent immunosensor and application of the double-signal intelligent immunosensor in silk fibroin detection. The preparation method comprises the following steps: (1) preparing the INV / HRP-ZIF-90 nanoparticles; (2) preparing an antibody functionalized magnetic bead probe solution; (3) preparing an Ab2-INV / HRP (at) ZIF-90 solution; and (4) preparing the double-signal intelligent immunosensor. According to the double-signal immunosensor, on the basis of an immunomagnetic bead ZIF-90 sandwich structure, ATP is used for triggering release of double enzymes (sucrose invertase and horse radish peroxidase), and a double-mode detection means of glucometer signal conversion and TMB color development-Image J analysis is combined, so that qualitative and semi-quantitative detection of a target antigen in silk can be realized, double signals can be mutually verified, and the detection sensitivity is high. And the detection accuracy is improved. The double-signal immunosensor not only has strong response specificity, but also has good portability, and can meet the requirements of on-site rapid detection and result verification of silk cultural relics.

Description

Technical Field

[0001] This invention relates to the field of immune sensors, and more particularly to a method for preparing a dual-signal intelligent immune sensor and its application in the detection of silk fibroin. Background Technology

[0002] Silk artifacts are frequently discovered in archaeological work, but the silk protein they contain is severely degraded due to long-term environmental erosion. Existing detection methods, such as microscopic observation and spectral analysis, not only rely on large, precision instruments and must be performed in a laboratory environment, but may also damage the artifacts, making real-time on-site analysis difficult. However, there is an urgent need for rapid identification of silk species at archaeological sites, which is crucial for the development of subsequent conservation plans. Existing dual-mode sensors typically require complex instruments (such as fluorescence-electrochemical coupling), failing to meet the need for portability on-site. Therefore, developing a portable, non-invasive detection technology that can be cross-validated using multiple methods, freeing it from dependence on large instruments, is key to meeting the needs of on-site detection.

[0003] Intelligent immunosensors based on personal blood glucose meters and smartphones integrate the high specificity of immunoassay technology and offer advantages such as convenient on-site operation, high level of intelligence, simple preparation process, fast detection speed, and low cost, showing broad application prospects in the field of trace residue detection. Therefore, developing a portable, non-invasive immunosensor capable of rapidly detecting silk proteins at archaeological sites will significantly improve the efficiency of silk artifact archaeology. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a dual-signal immunosensor based on dual-enzyme co-encapsulated ZIF-90 and its application in silk fibroin detection. This invention's dual-signal immunosensor utilizes an immunomagnetic bead-ZIF-90 sandwich structure, leveraging ATP to trigger the release of two enzymes (invertase and horseradish peroxidase). Combined with a dual-mode detection approach integrating glucometer signal conversion and TMB colorimetric-Image J analysis, it can achieve qualitative and semi-quantitative detection of target antigens in silk. Furthermore, the dual signals corroborate each other, improving detection accuracy. This dual-signal immunosensor not only exhibits strong response specificity but also boasts excellent portability, meeting the needs for rapid on-site detection and result verification of silk artifacts.

[0005] The specific technical solution of this invention is as follows:

[0006] First, this invention provides a method for preparing a dual-signal smart immunosensor based on dual-enzyme co-encapsulated ZIF-90, which includes the following steps:

[0007] (1) Preparation of INV / HRP@ZIF-90 nanoparticles: A tert-butanol / aqueous solution of zinc nitrate hexahydrate was mixed with a composite aqueous solution containing imidazole-2-carboxaldehyde, sucrose invertase, horseradish peroxidase, and polypyrrolidone, and then reacted. Zinc nitrate hexahydrate provided zinc ions, and imidazole-2-carboxaldehyde served as a ligand. The two combined to form a dodecahedral polyhedral ZIF-90 material. During formation, sucrose invertase and horseradish peroxidase were encapsulated within the material, resulting in synergistic protection of the two enzymes. After the reaction, the nanoparticles were centrifuged, washed, and dried to obtain the dual-enzyme-loaded INV / HRP@ZIF-90 nanoparticles.

[0008] (2) Preparation of antibody-functionalized magnetic bead probe solution: Take the carboxylated magnetic bead dispersion and add MES buffer (pH 5.8-6.2) containing N-hydroxysuccinimide (NHS) and 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC). The carboxyl groups on the surface of the magnetic beads react with NHS through EDC to generate highly active N-hydroxysuccinimide ester intermediates. After magnetic separation and washing to remove free reagents, the beads are resuspended in mouse anti-silk protein monoclonal antibody PBS solution. The amino groups of the antibody molecules undergo nucleophilic substitution reaction with the active ester to achieve covalent coupling between the antibody and the magnetic beads. After the reaction is completed, unbound antibody is removed by magnetic separation. Blocking agents such as BSA are added to block the residual active sites on the surface of the magnetic beads. After magnetic separation and washing again, the magnetic beads are resuspended in phosphate buffer (PBS) containing Tween-20 to obtain the functionalized magnetic bead probe solution with surface-directedly immobilized antibody.

[0009] (3) Preparation of Ab2-INV / HRP@ZIF-90 bifunctional probe solution: The INV / HRP@ZIF-90 nanoparticles obtained in step (1) were dispersed in a weakly alkaline borate buffer and sonicated to form a uniform suspension. Diluted Ab2 antibody solution was added dropwise to the system and reacted under isothermal shaking conditions to promote the Schiff base reaction between the aldehyde group on the ZIF-90 surface and the ε-amino group of the lysine residue of the antibody to form an imine bond intermediate. Subsequently, a mild reducing agent was added to carry out a reduction reaction to convert the imine bond into a stable alkylamine bond, thereby achieving covalent coupling between the antibody and ZIF-90. In this process, the weakly alkaline environment of the borate buffer can effectively activate the aldehyde group reactivity while maintaining the spatial conformational stability of the antibody; the mild reducing agent can selectively reduce the imine bond without affecting the disulfide bond structure of the antibody. After the reaction was completed, blocking buffer was added to block the unreacted aldehyde sites. The product was collected by centrifugation, washed with a buffer containing surfactant, and finally resuspended in PBS buffer to obtain the Ab2-directed modified dual-enzyme functionalized ZIF-90 probe solution, namely Ab2-INV / HRP@ZIF-90 bifunctional probe solution.

[0010] (4) Preparation of the immunosensor: The antibody-functionalized magnetic bead probe solution prepared in step (2) was added to a silanized centrifuge tube and incubated to allow the magnetic beads to be uniformly adsorbed onto the tube wall. After discarding the supernatant, the tube was washed with buffer solution. After each addition of buffer solution, the tube was shaken on a micro-shaker, and the washing solution was discarded after magnetic separation. Then, BSA solution was added and incubated to saturate the non-specific binding sites, and the tube was washed again. The silk fibroin antigen solution to be detected was added to the centrifuge tube and incubated at a constant temperature with shaking to allow the antigen to fully bind to the antibody on the surface of the magnetic beads. After incubation, the tube was magnetically separated and the supernatant was discarded. The tube was then washed to remove unbound antigen. Next, the Ab2-INV / HRP@ZIF-90 bifunctional probe solution prepared in step (3) was added and incubated to form an "antibody-functionalized magnetic bead-antigen-Ab2-INV / HRP@ZIF-90" sandwich complex. After the reaction was completed, the complex was separated by an external magnet and washed. During each wash, the centrifuge tube was placed on a rotary mixer and slowly rotated to ensure that unbound probe was completely removed. Finally, the complex was resuspended in PBS buffer to obtain a sandwich-structured dual-signal immunosensor.

[0011] This invention uses immunomagnetic beads coupled with capture antibodies as a solid-phase carrier and ZIF-90, co-encapsulated with invertase (INV) and horseradish peroxidase (HRP) and coupled with detection antibodies, as a bifunctional signal probe. When the target antigen is present, a sandwich immune complex of "immunomagnetic beads-antigen-dual-enzyme ZIF-90 probe" is formed through the specific binding of the antigen and antibody. The core mechanism is as follows: upon the addition of ATP, ATP competitively coordinates with zinc ions in ZIF-90, causing the ZIF-90 framework to disintegrate and simultaneously release the two encapsulated enzymes. Invertase catalyzes the conversion of sucrose to glucose, and the change in glucose concentration can be read using a portable blood glucose meter to achieve the first level of signal conversion. Horseradish peroxidase catalyzes the reaction of TMB with hydrogen peroxide to produce a color change, and the color intensity can be analyzed using ImageJ software to achieve the second level of signal verification.

[0012] The significant features and advantages of this invention are as follows: the magnetic core of the immunomagnetic beads can quickly separate the sandwich complex through an external magnetic field, greatly improving detection efficiency; the mesoporous structure of ZIF-90 can efficiently encapsulate the two enzymes and precisely trigger enzyme release through specific interaction with ATP, ensuring the specificity and stability of the signal; the dual-signal mode reduces detection errors through mutual verification, while retaining the advantage of portability, allowing detection to be completed without large instruments, providing a brand-new solution for rapid and reliable on-site detection of silk artifacts.

[0013] Further, step (1) specifically includes: first, adding imidazole-2-carboxaldehyde and polypyrrolidone to water and stirring until completely dissolved, then adding sucrose invertase and horseradish peroxidase and stirring to disperse; then adding tert-butanol / water solution containing zinc nitrate hexahydrate and stirring until the system is homogeneous; placing the resulting mixture in a water bath for reaction, cooling after the reaction, centrifuging to collect the solid product, and washing with anhydrous ethanol and water alternately to remove unreacted raw materials; finally, vacuum drying to obtain INV / HRP@ZIF-90 nanoparticles.

[0014] Furthermore, in step (1), the molar ratio of zinc nitrate hexahydrate to imidazole-2-formaldehyde is 1:(4-6); the volume ratio of tert-butanol to water is 1:(0.8-1.2); the molar ratio of polypyrrolidone to imidazole-2-formaldehyde is 1:(35-45); the molar ratio of polypyrrolidone to zinc nitrate hexahydrate is 1:(8-12); the mass ratio of sucrose invertase to imidazole-2-formaldehyde is 1:(15-20); and the mass ratio of horseradish peroxidase to imidazole-2-formaldehyde is 1:(12-22).

[0015] The core reason for controlling the proportions of the aforementioned substances within the specified range in this invention is that the crystal growth state and particle size of ZIF-90 are jointly determined by the ratio of the central coordinating ion and the ligand. If the ratio is too high, the ZIF-90 particles will be too large, hindering subsequent antibody conjugation and dual enzyme release; conversely, if the ratio is too low, the crystal structure will be loose, affecting the encapsulation stability of the dual enzymes. Simultaneously, ZIF-90 has a limited co-encapsulation capacity for sucrase and horseradish peroxidase, and the enzyme dosage must be matched with the carrier: excessive dosage will result in residual free enzymes, interfering with the detection signal; insufficient dosage will reduce the catalytic capacity per unit carrier, affecting the dual-mode signal intensity.

[0016] Furthermore, in step (1), the temperature of the reaction is controlled at 20-30℃ and the reaction time is controlled at 5-15 minutes.

[0017] Under the above conditions, the normal growth of ZIF-90 crystals can be ensured while avoiding the destruction of dual enzyme activity by high temperature, thus ensuring the stability of the enzyme's catalytic function.

[0018] Preferably, in step (2), the method for preparing the carboxylated magnetic bead dispersion is as follows: the carboxylated magnetic beads are repeatedly washed with PBS at pH=7-7.5 and then resuspended to remove impurities and balance the surface environment of the magnetic beads, so as to provide a stable basis for subsequent antibody conjugation.

[0019] Further, in step (2), the concentration of N-hydroxysuccinimide in the MES buffer is 3-7 mM, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 8-12 mM, and the pH of the MES buffer is 6.0-6.5.

[0020] Further, in step (2), the conditions for the oscillation activation are: the temperature is room temperature and the time is 20-40 minutes; the incubation temperature is 35-40℃ and the time is 1.5-2.5h.

[0021] The above-mentioned oscillation activation conditions are conducive to activating the carboxyl groups on the surface of magnetic beads; applying temperature can promote the full covalent binding of antibodies and magnetic beads, and enhance the specific binding ability of probes.

[0022] Further, in step (3), the Ab2 antibody is diluted with sodium bicarbonate solution with pH=7.8-8.2, and the concentration after dilution is 0.8-1.2 mg / mL.

[0023] A weakly alkaline environment can enhance the reactivity of the aldehyde groups on the ZIF-90 surface with the amino groups of the antibody.

[0024] Furthermore, in step (3), the reducing agent is a NaOH solution containing Na[BH3(CN)]; the reduction reaction takes 3-4 hours.

[0025] The reduction reaction lasts for 3-4 hours to ensure the formation of a stable covalent coupling while preserving the catalytic activity of both enzymes.

[0026] Further, in step (4), the volume ratio of the antibody-functionalized magnetic bead probe solution, BSA solution, silk fibroin antigen solution to be detected, and Ab2-INV@ZIF-90 solution is 80-100:180-220:100:80-100.

[0027] The above ratio can balance the binding efficiency of each component and ensure the stable formation of the sandwich structure.

[0028] Furthermore, in step (4), the concentration range of the antibody-functionalized magnetic bead probe solution is 3-4 mg / mL; the concentration of the BSA solution is 1-3 wt%, which can effectively block non-specific binding sites; the concentration range of the silk fibroin antigen solution to be detected is 10 ng / mL-100 μg / mL, which is suitable for the detection of samples with different contents; the concentration of the Ab2-INV / HRP@ZIF-90 solution is 8-10 μg / mL, which ensures the effective output of dual-mode signals.

[0029] Furthermore, in step (4), the incubation time is 0.5-3 hours.

[0030] Secondly, this invention provides the application of the dual-signal immunosensor prepared by the above method in the detection of silk fibroin or on-site detection of silk artifacts.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] (1) The dual-signal immunosensor of the present invention relies on the magnetic separation capability of immunomagnetic beads and the characteristic of ZIF-90 releasing dual enzymes triggered by ATP. Combined with the dual detection mode of portable blood glucose meter and TMB colorimetric-Image J analysis, it can be used to detect target antigens in silk fibroin or silk artifacts. It has the characteristics of fast response speed, low detection limit, wide linear range and good stability. In particular, it can realize simple, rapid, highly sensitive and specific detection of on-site samples and mutual verification of results, which can provide a more reliable POCT analysis path for the detection of silk artifacts.

[0033] (2) This invention utilizes the synergistic effect of the immunomagnetic beads and the ZIF-90 co-encapsulated dual enzyme composite system to integrate the rapid separation advantage of magnetic materials, the specific response characteristics of ZIF-90 materials, and the cascade catalytic amplification function of dual enzymes, thereby achieving efficient enhancement of detection signals. At the same time, the signal deviation problem of traditional single-mode sensors is avoided through dual-signal complementary verification, significantly improving detection efficiency and result reliability.

[0034] (3) The dual-function detection probe obtained by the present invention does not require the addition of conductive agent or activating reagent. Immunomagnetic beads and ZIF-90 surface can be directly coupled with antibody through covalent bond. Compared with traditional methods, the loaded material is more uniformly dispersed, the synergistic effect of dual enzymes and antibody is more stable, the operation steps are simpler, and the signal transmission efficiency is higher, making it suitable for on-site rapid preparation and instant detection scenarios.

[0035] (4) The materials used in this invention have excellent biocompatibility, the preparation process is green and environmentally friendly, the self-assembly process of dual enzyme co-embedding and antibody conjugation can be tracked in real time, the modification sites are clear, and it can adapt to the on-site detection needs of silk samples (such as fragments and fibers) in different preservation states, and has wider applicability and universality.

[0036] (5) The dual-response immunosensor of this invention further reduces the limit of detection for target antigens in silk samples compared to the original single-mode method, improves sensitivity by more than 15 times compared to colloidal gold immunochromatography, and shortens the detection time by 60% compared to traditional ELISA. Under ATP-triggered conditions, the synergistic catalytic efficiency of the two enzymes released by ZIF-90 reaches 90% of that of the free enzyme. Both the blood glucose meter detection results and the ImageJ grayscale analysis results show a good linear relationship with the antigen concentration, and the correlation between the two signals is over 95%. Compared with time-resolved fluorescence immunoassay and electrochemiluminescence immunoassay, this sensor maintains portability (requiring only a blood glucose meter and a regular camera) while improving detection accuracy through dual-signal verification. The detection performance retention rate exceeds 85% after 30 days of storage at room temperature. Under optimal conditions, the cross-reactivity rate to interfering proteins is <2%, and the relative standard deviation of repeated detections is <4%, demonstrating superior stability, selectivity, and repeatability. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail, but this detailed description should not be construed as limiting the present invention.

[0038] This is not a description, but rather a more detailed description of certain aspects, features, and embodiments of the invention.

[0039] Example 1

[0040] (1) Preparation of INV / HRP@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde and polypyrrolidone were first added to water and stirred until completely dissolved. Then, sucrose invertase and horseradish peroxidase (mass ratio 1:1, 30 mg each) were added and mixed evenly. Subsequently, a tert-butanol solution containing zinc nitrate hexahydrate was added and stirred to dissolve. The volume ratio of tert-butanol to deionized water was 1:1, 12.5 mL each; the amount of imidazole-2-carboxaldehyde was 480 mg, zinc nitrate hexahydrate was 371 mg, and polypyrrolidone was 500 mg. The mixture was heated and stirred at 26 °C for 5 min. After naturally cooling to room temperature, it was filtered, washed, and vacuum dried to obtain INV / HRP@ZIF-90 nanoparticles co-encapsulated with the two enzymes.

[0041] (2) Preparation of antibody-functionalized magnetic bead probe solution: Take 100µL of a 50mg / mL carboxylated magnetic bead solution, transfer it to a 1.5mL microcentrifuge tube, and wash three times with 200µL of pH 7.4 PBS buffer; after washing, resuspend the magnetic beads in 400µL of pH 7.4 PBS. Take another 20mL of pH 6.0 MES buffer, add 0.5209g NaCl, 24mg NHS and 16mg EDC, dissolve and prepare an activation buffer. Add 100μL of the above activation buffer to the magnetic bead suspension, shake at room temperature for 30 minutes to activate the carboxyl groups on the surface of the magnetic beads with NHS. Magnetic separation and discard the supernatant, wash three times with MES buffer, and resuspend the magnetic beads in 500μL of PBS solution containing 10μg / mL mouse anti-silk monoclonal antibody, incubate overnight at 4℃ to allow the antibody to covalently couple with the activated carboxyl groups through the amino groups. The following day, the supernatant was magnetically removed, and 1% BSA blocking buffer was added. The mixture was then shaken at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was then stored at 4°C for later use.

[0042] (3) Preparation of Ab2-INV / HRP@ZIF-90 solution: The antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.0. 100 µL of the antibody solution was mixed with 10 µL of 1 mg / mL INV / HRP@ZIF-90 nanoparticles, and 1.5 mL of borate buffer at pH 8.0 was added. The mixture was ultrasonically dispersed for 5 min. Then, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was reacted at room temperature for 4 h to allow the aldehyde groups on the ZIF-90 surface to form stable covalent bonds with the amino groups of the antibody. After the reaction, the unbound sites were blocked with BSA, and after centrifugation and washing, the mixture was resuspended in PBS and stored at 4 °C.

[0043] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL to the centrifuge tube, wash, and then add 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution and incubate for 1.2 h. After washing with a magnet, add 50 μL of 1.5 mg / mL ATP to the solution and incubate for 30 min to trigger the signal, thus obtaining a dual-signal immunosensor.

[0044] Example 2

[0045] (1) Preparation of INV / HRP@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde and polypyrrolidone were first added to water and stirred to dissolve. Then, 30 mg of sucrose invertase and 45 mg of horseradish peroxidase were added and mixed evenly. Then, tert-butanol containing zinc nitrate hexahydrate was added and stirred to dissolve. The ratio of tert-butanol to deionized water was 1:1, and both were 14 mL. The amount of imidazole-2-carboxaldehyde was 480 mg, the amount of zinc nitrate hexahydrate was 371 mg, and the amount of polypyrrolidone was 500 mg. The mixture was heated and stirred at 28 °C for 7 min in a beaker, and then naturally cooled to room temperature. After filtration through a 0.45 μm filter membrane, washing three times with ultrapure water, and vacuum drying, INV / HRP@ZIF-90 nanoparticle material was obtained.

[0046] (2) Preparation of antibody-functionalized magnetic bead probe solution: 150 µL of a 50 mg / mL carboxylated magnetic bead (MB) solution was transferred into a 1.5 mL microcentrifuge tube, and then the magnetic beads were washed three times with 250 µL of pH 7.4 phosphate buffer (PBS) containing 0.02% Tween-20. After washing, the magnetic beads were redispersed and resuspended in 400 µL of pH 7.4 PBS. Next, 0.6209 g NaCl, 24 mg N-hydroxysuccinimide (NHS), and 16 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were dissolved in 25 mL of pH 6.0 morpholinoethanesulfonic acid monohydrate (MES) buffer, and the mixture was magnetically stirred for 15 min until completely dissolved to prepare the activation buffer. 100 μL of the above activation buffer was added to the magnetic bead suspension, and the mixture was shaken at room temperature for 30 min to activate the carboxyl groups on the surface of the magnetic beads with NHS. After magnetic separation and discarding the supernatant, the beads were washed three times with MES buffer and resuspended in 500 μL of PBS solution containing 10 μg / mL mouse anti-silk protein monoclonal antibody. The mixture was incubated overnight at 4°C to allow the antibody to covalently couple with the activated carboxyl group via the amino group. The next day, the supernatant was discarded by magnetic separation, and 1% BSA blocking buffer was added with shaking at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was stored at 4°C for later use.

[0047] (3) Preparation of Ab2-INV / HRP@ZIF-90 solution: First, the antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.1. Then, 100 µL of the above antibody solution and 12 µL of 1 mg / mL INV / HRP@ZIF-90 were ultrasonically dispersed in 1.5 mL borate buffer (pH 8.1) for 8 min. Then, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was placed in a shaker at 220 rpm at room temperature for 4 hours to reduce the aldehyde group on the ZIF-90 surface to a stable alkylamine covalent bond via an imine bond intermediate. After the reaction, 2 wt% BSA solution was added to block the unbound sites. The mixture was allowed to stand at room temperature for 40 min, washed three times by centrifugation at 9000 rpm for 8 min, and finally resuspended in PBS and stored at 4 °C.

[0048] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL to the centrifuge tube, wash, and then add 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution and incubate for 1.2 h. After washing with a magnet, add 50 μL of 1.5 mg / mL ATP to the solution and incubate for 30 min to trigger the signal, thus obtaining a dual-signal immunosensor.

[0049] Example 3

[0050] (1) Preparation of INV / HRP@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde and polypyrrolidone were first added to water and stirred to dissolve. Then, 30 mg of sucrose invertase and 45 mg of horseradish peroxidase (mass ratio of 1:1.5) were added and mixed evenly. Then, tert-butanol containing zinc nitrate hexahydrate was added and stirred to dissolve. The ratio of tert-butanol to deionized water was 1:1, and both were 14 mL. The amount of imidazole-2-carboxaldehyde was 480 mg, the amount of zinc nitrate hexahydrate was 371 mg, and the amount of polypyrrolidone was 500 mg. The mixture was heated and stirred at 28 °C for 7 min in a beaker, then naturally cooled to room temperature, washed three times with ultrapure water, and vacuum dried to obtain INV / HRP@ZIF-90 nanoparticle material.

[0051] (2) Preparation of antibody-functionalized magnetic bead probe solution: 250 µL of a 50 mg / mL carboxylated magnetic bead (MB) solution was transferred into a 1.5 mL microcentrifuge tube, and then the magnetic beads were washed three times with 250 µL of pH 7.4 phosphate buffer (PBS) containing 0.02% Tween-20. After washing, the magnetic beads were redispersed and resuspended in 400 µL of pH 7.4 PBS. Next, 0.7358 g NaCl, 24 mg N-hydroxysuccinimide (NHS), and 23 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were dissolved in 25 mL of pH 6.0 morpholinoethanesulfonic acid monohydrate (MES) buffer, and the mixture was magnetically stirred for 15 min until completely dissolved to prepare the activation buffer. 100 μL of the above activation buffer was added to the magnetic bead suspension, and the mixture was shaken at room temperature for 30 min to activate the carboxyl groups on the surface of the magnetic beads with NHS. After magnetic separation and discarding the supernatant, the beads were washed three times with MES buffer and resuspended in 500 μL of PBS solution containing 10 μg / mL mouse anti-silk monoclonal antibody. The mixture was incubated overnight at 4°C to allow the antibody to covalently couple with the activated carboxyl group via the amino group. The next day, the supernatant was discarded by magnetic separation, and 1% BSA blocking buffer was added with shaking at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was stored at 4°C for later use.

[0052] (3) Preparation of Ab2-INV / HRP@ZIF-90 solution: First, the antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.1. Then, 100 µL of the above antibody solution and 12 µL of 1 mg / mL INV / HRP@ZIF-90 were ultrasonically dispersed in 1.5 mL borate buffer (pH 8.1) for 8 min. Then, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was placed in a shaker at 220 rpm at room temperature for 4 hours to reduce the aldehyde group on the ZIF-90 surface to a stable alkylamine covalent bond via an imine bond intermediate. After the reaction, 2 wt% BSA solution was added to block the unbound sites. The mixture was allowed to stand at room temperature for 40 min, washed three times by centrifugation at 9000 rpm for 8 min, and finally resuspended in PBS and stored at 4 °C.

[0053] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL to the centrifuge tube, wash, and then add 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution and incubate for 1.2 h. After washing with a magnet, add 50 μL of 1.5 mg / mL ATP to the solution and incubate for 30 min to trigger the signal, thus obtaining a dual-signal immunosensor.

[0054] Comparative Example 1:

[0055] The difference from Example 1 is that ATP was not added to trigger the signal, and the specific steps include:

[0056] (1) Preparation of INV / HRP@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde and polypyrrolidone were first added to water and stirred until completely dissolved. Then, sucrose invertase and horseradish peroxidase (mass ratio 1:1, 30 mg each) were added and mixed evenly. Subsequently, an aqueous solution containing zinc nitrate hexahydrate was added and stirred to dissolve. The volume ratio of tert-butanol to deionized water was 1:1, 12.5 mL each; the amount of imidazole-2-carboxaldehyde was 480 mg, zinc nitrate hexahydrate was 371 mg, and polypyrrolidone was 500 mg. The mixture was heated and stirred at 26 °C for 5 min. After naturally cooling to room temperature, it was filtered, washed, and vacuum dried to obtain INV / HRP@ZIF-90 nanoparticles with dual enzyme co-encapsulation.

[0057] (2) Preparation of antibody-functionalized magnetic bead probe solution: Take 100µL of a 50mg / mL carboxylated magnetic bead solution and transfer it to a 1.5mL microcentrifuge tube. Wash three times with 200µL of pH 7.4 PBS buffer. After washing, resuspend the magnetic beads in 400µL of pH 7.4 PBS. Take another 20mL of pH 6.0 MES buffer, add 0.5209g NaCl, 24mg NHS and 16mg EDC, dissolve and prepare an activation buffer. Add 100μL of the above activation buffer to the magnetic bead suspension and shake at room temperature for 30 minutes to activate the carboxyl groups on the surface of the magnetic beads with NHS. Magnetic separation and discard the supernatant. Wash three times with MES buffer and resuspend the magnetic beads in 500μL of PBS solution containing 10μg / mL mouse anti-silk protein monoclonal antibody. Incubate overnight at 4℃ to allow the antibody to covalently couple with the activated carboxyl groups via amino groups. The following day, the supernatant was magnetically removed, and 1% BSA blocking buffer was added. The mixture was then shaken at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was then stored at 4°C for later use.

[0058] (3) Preparation of Ab2-INV / HRP@ZIF-90 solution: The antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.0. 100 µL of the antibody solution was mixed with 10 µL of 1 mg / mL INV / HRP@ZIF-90 nanoparticles, and 1.5 mL of borate buffer at pH 8.0 was added. The mixture was then sonicated for 5 min. Subsequently, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was reacted at room temperature for 4 h to allow the aldehyde groups on the ZIF-90 surface to form stable covalent bonds with the amino groups of the antibody. After the reaction, the unbound sites were blocked with BSA, and after centrifugation and washing, the mixture was resuspended in PBS and stored at 4 °C.

[0059] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL respectively to the centrifuge tube, wash, and then add 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution and incubate for 1.2 h, and then separate with a magnet to obtain a dual-signal immunosensor.

[0060] Comparative Example 2

[0061] The difference from Example 1 is that the two enzymes are encapsulated separately instead of being mixed, specifically including the following steps:

[0062] (1) Preparation of stepwise encapsulated INV / HRP@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde (480 mg) and polypyrrolidone (500 mg) were added to 12.5 mL of water and stirred to dissolve. Then, sucrose invertase (30 mg) was added and stirred at room temperature for 30 min to disperse it evenly. Then, 12.5 mL of tert-butanol solution containing zinc nitrate hexahydrate (371 mg) was added to the above mixture and stirred at 26 °C for 2 h to form INV@ZIF-90 intermediate. Subsequently, horseradish peroxidase (45 mg, mass ratio of 1.5:1 to sucrose invertase) was added to the system, and 100 mg of imidazole-2-carboxaldehyde and 60 mg of zinc nitrate hexahydrate were added. The mixture was stirred for 5 min to allow HRP to grow and encapsulate further on the outer layer of the already formed ZIF-90. After the reaction was completed, the nanoparticles were naturally cooled to room temperature, washed five times alternately with ultrapure water and anhydrous ethanol, and then dried under vacuum to obtain stepwise embedded INV / HRP@ZIF-90 nanoparticles.

[0063] (2) Preparation of antibody-functionalized magnetic bead probe solution: Take 100µL of a 50mg / mL carboxylated magnetic bead solution and transfer it to a 1.5mL microcentrifuge tube. Wash three times with 200µL of pH 7.4 PBS buffer. After washing, resuspend the magnetic beads in 400µL of pH 7.4 PBS. Take another 20mL of pH 6.0 MES buffer, add 0.5209g NaCl, 24mg NHS and 16mg EDC, dissolve and prepare an activation buffer. Add 100μL of the above activation buffer to the magnetic bead suspension and shake at room temperature for 30 minutes to activate the carboxyl groups on the surface of the magnetic beads with NHS. Magnetic separation and discard the supernatant. Wash three times with MES buffer and resuspend the magnetic beads in 500μL of PBS solution containing 10μg / mL mouse anti-silk protein monoclonal antibody. Incubate overnight at 4℃ to allow the antibody to covalently couple with the activated carboxyl groups via amino groups. The following day, the supernatant was magnetically removed, and 1% BSA blocking buffer was added. The mixture was then shaken at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was then stored at 4°C for later use.

[0064] (3) Preparation of Ab2-INV / HRP@ZIF-90 solution: The antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.0. 100 µL of the antibody solution was mixed with 10 µL of 1 mg / mL INV / HRP@ZIF-90 nanoparticles, and 1.5 mL of borate buffer at pH 8.0 was added. The mixture was ultrasonically dispersed for 5 min. Then, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was reacted at room temperature for 4 h to allow the aldehyde groups on the ZIF-90 surface to form stable covalent bonds with the amino groups of the antibody. After the reaction, the unbound sites were blocked with BSA, and after centrifugation and washing, the mixture was resuspended in PBS and stored at 4 °C.

[0065] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL to the centrifuge tube, wash, and then add 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution and incubate for 1.2 h. After washing with a magnet, add 50 μL of 1.5 mg / mL ATP to the solution and incubate for 30 min to trigger the signal, thus obtaining a dual-signal immunosensor.

[0066] Comparative Example 3

[0067] The difference from Example 1 is that a single-mode enzyme catalysis is used and the silk fibroin solution used contains interfering antibodies (bovine serum albumin). Specifically, the following steps are included:

[0068] (1) Preparation of INV@ZIF-90 nanoparticles: Imidazole-2-carboxaldehyde and polypyrrolidone were first added to water and stirred until completely dissolved. Then, 30 mg of sucrose invertase was added and mixed evenly. Subsequently, a tert-butanol solution containing zinc nitrate hexahydrate was added and stirred to dissolve. The volume ratio of tert-butanol to deionized water was 1:1, with 12.5 mL of each. The amount of imidazole-2-carboxaldehyde was 480 mg, zinc nitrate hexahydrate was 371 mg, and polypyrrolidone was 500 mg. The mixture was heated and stirred at 26 °C for 5 min. After naturally cooling to room temperature, it was filtered, washed, and vacuum dried to obtain INV@ZIF-90 nanoparticles.

[0069] (2) Preparation of antibody-functionalized magnetic bead probe solution: Take 100µL of a 50mg / mL carboxylated magnetic bead solution and transfer it to a 1.5mL microcentrifuge tube. Wash three times with 200µL of pH 7.4 PBS buffer. After washing, resuspend the magnetic beads in 400µL of pH 7.4 PBS. Take another 20mL of pH 6.0 MES buffer, add 0.5209g NaCl, 24mg NHS and 16mg EDC, dissolve and prepare an activation buffer. Add 100μL of the above activation buffer to the magnetic bead suspension and shake at room temperature for 30 minutes to activate the carboxyl groups on the surface of the magnetic beads with NHS. Magnetic separation and discard the supernatant. Wash three times with MES buffer and resuspend the magnetic beads in 500μL of PBS solution containing 10μg / mL mouse anti-silk protein monoclonal antibody. Incubate overnight at 4℃ to allow the antibody to covalently couple with the activated carboxyl groups via amino groups. The following day, the supernatant was magnetically removed, and 1% BSA blocking buffer was added. The mixture was then shaken at room temperature for 1 hour to block unreacted active sites. Finally, the beads were washed three times with PBS and redispersed in 1 mL of PBS buffer containing 0.05% Tween-20. The mixture was then stored at 4°C for later use.

[0070] (3) Preparation of Ab2-INV@ZIF-90 solution: The antibody was diluted to 1 mg / mL with sodium bicarbonate solution at pH 8.0. 100 µL of the antibody solution was mixed with 10 µL of 1 mg / mL INV@ZIF-90 nanoparticles, and 1.5 mL of borate buffer at pH 8.0 was added. The mixture was ultrasonically dispersed for 5 min. Then, 50 µL of 5 M Na[BH3(CN)] solution (dissolved in 1 M NaOH) was added, and the mixture was reacted at room temperature for 4 h to allow the aldehyde groups on the ZIF-90 surface to form stable covalent bonds with the amino groups of the antibody. After the reaction, the unbound sites were blocked with BSA, and after centrifugation and washing, the mixture was resuspended in PBS and stored at 4 °C.

[0071] (4) Preparation of immunosensor: Take 100 μL of antibody-functionalized magnetic bead probe solution with a concentration of 5 mg / mL, drop it into a 5 mL centrifuge tube, incubate for 0.5 h, and wash the centrifuge tube 3 times with PBS at pH 7.4; drop 200 μL of 1 wt% BSA solution into a microplate, wash and set aside; add 100 μL of silk fibroin antigen solution with concentrations of 1 mg / mL and 1 μg / mL to the centrifuge tube, wash, then add 80 μL of 1 mg / mL Ab2-INV@ZIF-90 solution and incubate for 1.2 h, add 50 μL of 1.5 mg / mL ATP to the solution after washing with a magnet and incubate for 30 min to trigger the signal and obtain the immunosensor.

[0072] Test Analysis

[0073] The immunosensors of each embodiment and comparative example were tested using the following methods: 100 μL of antibody-functionalized magnetic bead probe solution (5 mg / mL) was added to a 5 mL centrifuge tube and incubated for 0.5 h. The centrifuge tube was then washed three times with PBS buffer (pH 7.4). 1 wt% (200 μL) of BSA solution was added to a microplate and washed with PBS buffer. The target silk fibroin antigen was added to a centrifuge tube and washed with PBS buffer. Then, 80 μL of 1 mg / mL Ab2-INV / HRP@ZIF-90 solution was added to a centrifuge tube and incubated at room temperature for 1 h to form an antibody-functionalized magnetic bead probe-silk fibroin-Ab2-INV / HRP@ZIF-90 complex. The centrifuge tube was then washed three times with PBS buffer (pH 7.4) to remove unbound components. Transfer 450 μL of the complex from the centrifuge tube into an EP tube. Depending on the specific case, add 50 μL of 1.5 mg / mL ATP solution. Incubate at 37°C for 30 min to trigger ZIF-90 deconstruction. Then centrifuge the solution in the EP tube at 12000 rpm for 5 min. Mix 100 μL of the supernatant with 100 μL of 5 mol / L sucrose-PBS solution. Incubate at 37°C for 30 min. Use a 10 μL sample of the reaction solution to measure glucose concentration using a glucometer. Simultaneously, mix 50 μL of the supernatant with 50 μL of TMB chromogenic solution. React at 37°C for 15 min. Analyze the color intensity using ImageJ. The steps and principle for analyzing the color intensity are as follows: First, convert the RGB values ​​to grayscale values ​​using the following formula:

[0074] Grayscale value = 0.299×R + 0.587×G + 0.114×B (weighted average method, simulating the human eye's sensitivity to different colors). The darker the color (such as blue or yellow after TMB color rendering), the lower the pixel brightness and the smaller the grayscale value; conversely, the lighter the color, the larger the grayscale value.

[0075] Table 1

[0076] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Blood glucose meter reading mmol / L 14.3 13.4 12.1 2.2 7.5 13.6 TMB grayscale value (450nm) 57 59 65 183 164 0

[0077] A comparison of the data in Table 1 shows that:

[0078] Compared with Example 1, since ATP was not used for signal triggering, the results showed that the catalytic efficiency per unit weight of probe was reduced, indicating that ATP-triggered signals are beneficial to improving sensor sensitivity.

[0079] Compared with Example 1, Comparative Example 2 showed a significant decrease in probe catalytic efficiency per unit weight due to the absence of a one-step encapsulation method using mixed enzymes. This indicates that one-step encapsulation with mixed enzymes is beneficial for improving sensor sensitivity. Stepwise encapsulation is less effective than one-step encapsulation, primarily because the ZIF-90 framework may develop structural defects or heterogeneous interfaces due to multiple synthesis processes, leading to decreased integrity. Furthermore, the enzymes require repeated exposure to the synthesis environment and centrifugation / washing, making them susceptible to stability degradation. Encapsulating multiple enzymes can also increase substrate diffusion resistance due to uneven spatial distribution, and enzymes non-specifically adsorbed on the surface are easily eluted, reducing encapsulation efficiency and ultimately affecting overall performance.

[0080] Comparing Comparative Example 3 and Example 1, the results showed that false positives occurred because the dual-mode strategy was not adopted, indicating that the dual-signal strategy is beneficial to improving sensor accuracy.

[0081] The above embodiments of the present invention are merely illustrative examples and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a dual-signal intelligent immune sensor, characterized in that... include: (1) The tert-butanol / aqueous solution of zinc nitrate hexahydrate was mixed with a composite aqueous solution containing imidazole-2-carboxaldehyde, sucrose invertase, horseradish peroxidase and polypyrrolidone, reacted, centrifuged, washed and dried to obtain dual-enzyme-loaded INV / HRP@ZIF-90 nanoparticles. (2) Add MES buffer containing N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the carboxylated magnetic bead dispersion, shake to activate, magnetically separate, wash, resuspend the magnetic beads in mouse anti-silk protein monoclonal antibody PBS solution, incubate, magnetically block unreacted active sites, wash, disperse the magnetic beads in Tween-20 PBS solution to obtain antibody-functionalized magnetic bead probe solution; (3) INV / HRP@ZIF-90 nanoparticles were dispersed in borate buffer to form a suspension; Ab2 antibody was diluted and added to the suspension; a reducing agent was added, and through the reduction reaction of the imine bond intermediate, the aldehyde group on the surface of ZIF-90 was covalently linked to the amino group of the lysine residue of the antibody to form a stable alkylamine; and the unreacted active site was blocked. Separate the precipitate, wash it, and resuspend it in PBS solution containing preservative to obtain Ab2-INV / HRP@ZIF-90 solution; (4) The antibody-functionalized magnetic bead probe solution, BSA solution, silk fibroin antigen solution to be detected, and Ab2-INV / HRP@ZIF-90 solution are combined with a magnet to obtain a dual-signal smart immunosensor.

2. The preparation method according to claim 1, characterized in that: Step (1) specifically includes: first, adding imidazole-2-carboxaldehyde and polypyrrolidone to water and stirring until completely dissolved, then adding sucrose invertase and horseradish peroxidase and stirring to disperse; then adding tert-butanol / water solution containing zinc nitrate hexahydrate and stirring until the system is homogeneous; placing the resulting mixture in a water bath for reaction, cooling after the reaction, centrifuging to collect the solid product, and washing with anhydrous ethanol and water alternately to remove unreacted raw materials; finally, vacuum drying to obtain INV / HRP@ZIF-90 nanoparticles.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), The molar ratio of zinc nitrate hexahydrate to imidazole-2-carbaldehyde is 1:(4-6); The volume ratio of tert-butanol to water is 1:(0.8-1.2); The molar ratio of the polypyrrolidone to imidazole-2-carboxaldehyde is 1:(35-45); The molar ratio of the polypyrrolidone to zinc nitrate hexahydrate is 1:(8-12); The mass ratio of the sucrose invertase to imidazole-2-formaldehyde is 1:(15-20); The mass ratio of horseradish peroxidase to imidazole-2-formaldehyde is 1:(12-22).

4. The preparation method according to claim 1 or 2, characterized in that: In step (1), the temperature of the reaction is controlled at 20-30℃ and the reaction time is controlled at 5-15 minutes.

5. The preparation method according to claim 1, characterized in that: In step (2), The concentration of N-hydroxysuccinimide in the MES buffer is 3-7 mM, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 8-12 mM. The pH of the MES buffer solution is 6.0-6.

5.

6. The preparation method according to claim 1, characterized in that: In step (2), The conditions for the oscillation activation are: room temperature and 20-40 minutes. The incubation temperature is 35-40℃, and the time is 1.5-2.5h.

7. The preparation method according to claim 1, characterized in that: In step (3), The concentration of the Ab2 antibody after dilution is 0.8-1.2 mg / mL; The reducing agent is a NaOH solution containing Na[BH3(CN)]; The reduction reaction takes 3-4 hours.

8. The preparation method according to claim 1, characterized in that: In step (4), The concentration of the antibody-functionalized magnetic bead probe solution is 3-4 mg / mL; The concentration of the BSA solution is 1-3 wt%; The concentration of the silk fibroin antigen solution to be tested is 10 ng / mL-100 μg / mL; The concentration of the Ab2-INV@ZIF-90 solution is 8-10 g / mL; The volume ratio of the antibody-functionalized magnetic bead probe solution, BSA solution, silk fibroin antigen solution to be detected, and Ab2-INV@ZIF-90 solution is 80-100:180-220:100:80-100.

9. The application of the dual-signal intelligent immunosensor prepared by the preparation method according to any one of claims 1-8 in the detection of silk fibroin.

10. The application of the dual-signal intelligent immunosensor prepared by the preparation method according to any one of claims 1-8 in the on-site detection of silk cultural relics.