Programmable perforated nanoscale enzyme probe, preparation method and application thereof

Abstract

This invention belongs to the field of nanobiosensing technology, specifically relating to a programmable perforated nanozyme probe, its preparation method, and its application. The programmable perforated nanozyme probe comprises a metal-organic framework (MOF) material, within which heme chloride is loaded. The MOF material is modified with polydopamine, and platinum nanoparticles are loaded on its surface. The preparation method includes: adding heme chloride and cobalt nitrate hexahydrate to a sodium dodecyl sulfate solution for reaction, followed by the addition of 2-methylimidazole solution for further reaction; centrifuging to collect the product after the reaction to obtain Hemin@ZIF; preparing solutions of Hemin@ZIF and dopamine separately; adding ethanolamine solution to the Hemin@ZIF solution, followed by the addition of dopamine solution; stirring and reacting; centrifuging to collect the product to obtain Hemin@ZIF@PDA; mixing Hemin@ZIF@PDA with a platinum nanoparticle dispersion; stirring and reacting; and centrifuging to collect the product to obtain Hemin@ZIF@PDA@Pt. This invention has significant advantages such as extremely high sensitivity, strong platform versatility, rapid detection adaptation, result visualization, and low cost and stability.

Description

Technical Field

[0001] This invention belongs to the field of nanobiosensing technology, specifically relating to a programmable perforated nanoenzyme probe, its preparation method, and its application. Background Technology

[0002] Tuberculosis (TB) and AIDS (Acute Respiratory Syndrome) are major infectious diseases that pose a serious threat to human health and public health security worldwide. Mycobacterium tuberculosis (MTB) nucleic acid provides direct molecular evidence of pathogen infection and is a low-copy target; its accurate detection is crucial for TB diagnosis. HIV-P24 antigen, a core structural protein of the HIV virus, appears in the body early in infection and is a low-abundance protein target, making it a core biomarker for early HIV screening. Reports indicate that in 2023, there were 10.8 million new TB cases globally, with my country ranking third in the world in terms of incidence. Globally, there are approximately 39.9 million people living with HIV, and my country has reported over 1.22 million living HIV-infected individuals. Both diseases face the challenge of atypical early symptoms and limited screening methods at the grassroots level, urgently requiring highly sensitive, rapid, and convenient point-of-care testing (POCT) technologies to support early diagnosis and prevention.

[0003] However, the detection of both types of targets faces significant technical bottlenecks: for Mycobacterium tuberculosis nucleic acid detection, traditional culture methods, as the "gold standard," require several weeks, making it difficult to meet the needs of rapid diagnosis; while molecular biology methods such as PCR shorten the detection cycle, they rely on expensive instruments and specialized technicians, making them difficult to promote in grassroots settings. For HIV-P24 antigen detection, existing ELISA kits have a sensitivity of only 30%-90%, insufficient detection rate for low-abundance antigens in the early stages of infection, and are cumbersome and time-consuming, making them unsuitable for large-scale grassroots screening. Existing detection methods are generally limited by probe performance. Conventional probes such as traditional gold nanoparticles (AuNPs) have inherent defects such as low molar extinction coefficients, weak colorimetric signals, and insufficient catalytic activity, resulting in limited signal amplification capabilities and detection sensitivity that is 1-3 orders of magnitude lower than laboratory techniques. This makes it difficult to achieve ultrasensitive and visual detection of low-copy nucleic acids and low-abundance proteins. Developing novel high-sensitivity signal probes is the core key to overcoming this technical bottleneck.

[0004] Current mainstream detection technologies still have many shortcomings: First, although laboratory detection technologies such as enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qPCR) have high sensitivity, they rely on large-scale instruments, professional laboratory environments, and skilled technicians, resulting in high detection costs and long cycles, which cannot meet the real-time and universal requirements of POCT scenarios. Second, lateral flow immunochromatography (LFIA), as a mainstream technology in the POCT field, has the advantages of simple operation, low cost, and visualized results, but its detection sensitivity is limited by the performance defects of traditional probes, making it difficult to match the detection requirements of low-abundance targets. Third, existing metal-organic framework (MOF) modification technologies, as potential probe carrier solutions, still face multiple technical challenges such as cumbersome processes, poor structural stability, low mass transfer efficiency, and limited application scenarios, failing to achieve efficient and low-cost preparation of high-performance probe carriers. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a programmable perforated nanozyme probe, its preparation method, and its application. By introducing programmable perforated nanomaterials with enzyme-like catalytic activity into a side-flow chromatography system, ultrasensitive quantitative or qualitative detection of trace disease biomarkers can be achieved. This reduces operational difficulty and preparation costs, provides high detection sensitivity, strong platform versatility, rapid detection adaptation, and result visualization.

[0006] The technical problem solved by this invention is achieved by the following technical solution: The present invention aims to provide a programmable perforated nanozyme probe, comprising a metal-organic framework material, wherein the metal-organic framework material is loaded with heme chloride, and the surface of the metal-organic framework material is loaded with platinum nanoparticles after being modified with polydopamine.

[0007] A method for preparing a programmable perforated nanozyme probe includes the following steps: S1 metal-organic framework material loaded with heme chloride: Heme chloride and cobalt nitrate hexahydrate were added to sodium dodecyl sulfate solution and reacted. Then, 2-methylimidazole solution was added and the reaction continued. After the reaction was completed, the product was collected by centrifugation to obtain Hemin@ZIF. S2 polydopamine-modified metal-organic framework material: Hemin@ZIF and dopamine were prepared into solutions respectively. Ethanolamine solution was added to the Hemin@ZIF solution, followed by dopamine solution. After stirring and reacting, the product was collected by centrifugation to obtain Hemin@ZIF@PDA. S3-loaded platinum nanoparticles: Hemin@ZIF@PDA was mixed with a platinum nanoparticle dispersion and stirred to react; the product was collected by centrifugation to obtain Hemin@ZIF@PDA@Pt.

[0008] Furthermore, in S1, sodium dodecyl sulfate is dissolved in deionized water and the reaction is continued for 15 min; then heme chloride and cobalt nitrate hexahydrate are added and the reaction is continued for 15 min; then 2-methylimidazole solution is added and the reaction is carried out for 1 h.

[0009] Furthermore, in S2, Hemin@ZIF and dopamine were weighed in a 1:1 mass ratio, dissolved separately in anhydrous ethanol by ultrasonication, and prepared into solutions. After adding the dopamine solution, the mixture was stirred at 400-600 rpm for 3-7 h. After the reaction was completed, the product was collected by centrifugation.

[0010] Furthermore, the preparation method of the platinum nanoparticle dispersion includes: dissolving chloroplatinic acid solution in ethylene glycol, placing it in an oil bath environment, magnetically stirring and heating; adding polyvinylpyrrolidone pre-dissolved in ethylene glycol, heating to 100-120℃ and reacting for 1-5 h; after the reaction is completed, washing the product with acetone, and finally dispersing and storing the platinum nanoparticles in anhydrous ethanol.

[0011] Application of a programmable perforated nanozyme probe or a programmable perforated nanozyme probe and its preparation method in the preparation of a lateral flow immunochromatographic platform.

[0012] Furthermore, the method for constructing the lateral flow immunochromatographic platform includes: dissolving a programmable perforated nanozyme probe in PBS buffer and vortexing to mix; adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution and vortexing to mix; adding the detection antibody and incubating at a constant temperature; then adding casein solution and bovine serum albumin solution and blocking at room temperature; washing with PBS buffer and reconstituted in PBS buffer for later use.

[0013] Furthermore, the method for detecting MTB nucleic acid includes the following steps: Sample processing and nucleic acid amplification: Nucleic acid is directly extracted from the sample after thermal lysis; the lysis buffer is mixed with primers and RPA amplification reagent, and amplified at an isothermal temperature to obtain the target nucleic acid amplification product; CRISPR-specific cleavage and chromatography detection: The amplification product was mixed with the Cas protein reaction system and incubated to complete the specific cleavage reaction; then the CRISPR reaction solution, the lateral flow immunochromatography platform probe solution, the AuNPs@IgY Ab probe solution and the chromatography buffer were thoroughly mixed and dropped onto the prepared chromatography test strip for detection. Signal amplification and result reading: Read the initial signal after colorimetric chromatography; add substrate solution AEC and H2O2 to the reaction area of ​​the test strip, react, amplify the signal, and then read the result.

[0014] Furthermore, the preparation method of AuNPs@IgY Ab probe solution includes: adding chicken-derived IgY antibody to gold nanoparticle solution, stirring and reacting, then adding BSA solution and stirring to block; washing with PBS buffer and then reconstituted in PBS buffer for later use.

[0015] Furthermore, the method for detecting HIV-P24 antigen includes the following steps: Sample testing: Thoroughly mix the sample, lateral flow immunochromatography platform probe solution, and chromatography buffer, and drop the mixture onto the prepared chromatography test strip. The mixture flows sequentially through the sample pad, detection zone, and absorbent pad via capillary action. Signal amplification and result reading: Read the initial signal after colorimetric chromatography; add substrate solution AEC and H2O2 to the reaction area of ​​the test strip, react, amplify the signal, and then read the result.

[0016] This invention innovatively employs a one-pot in-situ synthesis method for perforated sheet-like MOF materials, abandoning the traditional complex modification process and significantly reducing the preparation time from 1-5 days in traditional etching processes to 1.5 hours, thereby significantly reducing operational difficulty and preparation costs. By precisely controlling key parameters such as the composition, temperature, and time of the reaction system, controllable synthesis of MOF materials from monopores to porous structures can be achieved, significantly increasing the specific surface area and contact area of ​​the material, allowing the internally loaded active ingredients to be fully exposed, while promoting the rapid penetration of substrates through the newly added interface, shortening the mass transfer distance, and effectively improving the catalytic reaction efficiency. Furthermore, this invention successfully constructs a programmable perforated nanozyme probe with both high colorimetric signal intensity and high catalytic activity by loading hemin chloride small molecules inside the perforated sheet-like MOF material and utilizing the high adsorption properties of the polydopamine (PDA) modified coating to increase the loading of platinum (Pt) nanoparticles in the outer layer. This probe was applied for the first time to a lateral flow immunochromatographic platform. By leveraging the high loading capacity and synergistic catalytic effect of MOF materials, ultrasensitive and rapid detection of Mycobacterium tuberculosis nucleic acid and HIV-P24 antigen was achieved, providing a novel technical solution for the early diagnosis and prevention of these two major infectious diseases.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1. Extremely high sensitivity: The nanoprobe synergistic catalysis amplifies the signal, with the detection limit of HIV-P24 by naked eye colorimetry reaching 1 pg / mL and MTB nucleic acid reaching 1 Copies / μL, accurately capturing low-abundance targets.

[0018] 2. Strong platform versatility: By changing the probe antibody and the test strip capture molecule, it can be adapted to nucleic acid (RPA-CRISPR mode) or antigen (sandwich mode) detection, with good scalability.

[0019] 3. Rapid and adaptable testing: Simplified sample processing (nucleic acid thermal lysis, antigens do not require complex pretreatment), the entire nucleic acid testing process is completed within 40 minutes, and antigen testing is completed within 15 minutes, adapting to the needs of different POCT scenarios.

[0020] 4. Visualized results: Supports dual modes of "qualitative analysis by visual inspection + quantitative analysis by mobile phone photography", eliminating the need for large instruments and providing flexible and convenient interpretation.

[0021] 5. Low cost and stable: One-pot in-situ synthesis of the carrier significantly reduces preparation time and cost, and the test strips exhibit excellent stability, which is conducive to large-scale promotion.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the step-by-step construction process of the Hemin@ZIF@PDA@Pt composite nanomaterial of the present invention.

[0024] Figure 2 The color development results of the MTB nucleic acid detection test strip based on Hemin@ZIF@PDA@Pt in this invention are shown.

[0025] Figure 3 The results show the color development of the HIV-P24 antigen test strip based on Hemin@ZIF@PDA@Pt according to this invention. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0027] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0028] The existing technology mainly faces the following technical challenges: 1. Challenges in ultrasensitive visualization detection: Traditional gold nanoparticles (AuNPs) and other probes have weak signals and insufficient catalytic activity, with sensitivity 1-3 orders of magnitude lower than laboratory techniques. This makes it difficult to achieve ultrasensitive visualization detection of low-copy MTB nucleic acids and low-abundance HIV-P24 antigens, thus failing to meet the needs of early diagnosis.

[0029] 2. Insufficient adaptability of testing technologies to various scenarios: Laboratory testing technologies rely on large instruments and professional personnel, which are costly and time-consuming; Although lateral flow immunochromatography (LFIA) is convenient and low-cost, it is limited by the performance of traditional probes and lacks a testing solution that balances high sensitivity and universality, making it difficult to adapt to POCT and primary screening.

[0030] 3. Bottlenecks in MOF material modification technology: Existing MOF material modification processes are cumbersome, have low mass transfer efficiency, and are costly, making it impossible to efficiently and on a large scale prepare high-performance probe carriers, which restricts the development of highly sensitive probes.

[0031] 4. Probe synergistic performance optimization issues: There is a lack of probe construction schemes that combine high loading capacity, high catalytic activity and signal amplification capabilities. The carrier cannot fully expose the active ingredients and improve mass transfer efficiency, resulting in poor probe detection performance.

[0032] This invention addresses the core need for ultrasensitive and visualized detection of low-copy nucleic acid (such as MTB nucleic acid) and low-abundance protein (such as HIV-P24 antigen) targets. It focuses on four major technical challenges: insufficient performance of traditional signal probes, poor adaptability of detection technologies to various scenarios, defects in MOF vector modification processes, and poor probe synergy. It innovatively designs a technical path of "high-performance vector synthesis - multifunctional probe construction - universal platform integration" to provide a lateral flow chromatography ultrasensitive immunoassay technology that combines high sensitivity and universality, breaking through the multiple limitations of existing detection technologies in terms of sensitivity, scenario adaptability, and universality.

[0033] Example 1: Construction of a programmable perforated nanozyme probe Hemin@ZIF@PDA@Pt I. Synthesis of Hemin@ZIF materials 0.2 g of sodium dodecyl sulfate (SDS) was dissolved in 45 mL of deionized water under vigorous stirring at 25 °C, and the reaction was continued for 15 min. Then, 1 mg of hemin chloride (Hemin) and 353.75 mg of cobalt nitrate hexahydrate were added sequentially, and the reaction was continued for another 15 min. 798.75 mg of 2-methylimidazole was dissolved in 5 mL of deionized water by sonication and added to the above system, and the reaction was carried out for 1 h. After the reaction was completed, the product was collected by centrifugation and washed three times with anhydrous ethanol.

[0034] II. Synthesis of Hemin@ZIF@PDA Materials Hemin@ZIF and dopamine were weighed in a 1:1 mass ratio and dissolved separately in anhydrous ethanol by sonication to prepare a solution with a concentration of 1 mg / mL. Three volumes of ethanolamine solution with a pH of 8.5 were added to the Hemin@ZIF solution, and then dopamine solution was added dropwise. The mixture was stirred at 500 rpm for 5 h. After the reaction was completed, the product was collected by centrifugation and washed twice with anhydrous ethanol and deionized water alternately.

[0035] III. Synthesis of Pt Nanoparticles Take 3.185 mL of 100 mM chloroplatinic acid (H2PtCl6) solution, dissolve it in 10 mL of ethylene glycol, place it in an oil bath environment, stir magnetically and heat it; when the system is close to boiling (e.g., 90-99℃), add 0.225 g of polyvinylpyrrolidone (PVP, molecular weight 29000) that has been dissolved in 10 mL of ethylene glycol, raise the temperature to 110℃ and react for 3 h; after the reaction is completed, wash the product with acetone, and finally disperse and store the Pt nanoparticles in 20 mL of anhydrous ethanol.

[0036] IV. Synthesis of Hemin@ZIF@PDA@Pt Nanoprobes Mix Hemin@ZIF@PDA with 1 mL of Pt nanoparticle dispersion at a ratio of 8 mg of Hemin@ZIF@PDA and stir continuously for 12 h. After centrifugation to collect the product, wash it three times with anhydrous ethanol to obtain the Hemin@ZIF@PDA@Pt composite nanomaterial. Figure 1 This is a schematic diagram illustrating the synthesis of Hemin@ZIF@PDA@Pt nanoprobes.

[0037] Example 2: Detection of MTB nucleic acid using a chromatography platform based on Hemin@ZIF@PDA@Pt programmable perforated nanoprobes I. Preparation of MTB Nucleic Acid Immunochromatographic Test Strips Preparation of nitrocellulose membranes for MTB nucleic acid detection: Streptavidin (Aladdin), goat anti-mouse antibody (Jingda Biotechnology), and goat anti-chicken antibody (Jingda Biotechnology) were dissolved in buffer solutions (containing 4% sucrose, 4% sodium chloride, and 0.01M phosphate buffer) to prepare spray solutions with final concentrations of 5 mg / mL, 4 mg / mL, and 1 mg / mL, respectively. The nitrocellulose membrane (NC membrane) was fixed onto a substrate, and streptavidin solution was sprayed onto the detection line (T1 line), goat anti-mouse antibody solution onto the detection line (T2 line), and goat anti-chicken antibody solution onto the control line (C line) of the NC membrane using a membrane scrubbing instrument. The membrane was then dried overnight at 37°C for later use.

[0038] Assembly of MTB nucleic acid immunochromatographic test strips: The pretreated glass fiber sample pad and absorbent pad are overlapped at both ends of the NC membrane (overlap width is 2 mm) to construct a complete lateral chromatography strip structure; the chromatography strip is cut into test strips with a width of 4 mm using a special cutting instrument, sealed and stored in a dry environment for later use.

[0039] II. Synthesis of Hemin@ZIF@PDA@Pt@anti-FAM Ab nanolabeled probes 1 mg of Hemin@ZIF@PDA@Pt material was dissolved in 1 mL of PBS buffer (10 mM, pH=7.4) and vortexed to mix. 10 μL (10 mg / mL) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added to the solution, and after further vortexing, 50 μg of anti-FAM antibody was added. The mixture was incubated at room temperature for 4 h to achieve conjugation between the nanomaterial and the detection antibody via covalent bonding of carboxyl and amino groups. Subsequently, 200 μL (10 wt%) casein solution and 200 μL (10 wt%) bovine serum albumin (BSA) solution were added sequentially, and each was blocked at room temperature for 1 h. After washing three times with PBS buffer, the mixture was reconstituted in 250 μL of PBS buffer (10 mM, pH=7.4) and stored at 4°C for later use.

[0040] III. Synthesis of AuNPs@IgY Ab nanolabeled probes Add 50 μg of chicken-derived IgY antibody to 1 mL of gold nanoparticle (AuNPs) solution and stir at room temperature for 20 min. The AuNPs are conjugated to the detection antibody through the interaction between the thiol groups on the colloidal gold surface and the amino groups of the antibody molecules. Then add 200 μL (10 wt%) BSA solution and stir at room temperature for 30 min to block the reaction. After washing three times with PBS buffer, the solution is reconstituted in 250 μL of PBS buffer (10 mM, pH=7.4) and stored at 4°C for later use.

[0041] IV. Detection Procedure and Method for MTB Nucleic Acid in Bronchoalveolar Lavage Fluid (1) Sample processing and nucleic acid amplification: Take bronchoalveolar lavage fluid samples, and extract nucleic acid directly after thermal lysis at 95℃ for 5 min; take 10 μL of lysis buffer and mix it with 10 μM primers P and F (2.5 μL each) and RPA amplification reagent, and amplify at 39℃ for 10 min to obtain the target nucleic acid product.

[0042] (2) CRISPR specific cleavage and chromatography detection: 2 μL of RPA amplification product was mixed with the Cas protein reaction system and incubated at 48℃ for 10 min to complete the specific cleavage reaction; then, the CRISPR reaction solution, 1.5 μL of Hemin@ZIF@PDA@Pt@anti-FAM Ab probe solution, 1.5 μL of AuNPs@IgY Ab probe solution and 40 μL of chromatography buffer were thoroughly mixed and dropped onto the prepared chromatography test strip.

[0043] In the detection system, single-stranded DNA (FB) modified with FAM-Biotin groups at both ends serves as a reporter: when there is no target MTB nucleic acid, the Hemin@ZIF@PDA@Pt@anti-FAM-Ab@FB complex specifically binds to streptavidin (SA) at the T1 line via biotin; when the target MTB nucleic acid is present, FB is specifically cleaved by Cas protein, and the resulting Hemin@ZIF@PDA@Pt@anti-FAM-Ab@FAM complex migrates to the T2 line and is specifically intercepted by goat anti-mouse IgG; at the same time, AuNPs@IgY-Ab is captured by goat anti-chicken IgG at the C line, thus achieving detection quality control.

[0044] (3) Signal amplification and result reading: The initial signal was read after chromatographic color development for 10 min; to further improve the detection sensitivity, substrate solution (AEC and H2O2) was added to the reaction area of ​​the test strip and reacted for 5 min. The peroxidase of Hemin@ZIF@PDA@Pt was used to simulate the catalytic activity, which promoted the formation of insoluble brown-red products of AEC and their deposition on the T1 and T2 lines, thus achieving significant amplification of the color signal.

[0045] The results were read in two modes: (i) direct visual interpretation for qualitative or semi-quantitative analysis; (ii) image of the test strip taken with a smartphone, and the gray values ​​of the T1 and T2 lines were quantitatively analyzed using ImageJ software, with the T2 / T1 gray value ratio used as the quantitative detection result.

[0046] Detection limit verification The MTB plasmid was serially diluted with enzyme-free water to prepare a concentration of 10. 9 Copies / μL, 10 8 Copies / μL, 10 7 Copies / μL, 10 6 Copies / μL, 10 5 Copies / μL, 10 4 A series of standards were prepared using samples with concentrations of 10 copies / μL, 10³ copies / μL, 10² copies / μL, 10 copies / μL, 8 copies / μL, 6 copies / μL, 4 copies / μL, 2 copies / μL, and 1 copies / μL. Experiments were performed following the aforementioned RPA amplification, Cas-specific cleavage, and chromatographic detection procedures.

[0047] Figure 2Images of the MTB nucleic acid detection chromatography strip constructed based on Hemin@ZIF@PDA@Pt are shown. Image A shows the color development result before AEC and H2O2 catalytic amplification, and image B shows the color development result after catalytic amplification. Experimental results show that the detection platform can achieve a naked-eye detection limit of 1 Copies / μL before and after catalytic amplification.

[0048] Example 3: Detection of HIV-P24 antigen using a chromatography platform based on Hemin@ZIF@PDA@Pt programmable perforated nanoprobes I. Preparation of HIV-P24 antigen immunochromatographic test strips Preparation of nitrocellulose membrane for HIV-P24 antigen detection: HIV-P24 capture antibody (Huakui Jinpei Biotechnology) and goat anti-mouse antibody (Jingda Biotechnology) were dissolved separately in buffer solutions (containing 4% sucrose, 4% sodium chloride, and 0.01M phosphate buffer) to prepare spray solutions with a final concentration of 1 mg / mL. The nitrocellulose membrane (NC membrane) was fixed on a substrate, and the HIV-P24 capture antibody solution was sprayed onto the detection line (T line) of the NC membrane using a membrane scrubbing instrument. The goat anti-mouse antibody solution was sprayed onto the control line (C line). The membrane was then dried overnight at 37°C for later use.

[0049] Assembly of HIV-P24 antigen immunochromatographic test strips: The pretreated glass fiber sample pad and absorbent pad are overlapped at both ends of the NC membrane (overlap width is 2 mm) to construct a complete lateral chromatography strip structure; the chromatography strip is cut into test strips with a width of 4 mm using a special cutting instrument, sealed and stored in a dry environment for later use.

[0050] II. Synthesis of Hemin@ZIF@PDA@Pt@anti-HIV-P24 Ab nanolabeled probes 1 mg of Hemin@ZIF@PDA@Pt material was dissolved in 1 mL of PBS buffer (10 mM, pH=7.4) and vortexed to mix. 10 μL (10 mg / mL) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added to the solution, and after further vortexing, 50 μg of anti-HIV-P24 antibody was added. The mixture was incubated at room temperature for 4 h to achieve conjugation between the nanomaterial and the detection antibody via covalent bonding of carboxyl and amino groups. Subsequently, 200 μL (10 wt%) casein solution and 200 μL (10 wt%) bovine serum albumin (BSA) solution were added sequentially, and each was blocked at room temperature for 1 h. After washing three times with PBS buffer, the mixture was reconstituted in 250 μL of PBS buffer (10 mM, pH=7.4) and stored at 4°C for later use.

[0051] III. Detection Procedures and Methods for HIV-P24 Antigen in Serum (1) Sample detection and reaction principle: Take 20 μL of serum sample, 5 μL of Hemin@ZIF@PDA@Pt@anti-HIV-P24 Ab probe solution and 35 μL of chromatography buffer, mix thoroughly, and drop onto the prepared chromatography test strip. The mixture flows sequentially through the sample pad, detection area (T line, C line) and absorbent pad by capillary action: If the sample contains HIV-P24 antigen, it will first bind to the nanolabeled probe to form an antigen-probe complex. When this complex migrates to the T line, it specifically binds to the HIV-P24 capture antibody fixed on the T line, forming a sandwich-type immune complex of "capture antibody-antigen-detection antibody-nanomaterial"; the remaining unbound nanolabeled probe continues to migrate to the C line and binds to the goat anti-mouse antibody, and finally, colored bands appear on both the T line and the C line. If the sample does not contain HIV-P24 antigen, the nanolabeled probe will not bind to the T line, but will only migrate to the C line and bind to the goat anti-mouse antibody, and only the C line will show a colored band.

[0052] (2) Signal amplification and result reading: The initial signal was read after chromatographic color development for 10 min; to further improve the detection sensitivity, substrate solution (AEC and H2O2) was added to the reaction area of ​​the test strip and reacted for 5 min. The peroxidase of Hemin@ZIF@PDA@Pt was used to simulate the catalytic activity, which promoted the formation of insoluble brown-red products of AEC and their deposition on the T line and C line, thus achieving significant amplification of the color development signal.

[0053] The results were read in two modes: (i) direct visual interpretation for qualitative or semi-quantitative analysis; (ii) image of the test strip taken with a smartphone, and the gray value of the T line was quantitatively analyzed using ImageJ software, with the gray value of the T line used as the quantitative detection result.

[0054] Detection limit verification HIV-P24 standard antigen was serially diluted to prepare a series of standards with concentrations of 1000 pg / mL, 750 pg / mL, 500 pg / mL, 250 pg / mL, 100 pg / mL, 75 pg / mL, 50 pg / mL, 25 pg / mL, 10 pg / mL, 5 pg / mL, and 1 pg / mL. 20 μL of each concentration of standard was taken and the experiment was performed according to the chromatographic detection procedure described above.

[0055] Figure 3Images of the HIV-P24 antigen detection chromatographic test strip constructed based on Hemin@ZIF@PDA@Pt are shown. Image A shows the color development result before AEC and H2O2 catalytic amplification, and image B shows the color development result after catalytic amplification. Experimental results show that the detection limit of this detection platform before catalytic amplification is 5 pg / mL, which can be reduced to 1 pg / mL after catalytic amplification, indicating a significant signal amplification effect.

[0056] This invention has the following characteristics: 1. Efficient synthesis of perforated sheet-like MOF supports: An innovative one-pot in-situ synthesis process shortens the preparation time to 1.5 hours, enables controllable adjustment of the porous structure, significantly improves the specific surface area and mass transfer efficiency of the support, and successfully solves the technical pain points of cumbersome processes and high preparation costs of traditional MOF materials.

[0057] 2. Multifunctional integration of Hemin@ZIF@PDA@Pt nanoprobes: Constructing an integrated synergistic system of "recognition-catalysis-signal amplification", which combines high target specificity recognition capability with signal amplification efficiency, effectively overcoming the performance bottlenecks of insufficient sensitivity and inefficient signal transmission of traditional probes.

[0058] 3. Targeted Adaptation Innovation of Universal Chromatography System: A flexibly switchable chromatography detection architecture is built. The "RPA-CRISPR-dual detection line" mode is customized for MTB nucleic acid (low copy), and the sandwich immunochromatographic capture mode is matched for HIV-P24 antigen (low abundance). Both are two-step processes of "initial color development + catalytic signal amplification", which can accurately adapt to the detection of different types of low abundance targets.

[0059] 4. Convenient design enhances universality: Sample processing is greatly simplified (MTB nucleic acid is directly extracted via thermal lysis, and HIV-P24 antigen requires no complex pretreatment), supporting a dual interpretation mode of "visual qualitative analysis + ImageJ software quantitative analysis"; antigen detection is completed within 15 minutes, and the entire nucleic acid detection process is completed within 40 minutes. The test strip has excellent stability and is suitable for efficient application in primary healthcare and POCT scenarios.

[0060] 5. The value of the general platform is highlighted: The core probe and chromatography architecture have strong compatibility and can be quickly adapted to nucleic acid / antigen target detection, taking into account both high sensitivity and convenience in POCT scenarios, providing efficient and universal technical support for the early screening and prevention of major infectious diseases.

[0061] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0062] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A programmable perforated nanozyme probe, characterized in that, This includes metal-organic framework materials, which contain heme chloride, and metal-organic framework materials modified with polydopamine with platinum nanoparticles loaded on the surface.

2. The method for preparing a programmable perforated nanozyme probe as described in claim 1, characterized in that: Includes the following steps: S1 metal-organic framework material loaded with heme chloride: Heme chloride and cobalt nitrate hexahydrate were added to sodium dodecyl sulfate solution and reacted. Then, 2-methylimidazole solution was added and the reaction continued. After the reaction was completed, the product was collected by centrifugation to obtain Hemin@ZIF. S2 polydopamine-modified metal-organic framework material: Hemin@ZIF and dopamine were prepared into solutions respectively. Ethanolamine solution was added to the Hemin@ZIF solution, followed by dopamine solution. After stirring and reacting, the product was collected by centrifugation to obtain Hemin@ZIF@PDA. S3-loaded platinum nanoparticles: Hemin@ZIF@PDA was mixed with a platinum nanoparticle dispersion and stirred to react; the product was collected by centrifugation to obtain Hemin@ZIF@PDA@Pt.

3. The method for preparing a programmable perforated nanozyme probe as described in claim 2, characterized in that: In S1, sodium dodecyl sulfate was dissolved in deionized water and reacted for 15 min; then heme chloride and cobalt nitrate hexahydrate were added and reacted for another 15 min; then 2-methylimidazole solution was added and reacted for 1 h.

4. The method for preparing a programmable perforated nanozyme probe as described in claim 2, characterized in that: In S2, Hemin@ZIF and dopamine were weighed in a 1:1 mass ratio, dissolved separately in anhydrous ethanol by ultrasonication, and prepared into solutions. After adding the dopamine solution, the mixture was stirred at 400-600 rpm for 3-7 h. After the reaction was completed, the product was collected by centrifugation.

5. The method for preparing a programmable perforated nanozyme probe as described in claim 2, characterized in that: The preparation method of platinum nanoparticle dispersion includes: dissolving chloroplatinic acid solution in ethylene glycol, placing it in an oil bath environment, magnetically stirring and heating; adding polyvinylpyrrolidone that has been dissolved in ethylene glycol beforehand, heating to 100-120℃ and reacting for 1-5 h; after the reaction is completed, washing the product with acetone, and finally dispersing and storing the platinum nanoparticles in anhydrous ethanol.

6. The application of the programmable perforated nanozyme probe as described in claim 1 or the method for preparing the programmable perforated nanozyme probe as described in any one of claims 2-5 in the preparation of a lateral flow immunochromatographic platform.

7. The application as described in claim 6, characterized in that: The method for constructing a lateral flow immunochromatographic platform includes: dissolving a programmable perforated nanozyme probe in PBS buffer and vortexing to mix; adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the solution and vortexing to mix; adding detection antibody and incubating at a constant temperature; then adding casein solution and bovine serum albumin solution and blocking at room temperature; washing with PBS buffer and reconstituted in PBS buffer for later use.

8. The application as described in claim 7, characterized in that: The method for detecting MTB nucleic acid includes the following steps: Sample processing and nucleic acid amplification: Nucleic acid is directly extracted from the sample after thermal lysis; the lysis buffer is mixed with primers and RPA amplification reagent, and amplified at an isothermal temperature to obtain the target nucleic acid amplification product; CRISPR-specific cleavage and chromatography detection: The amplification product was mixed with the Cas protein reaction system and incubated to complete the specific cleavage reaction; then the CRISPR reaction solution, the lateral flow immunochromatography platform probe solution, the AuNPs@IgY Ab probe solution and the chromatography buffer were thoroughly mixed and dropped onto the prepared chromatography test strip for detection. Signal amplification and result reading: Read the initial signal after colorimetric chromatography; add substrate solution AEC and H2O2 to the reaction area of ​​the test strip, react, amplify the signal, and then read the result.

9. The application as described in claim 8, characterized in that: The preparation method of AuNPs@IgY Ab probe solution includes: adding chicken-derived IgY antibody to gold nanoparticle solution, stirring and reacting, then adding BSA solution and stirring to block; washing with PBS buffer and then reconstituted in PBS buffer for later use.

10. The application as described in claim 7, characterized in that: Used to detect HIV-P24 antigen, to detect HIV-P24 The antigen method includes the following steps: Sample testing: Thoroughly mix the sample, lateral flow immunochromatography platform probe solution, and chromatography buffer, and drop the mixture onto the prepared chromatography test strip. The mixture flows sequentially through the sample pad, detection zone, and absorbent pad via capillary action. Signal amplification and result reading: Read the initial signal after colorimetric chromatography; add substrate solution AEC and H2O2 to the reaction area of ​​the test strip, react, amplify the signal, and then read the result.

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