Preparation method of multifunctional nanoscale enzyme probe and application of multifunctional nanoscale enzyme probe in tuberculosis-aids co-infection

Abstract

This invention relates to the field of nanobiosensing technology, and particularly to a method for preparing a multifunctional nanozyme probe and its application in tuberculosis and HIV comorbidities. The nanozyme probe comprises a covalent organic framework (COF) material as a carrier substrate, and first and second noble metal nanoparticles loaded on the surface of the carrier substrate. The first noble metal nanoparticles enhance the Raman signal of the carrier substrate, and the second noble metal nanoparticles provide enzyme activity. This nanozyme probe possesses excellent colorimetric response performance, peroxidase-like activity, and strong SERS performance, making it suitable as a multifunctional signal probe. When applied to bidirectional lateral flow immunochromatography (LFIA), by modifying it with specific antibodies against tuberculosis and HIV biomarkers, simultaneous and bidirectional detection of TB and HIV biomarkers can be achieved. This test strip supports three detection modes: visual colorimetry, catalytic amplification colorimetry, and precise SERS quantification, improving the sensitivity, dynamic range, and reliability of the detection.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensing technology, and in particular to a method for preparing a multifunctional nanozyme probe and its application in tuberculosis and HIV comorbidity. Background Technology

[0002] Currently, the gold standard for tuberculosis diagnosis is microbial culture, but this process can take several weeks. Molecular diagnostics (such as XpertMTB / RIF), while rapid and sensitive, require sophisticated equipment, high costs, and skilled operators. In serological testing, culture filtrate protein 10 (CFP-10), a secreted protein specific to the Mycobacterium tuberculosis complex, is absent from BCG and most non-tuberculous mycobacteria. Therefore, it effectively distinguishes tuberculosis infection from BCG inoculation or non-tuberculous mycobacterial infection, exhibiting good specificity and serving as an important target for the serological diagnosis of tuberculosis. The definitive diagnosis of HIV primarily relies on combined HIV antibody / antigen testing. HIV-1 p24 protein is the core capsid protein of the virus, appearing in the blood during the post-infection window period, and its level is closely related to viral replication activity. Therefore, p24 protein detection has irreplaceable value for early diagnosis, neonatal infection diagnosis, and monitoring the effectiveness of antiviral therapy. For the detection of TB-HIV co-infection, the existing strategies mostly involve performing two separate tests, which is cumbersome, time-consuming, and resource-intensive, and cannot achieve true synchronous and integrated analysis.

[0003] Traditional colloidal gold test strips, used as point-of-care testing (POCT) platforms, have been applied to the detection of many pathogenic microorganisms. However, because they usually rely on a single signal probe such as colloidal gold, their detection sensitivity is insufficient, and they are mostly limited to qualitative or semi-quantitative interpretation by the naked eye, making it difficult to meet the needs for accurate quantification of low-abundance biomarkers.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing a multifunctional nanozyme probe and its application in tuberculosis-AIDS comorbidity, aiming to solve the problems of insufficient sensitivity and reliability and single signal mode of existing probes for detecting TB-HIV co-infection.

[0006] The technical solution of the present invention is as follows: A multifunctional nanozyme probe includes a covalent organic framework material (COFs) as a carrier substrate, and a first noble metal nanoparticle and a second noble metal nanoparticle loaded on the surface of the carrier substrate; the first noble metal nanoparticle is used to enhance the Raman signal of the carrier substrate; and the second noble metal nanoparticle is used to provide enzyme activity.

[0007] The multifunctional nanozyme probe, wherein the first noble metal nanoparticle includes one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles; and the second noble metal nanoparticle includes one or more of Pt nanoparticles, Pd nanoparticles, Au nanoparticles, Ag nanoparticles, Ir nanoparticles, Ru nanoparticles, and Rh nanoparticles.

[0008] The multifunctional nanozyme probe further adsorbs specific antibodies corresponding to CFP-10 and p24 proteins.

[0009] The multifunctional nanozyme probe has a particle size of 350 nm-450 nm.

[0010] A method for preparing a multifunctional nanozyme probe includes the following steps: 2,5-Dimethoxytetraphenylbenzene, 1,3,5-tris(4-aminophenyl)benzene, solvent, regulator and catalyst are mixed and reacted to obtain a COFs solution; The COFs solution is mixed with a first noble metal salt solution, a first reducing agent and a stabilizer to obtain a COFs nanoparticle solution loaded with the first noble metal. The COF nanoparticle solution loaded with the first noble metal is mixed with a surfactant and a second reducing agent, and then mixed with a second noble metal salt solution to obtain a multifunctional nanozyme probe.

[0011] The method for preparing the multifunctional nanozyme probe, wherein the concentration of 2,5-dimethoxy-terephthalaldehyde is 25 mmol / L-35 mmol / L; the concentration of 1,3,5-tris(4-aminophenyl)benzene is 35 mmol / L-50 mmol / L; and the volume ratio of 2,5-dimethoxy-terephthalaldehyde to 1,3,5-tris(4-aminophenyl)benzene is (4-6):(5-7).

[0012] The method for preparing the multifunctional nanozyme probe, wherein the concentration of the COFs solution is 25 mmol / L-35 mmol / L; the concentration of the first noble metal salt solution is 7 mmol / L-14 mmol / L; and the volume ratio of the COFs solution to the first noble metal salt solution is (1-25):1.

[0013] The method for preparing the multifunctional nanozyme probe, wherein the concentration of the COF nanoparticle solution loaded with the first noble metal is 25 mmol / L-35 mmol / L; the concentration of the second noble metal salt solution is 40 mmol / L-60 mmol / L; and the volume ratio of the COF nanoparticle solution loaded with the first noble metal to the second noble metal salt solution is (0.75-6):1.

[0014] Application of a multifunctional nanozyme probe in a two-way rapid test strip platform for tuberculosis and HIV comorbidities.

[0015] In the aforementioned application, the tuberculosis and HIV comorbidity two-way rapid test strip platform includes a test strip and a reaction system; the test strip includes a base plate, a first nitrocellulose membrane and a second nitrocellulose membrane located on one side of the base plate and spaced apart, and a sample pad located between the first nitrocellulose membrane and the second nitrocellulose membrane; the first nitrocellulose membrane is provided with a first control line and a first detection line, and the second nitrocellulose membrane is provided with a second control line and a second detection line; the first detection line and the second detection line are positioned close to the sample pad relative to the first control line and the second control line; the first detection line is loaded with anti-CFP-10 protein-specific monoclonal antibody Ab2, and the second detection line is loaded with anti-p24 protein-specific monoclonal antibody Ab2; the reaction system includes a nanozyme probe adsorbed with CFP-10 protein polyclonal antibody Ab1 and anti-p24 protein monoclonal antibody Ab1.

[0016] Beneficial Effects: This invention provides a method for preparing a multifunctional nanozyme probe and its application in tuberculosis-AIDS comorbidity. The multifunctional nanozyme probe comprises a covalent organic framework (COF) material as a carrier substrate, and first and second noble metal nanoparticles loaded on the surface of the carrier substrate. The first noble metal nanoparticles enhance the Raman signal of the carrier substrate; the second noble metal nanoparticles provide enzyme activity. The nanozyme probe provided by this invention uses a covalent organic framework (COF) material as a substrate and forms a composite nanomaterial by loading first and second noble metal nanoparticles. This material possesses both excellent peroxidase-like activity and strong SERS (Surface-Enhanced Raman Spectroscopy) performance, and can serve as a multifunctional signal probe. Furthermore, applying this nanozyme probe to two-way lateral flow immunoassay (LFIA) can avoid potential interference between multiple targets. By modifying it with specific antibodies against tuberculosis (TB) and HIV biomarkers, simultaneous and bidirectional detection of TB and HIV biomarkers can be achieved. At the same time, the test strip supports three detection modes: visual colorimetry, catalytic amplification colorimetry, and SERS precise quantification, which significantly improves the sensitivity, dynamic range, and reliability of the detection. It is especially suitable for early screening, efficacy monitoring, and epidemic prevention and control of TB-HIV co-infection in resource-limited areas. Attached Figure Description

[0017] Figure 1 This is a transmission electron microscope image of COFs@Au@Pt NPs prepared in Example 1 of the present invention; Figure 2 The catalytic color development and absorption spectra of each solution are shown. Figure 3 A comparison chart of SERS performance with different Au NPs loadings; Figure 4 A comparison of SERS performance for different Pt NPs loads under the same Au NPs load. Figure 5 A diagram of the LFIA model for two-way side-flow chromatography; Figure 6 This is the colorimetric diagram of Example 3; Figure 7 The images show the detection results of p24 test strips at a series of concentration gradients, the catalytic color development results, and the corresponding Raman spectra collected on the T line. Figure 8 The images show the detection results of CFP-10 test strips at a series of concentration gradients, the catalytic color development results, and the Raman spectra collected on the corresponding T lines. Detailed Implementation

[0018] This invention provides a method for preparing a multifunctional nanozyme probe and its application in tuberculosis and HIV comorbidities. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Co-infection with tuberculosis (TB) and HIV is a major challenge in global public health. HIV infection severely impairs cellular immunity, greatly increasing the risk of latent TB infection developing into active TB, while also making TB diagnosis more difficult, its progression faster, and its mortality rate higher. Conversely, active TB can also accelerate the disease progression in HIV-infected individuals. Therefore, establishing a technological platform capable of rapidly, accurately, and simultaneously detecting TB-HIV co-infection is crucial for early intervention, optimizing treatment strategies, and blocking disease transmission.

[0021] SERS technology, due to its ability to significantly enhance the detection signal of molecules and its characteristics such as ultrasensitivity and fingerprinting, has been applied to the analysis and detection of biomarkers for bacteria, cells, proteins, and nucleic acids. While using noble metals such as Au and Ag to construct SERS substrates can amplify signals through electromagnetic field enhancement (EM) via plasmon resonance, it still suffers from drawbacks such as poor stability and uniformity, low selectivity, weak enhancement effect, and susceptibility to oxidation and denaturation, hindering its further development and application. Nanoscale COFs, on the other hand, are novel porous crystalline materials composed of purely organic structural units linked by covalent bonds. These covalent bonds give COFs high chemical stability and generate characteristic signals, making them ideal substrates for loading nanoparticles. By introducing noble metals such as Au and Ag, the SERS-enhanced substrate effect can be utilized to enhance the Raman signal of the COFs themselves.

[0022] Furthermore, COFs are important materials for constructing nanozymes. Nanozymes are artificial enzymes that possess both the properties of nanomaterials and catalytic functions, enabling specific catalytic reactions and generating corresponding signals, such as chemical, optical, and electrical signals. To date, numerous reports have demonstrated that many nanomaterials exhibit catalase and peroxidase-like activities, with noble metal materials such as Pd and Pt being the most representative.

[0023] Based on this, the present invention provides a multifunctional nanozyme probe, comprising a covalent organic framework material (COFs) as a carrier substrate, and a first noble metal nanoparticle and a second noble metal nanoparticle loaded on the surface of the carrier substrate; the first noble metal nanoparticle is used to enhance the Raman signal of the carrier substrate; and the second noble metal nanoparticle is used to provide enzyme activity.

[0024] In this embodiment, the provided nanozyme probe uses covalent organic framework materials (COFs) as a substrate and forms a composite nanomaterial by loading first and second noble metal nanoparticles. This material possesses excellent colorimetric response performance, peroxidase-like activity, and strong SERS (Surface-Enhanced Raman Spectroscopy) performance, making it suitable as a multifunctional signal probe. Furthermore, applying this nanozyme probe to two-way lateral flow immunoassay (LFIA) can avoid potential interference between multiple targets. By modifying it with specific antibodies against tuberculosis (TB) and HIV biomarkers, simultaneous and bidirectional detection of TB and HIV biomarkers can be achieved. Simultaneously, this test strip supports three detection modes: visual colorimetry, catalytic amplification colorimetry, and precise SERS quantification, significantly improving the sensitivity, dynamic range, and reliability of the detection. It is particularly suitable for early screening, treatment monitoring, and epidemic prevention and control of TB-HIV co-infection in resource-limited areas.

[0025] Specifically, this invention uses COFs as a carrier to load first and second noble metal nanoparticles, forming a composite probe structure that is stable and well-dispersed, ensuring the consistency and reliability of the detection signal and improving the batch-to-batch reproducibility of the test strip. Furthermore, the bidirectional lateral flow immunochromatographic test strip constructed using this nanozyme probe can achieve simultaneous, bidirectional rapid detection, avoiding the cumbersome process of performing two separate tests in traditional methods, significantly improving detection efficiency, and is particularly suitable for rapid initial screening of TB-HIV co-infection. The test strip based on this nanozyme probe has a simple structure and low cost. This nanozyme probe enables preliminary screening without the need for complex instruments. By combining SERS and catalytic dual-signal amplification mechanisms, it significantly reduces the detection limit and improves the detection capability for low-abundance biomarkers. It also has a wide linear detection range, making it suitable for analysis throughout the entire process from early infection to disease monitoring. It is especially suitable for use in resource-limited areas, primary healthcare institutions, and on-site prevention and control. The nanozyme probe uses CFP-10 protein as the TB detection target, avoiding cross-reaction with BCG and most non-tuberculous bacilli. Furthermore, it combines p24 antigen detection for HIV, ensuring specific identification of TB-HIV co-infection.

[0026] In some embodiments, the first noble metal nanoparticles include, but are not limited to, one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles. Selecting the aforementioned first noble metal nanoparticles and loading them onto the surface of COFs can enhance the SERS-enhanced substrate, thereby amplifying the Raman signal of the COFs themselves.

[0027] In a preferred embodiment, the first noble metal nanoparticle is Au nanoparticle; Au nanoparticles possess strong plasma enhancement effect, excellent chemical stability, good biocompatibility and highly controllable morphology and structure, and can provide stable, efficient and reproducible SERS enhancement effect.

[0028] In some embodiments, the second noble metal nanoparticles include, but are not limited to, one or more of Pt nanoparticles, Pd nanoparticles, Au nanoparticles, Ag nanoparticles, Ir nanoparticles, Ru nanoparticles, and Rh nanoparticles. These second noble metal nanoparticles possess catalase and peroxidase-like activities. By co-loading them with the first noble metal nanoparticles onto the surface of COFs, the nanozyme probe exhibits both excellent peroxidase-like activity and strong SERS performance, making it suitable as a multifunctional signal probe.

[0029] In a preferred embodiment, the second noble metal nanoparticle is a Pt nanoparticle; compared with Pd, Au, Ag, etc., Pt is more stable under acidic, alkaline and high temperature conditions; compared with Ir, Ru and Rh, Pt has more mature synthesis methods and a wider range of applications.

[0030] In some embodiments, the nanozyme probe is also adsorbed with specific antibodies corresponding to CFP-10 and p24 proteins. By modifying the probe with specific antibodies against TB and HIV biomarkers, simultaneous, bidirectional detection of TB and HIV biomarkers can be achieved.

[0031] In some embodiments, the nanozyme probe has a particle size of 350 nm-450 nm; and the nanozyme probe is a composite star-shaped nanoparticle with a core of star-shaped COFs and an outer layer uniformly adhered with the first noble metal nanoparticle and the second noble metal nanoparticle.

[0032] In a preferred embodiment, the nanozyme probe has a particle size of 400 nm.

[0033] In addition, this invention also provides a method for preparing a multifunctional nanozyme probe, comprising the following steps: Step S10: 2,5-Dimethoxytetraphenylbenzene, 1,3,5-tris(4-aminophenyl)benzene, solvent, regulator and catalyst are mixed and reacted to obtain a COFs solution; Step S20: Mix the COFs solution with a first noble metal salt solution, a first reducing agent and a stabilizer to obtain a COFs nanoparticle solution loaded with the first noble metal; Step S30: The COFs nanoparticle solution loaded with the first noble metal is mixed with a surfactant and a second reducing agent, and then mixed with a second noble metal salt solution to obtain a multifunctional nanozyme probe.

[0034] In this embodiment, the preparation method synthesizes COF nanostars with high specific surface area, tunable pore structure, and excellent stability as a carrier substrate. A first noble metal nanoparticle is uniformly grown on the COF surface via in-situ reduction to form a composite material, which exhibits stronger Raman enhancement properties. Further controllable deposition of a second noble metal nanoparticle forms a ternary composite nanomaterial, providing a direct colorimetric signal and excellent peroxidase-like activity. Due to the high catalytic activity of this nanozyme probe, the detection capability of the test strip is improved by catalyzing the substrate colorimetric reaction at the T line.

[0035] Specifically, after modifying the surface of the nanozyme probe with specific antibodies (Ab1) of CFP-10 and p24 proteins to form a tag probe, the probe is enriched by centrifugation and resuspended in 500 μL of running buffer (0.01 M PBS solution containing 5% sucrose, 0.5% Tween 20, and 10% fetal bovine serum). Then, it is co-incubated with the target proteins CFP-10 and p24 and dropped onto the sample pad of the test strip. Through capillary action, the probe flows to both sides of the test strip. When the corresponding target proteins are present, the corresponding specific antibodies are captured and enriched on the T lines (Testlines) on both sides of the bidirectional test strip, thus presenting a black band for rapid colorimetric interpretation. Excess probes are captured by the secondary antibody on the C line (Control line) as a quality control evaluation of the test strip.

[0036] Because the nanozyme probe possesses high catalytic activity, it enhances the detection capability of the test strip by performing an enzyme-catalyzed substrate colorimetric reaction on the T line. Specifically, when the target analyte is present, the CFP-10 and p24 proteins labeled by the nanozyme probe are enriched on their respective T lines. By adding a pH 4.0 NaAC-HAC buffer and a mixed solution of 3-amino-9-ethylcarzole / hydrogen peroxide (AEC / H2O2) to the sample pad of the test strip, the nanozyme probe enriched on the T line can efficiently catalyze the oxidation of AEC by H2O2 to form a stable brown-red precipitate, thereby amplifying the colorimetric signal through enzyme catalysis and further improving the colorimetric sensitivity.

[0037] Simultaneously, based on the excellent SERS performance of the nanozyme probe, quantitative detection of the T-line Raman signal is possible. Specifically, when the target analyte is present, the CFP-10 and p24 proteins labeled by the nanozyme probe are enriched on the corresponding T-line. The Raman laser is then aligned with the T-line, and the Raman signal of the COFs is measured at 1575 cm⁻¹. -1 By analyzing the characteristic peak signals at specific locations, a linear relationship was established between SERS intensity and the concentrations of CFP-10 and p24 proteins, thereby achieving more sensitive qualitative and quantitative detection of SERS and effectively compensating for the shortcomings of visual colorimetry in terms of sensitivity and precise quantification. Therefore, the detection platform constructed using the nanozyme probes prepared by the above method integrates three detection modes: colorimetry, catalytic colorimetry, and SERS. It possesses outstanding advantages such as speed, high sensitivity, wide detection range, and cross-validation of results, making it suitable for high-performance rapid screening and quantitative analysis of TB-HIV co-infection.

[0038] In some embodiments, the concentration of 2,5-dimethoxy-terephthalaldehyde is 25 mmol / L-35 mmol / L; the concentration of 1,3,5-tris(4-aminophenyl)benzene is 35 mmol / L-50 mmol / L; and the volume ratio of 2,5-dimethoxy-terephthalaldehyde to 1,3,5-tris(4-aminophenyl)benzene is (4-6):(5-7). By controlling the concentration and volume ratio of 2,5-dimethoxy-terephthalaldehyde to 1,3,5-tris(4-aminophenyl)benzene within the above ranges, COF nanostars with high specific surface area, tunable pore structure, and excellent stability can be prepared at room temperature.

[0039] In a preferred embodiment, 250 µL of 30 mmol / L 2,5-dimethoxytetraphenylbenzene (DMTP), 300 µL of 40 mmol / L 1,3,5-tris(4-aminophenyl)benzene (TAPB), 1 mL of acetonitrile, and 3 mL of methanol (modifier) ​​were ultrasonically mixed, followed by the addition of 2 mL of ultrapure water and 500 µL of acetic acid (catalyst). The mixture was thoroughly shaken and reacted at room temperature for 12 h to obtain a yellow product. The product was washed twice by centrifugation with a 1:2 mixture of ethanol and acetone, then washed once by centrifugation with ultrapure water, and finally resuspended in 1 mL of ultrapure water and stored at 4°C for later use.

[0040] In some embodiments, the concentration of the COFs solution is 25 mmol / L-35 mmol / L; the concentration of the first noble metal salt solution is 7 mmol / L-14 mmol / L; and the volume ratio of the COFs solution to the first noble metal salt solution is (1-25):1. By controlling the concentration and volume ratio of the COFs solution and the first noble metal salt solution within the above ranges, in-situ growth of the first noble metal nanoparticles on the COFs surface can be achieved at room temperature, resulting in COFs nanoparticles loaded with the first noble metal.

[0041] In some embodiments, the first noble metal salt solution includes, but is not limited to, one or more salt solutions corresponding to Au, Ag, Pt, Pd, Ru, and Cu. Preferably, the first noble metal salt solution is a chloroauric acid (HAuCl4) solution.

[0042] In some embodiments, in step S20, the first reducing agent is, but is not limited to, one or more of sodium citrate and sodium borohydride; the stabilizer is, but is not limited to, sodium citrate.

[0043] In some embodiments, the concentration of the COF nanoparticle solution loaded with the first noble metal is 25 mmol / L-35 mmol / L; the concentration of the second noble metal salt solution is 40 mmol / L-60 mmol / L; and the volume ratio of the COF nanoparticle solution loaded with the first noble metal to the second noble metal salt solution is (0.75-6):1. By controlling the concentration and volume ratio of the COF nanoparticle solution loaded with the first noble metal and the second noble metal salt solution within the above range, the deposition of the second noble metal nanoparticles on the surface of the COF nanoparticles loaded with the first noble metal can be achieved at room temperature, thus obtaining a nanozyme probe.

[0044] In some embodiments, the second noble metal salt includes, but is not limited to, one or more salt solutions corresponding to Pt, Pd, Au, Ag, Ir, Ru, and Rh. Preferably, the second noble metal salt is a chloroplatinic acid (H₂PtCl₆) solution.

[0045] In some embodiments, in step S30, the surfactant is, but is not limited to, Tween 60; and the second reducing agent is, but is not limited to, ascorbic acid.

[0046] In addition, the present invention also provides an application of a multifunctional nanozyme probe in a two-way rapid test strip platform for tuberculosis-HIV comorbidity.

[0047] In this embodiment, the TB-HIV bidirectional rapid test strip platform developed based on the nanozyme probe supports three-mode output to solve the problem that existing TB and HIV tests must be performed independently and cannot be integrated for rapid screening. It overcomes the shortcomings of traditional single colorimetric probes such as colloidal gold, which have low sensitivity, are prone to missing low-concentration targets, and cannot accurately quantify. This TB-HIV bidirectional rapid test strip platform breaks through the contradiction between the single signal output mode of POCT platforms and the inability to balance screening convenience and result accuracy.

[0048] Specifically, this TB-HIV bidirectional rapid test strip platform can determine the presence of CFP-10 or p24 protein simply by visually observing the color development of the T-line. Furthermore, due to the surface-enhanced Raman spectroscopy (SERS) properties of the nanomaterials, quantitative detection of CFP-10 and p24 proteins can be achieved by acquiring Raman signals from the T-line and analyzing the intensity of the Raman signal. Since Raman detection is a highly sensitive method, it can compensate for the insufficient sensitivity of visual colorimetry. Similarly, because the nanozyme probe has excellent peroxidase-like activity, the colorimetric sensitivity can be further improved by catalyzing the substrate colorimetric reaction at the T-line, thus enhancing the test strip's detection capability. Ultimately, this TB-HIV bidirectional rapid test strip platform integrates multiple modes of detection—visual colorimetry, catalytic colorimetry, and Raman detection—offering significant advantages in speed and sensitivity.

[0049] In some embodiments, the TB-HIV bidirectional rapid test strip platform includes a test strip and a reaction system; the test strip includes a base plate, a first nitrocellulose membrane and a second nitrocellulose membrane located on one side of the base plate and spaced apart, and a sample pad located between the first nitrocellulose membrane and the second nitrocellulose membrane; the first nitrocellulose membrane is provided with a first control line and a first detection line, and the second nitrocellulose membrane is provided with a second control line and a second detection line; the first detection line and the second detection line are positioned close to the sample pad relative to the first control line and the second control line; the first detection line is loaded with anti-CFP-10 protein-specific monoclonal antibody Ab2, and the second detection line is loaded with anti-p24 protein-specific monoclonal antibody Ab2; the reaction system includes a nanozyme probe adsorbed with CFP-10 protein polyclonal antibody Ab1 and anti-p24 protein monoclonal antibody Ab1.

[0050] In some embodiments, Au nanoparticles are used as the first noble metal nanoparticles and Pt nanoparticles as the second noble metal nanoparticles to construct a COFs@Au@Pt nanomaterial integrating colorimetry, catalytic color development, and SERS. Specific antibodies (Ab1) for CFP-10 and p24 proteins are modified on the surface of this material to form tag probes. The specific detection of CFP-10 and p24 is achieved through the specific recognition of these proteins by the specific detection antibodies on the probes and the specific capture antibodies coated on the NC membrane. Therefore, the presence of CFP-10 or p24 protein can be determined simply by visually observing the color development of the T line on the test strip. Furthermore, the color development sensitivity can be further improved by using an enzyme-catalyzed color development reaction, thereby enhancing the detection capability of the test strip.

[0051] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0052] Example 1 This embodiment provides a colorimetric-catalytic-SERS multifunctional composite nanozyme probe COFs@Au@Pt NPs, the preparation steps and characterization of which are as follows: 1) In a 10 mL brown sample vial, add 250 μL of 30 mmol / L 2,5-dimethoxytetraphenylaldehyde (DMTP), 300 μL of 40 mmol / L 1,3,5-tris(4-aminophenyl)benzene (TAPB), 1 mL of acetonitrile, and 3 mL of methanol. After ultrasonic mixing, add 2 mL of ultrapure water and 500 μL of acetic acid. Shake the mixture thoroughly and react at room temperature for 12 h to obtain a yellow product. Wash twice with a 1:2 mixture of ethanol and acetone, then wash once with ultrapure water. Finally, resuspend in 1 mL of ultrapure water to obtain a COFs solution, which is stored at 4 °C for later use.

[0053] 2) Add 10 mL of ultrapure water to a 25 mL round-bottom flask, add 200 μL of the prepared COFs solution, sonicate for 10 min, place on a magnetic stirrer, add 10 mM HAuCl4 and 80 μL of 10 mM sodium citrate at room temperature, stir for 10 min, then quickly add 100 μL of 1 mM sodium borohydride, stir for 30 min, finally obtaining a dark red product. Wash twice with ultrapure water by centrifugation, and finally resuspend in 1 mL of ultrapure water to obtain the COFs@Au NPs solution, which is stored at 4℃ for later use.

[0054] 3) Add 2 mL of ultrapure water to a 25 mL round-bottom flask, add 300 μL of the prepared COFs@Au NPs solution, sonicate for 10 min, place on a magnetic stirrer, add 30 μL of Tween 60 at room temperature, sonicate for 1 min to dissolve, add 50 μL of 10 mM ascorbic acid, stir for 2 min, add 50 mM H2PtCl6, stir for 30 min, finally obtaining a black product. Wash twice with ultrapure water by centrifugation, and finally resuspend in 1 mL of ultrapure water to obtain the nanozyme probe COFs@Au@Pt NPs, which is stored at 4℃ for later use.

[0055] A series of COFs@Au NPs with different numbers of Au NPs were prepared by controlling the amount of HAuCl4 introduced during the preparation process; then, a series of COFs@Au@Pt NPs with different numbers of Pt NPs were synthesized by controlling the amount of H2PtCl6 introduced during the preparation process, so that the materials have both good morphology and Raman enhancement ability. According to the above method, the volumes of HAuCl4 added in step (2) were 8 μL, 20 μL, 50 μL, 100 μL, 150 μL, and 200 μL, respectively. The volumes of H2PtCl6 added in step (3) were 50 μL, 100 μL, 150 μL, 200 μL, 300 μL, and 400 μL, respectively.

[0056] The morphology of the COFs@Au@Pt NPs prepared above was characterized. The transmission electron microscopy (TEM) image of the COFs@Au@Pt NPs prepared with 50 μL of HAuCl4 and 200 μL of H2PtCl6 is shown below. Figure 1 As shown, the prepared COFs@Au@Pt NPs are composite star-shaped nanomaterials with a core of about 400 nm. The core is a star-shaped COFs, and the outer surface layer is uniformly adhered with Au@Pt, which has good dispersibility. The COFs@Au@Pt nanomaterials were successfully prepared.

[0057] Example 2 The three-mode performance of the COFs@Au@Pt NPs prepared in Example 1 was verified as follows: First, the classic enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB) was used as the chromogenic substrate, and the catalytic performance of COFs@Au@Pt NPs was investigated using UV absorption spectroscopy and enzyme reaction kinetics. A COFs@Au@Pt NPs solution prepared by adding 5 μL of HAuCl4 to 50 μL of H2PtCl6 was added to a mixed solution containing 500 μL of NaAC-HAC buffer (pH=4.0), 300 μL of 5M H2O2, and 300 μL of 5 mM TMB. The reaction was carried out at room temperature for 10 min, and then the UV absorption was measured.

[0058] Next, the SERS performance of the nanomaterials was verified: 2.5 μL of a COFs@Au@Pt NPs solution prepared by adding 50 μL of HAuCl4 and 200 μL of H2PtCl6 was dropped onto a glass slide. A micro Raman spectrometer was used with a 633 nm laser, a 50x telephoto objective lens, a collection time of 2 s, and one integration to collect and detect the Raman spectra.

[0059] The catalytic color development and absorption spectra of each solution are shown below. Figure 2 As shown, under the conditions of NaAC-HAC buffer at pH 4.0, with a TMB concentration of 5 mM and an H2O2 concentration of 5 M, COFs@Au@Pt NPs can catalytically oxidize TMB to oxTMB in the presence of H2O2, causing the solution to change from colorless to blue. A characteristic absorption peak at 650 nm can also be observed in the UV-Vis absorption spectrum. Without any one or two of COFs@Au@Pt NPs, H2O2, and TMB, the solution cannot be observed to turn blue, and the characteristic peak of oxTMB is not visible in the corresponding UV spectrum. These results indicate that COFs@Au@Pt NPs possesses excellent peroxidase-like catalytic activity.

[0060] Further investigation was conducted on the SERS performance of COFs@Au@Pt NPs. By introducing Au NPs, the Raman signal of the COFs themselves was enhanced. A comparison of the SERS performance of different Au NPs is shown in the figure below. Figure 3 As shown. Further introduction of Pt NPs reveals that excessive Pt layer growth may mask the plasmonic properties of the Au layer, thus affecting SERS signal amplification. The SERS signal intensity initially increases and then decreases with increasing Pt layer thickness, indicating that the Pt layer negatively impacts the SERS performance of multilayer tags. Figure 4 As shown. Considering the SERS / catalytic performance of COFs@Au@Pt NPs, subsequent experiments used 200 μL of Pt NPs to ensure that the prepared COFs@Au@Pt NPs exhibited the best SERS / catalytic signal.

[0061] Example 3 This embodiment provides a SERS nanotag conjugated with a two-way side-flow chromatography LFIA and an antibody, which is specifically prepared as follows: like Figure 5As shown, the two-dimensional flow chromatography test strip consists of four parts: a sample pad, an NC membrane, an absorbent pad, and a PVC base plate. First, the two sections of the NC membrane are adhered to the middle of both sides of the PVC base plate. After equilibration at 37°C for a period of time, a 1 mg / mL anti-CFP-10 protein-specific monoclonal antibody Ab2 and a 1 mg / mL goat anti-rabbit secondary antibody are sputtered at a rate of 1 µL / cm onto the left-side NC membrane to form the T and C lines, respectively. Similarly, a 0.8 mg / mL anti-p24 protein-specific monoclonal antibody Ab2 and a 1 mg / mL goat anti-mouse secondary antibody are sputtered at a rate of 1 µL / cm onto the right-side NC membrane to form the T and C lines, respectively. Next, the sample pad is fixed in the middle of the PVC base plate, and the absorbent pads are fixed at the two ends of the PVC base plate. Finally, the parts overlap, allowing the sample solution, once dropped onto the sample pad, to flow smoothly and continuously along each part under capillary action. Finally, cut the assembled test strips into 4 mm wide pieces, insert them into custom-made cartridges, and seal them with a desiccant for later use.

[0062] Anti-CFP-10 protein polyclonal antibody Ab1 and anti-p24 protein monoclonal antibody Ab1 were directly attached to the surface of COFs@Au@Pt NPs (prepared by adding 50 μL of HAuCl4 and 200 μL of H2PtCl6) through adsorption. First, adjust the pH of the COFs@Au@Pt NPs by adding an appropriate amount of 0.2 M K2CO3 solution. Then, mix the anti-CFP-10 protein detection antibody Ab1 (4 μL, 1.5 mg / mL) and the anti-p24 protein detection antibody Ab1 (3 μL, 1 mg / mL) with the COFs@Au@Pt NPs and react gently with the mixture at 25℃ and 220 rpm for 1 h. Next, add 20 μL of 20% BSA for blocking and react for 1 h. Centrifuge at 8000 rpm for 5 min and add 500 μL of running buffer (0.01 M PBS solution containing 5% sucrose, 0.5% Tween 20, and 10% FBS). Then, store the mixture at 4℃ protected from light for later use. Take 20 μL of the tag probe resuspended in the running buffer, mix it with 20 μL of 100 ng / mL CFP-10 protein and p24 protein, and add it to the sample pad. Observe the color development of the test strip after 15 min of chromatography.

[0063] See color development details Figure 6 After chromatography, the T line of the test strip containing the target protein solution appears clearly visible to the naked eye.

[0064] Example 4 This embodiment utilizes COFs@Au@Pt NPs combined with two-way side-flow chromatography (LFIA) for ultrasensitive and rapid detection of TB-HIV, as detailed below: By mixing 20 μL of running buffer (0.01 M PBS solution containing 5% sucrose, 0.5% Tween 20, and 10% fetal bovine serum) with 20 μL of the sample solution to be tested, when the target analyte CFP-10 / p24 protein is present in the sample solution, due to the specific recognition of the antigen and antibody, the tag and CFP-10 / p24 complex are first formed. This complex is then further added to the sample pad of LFIA for reaction. It can be seen that the presence of high concentrations of CFP-10 / p24 can be directly determined by visually observing the color change on the T line.

[0065] For low concentrations of target analytes, visual colorimetric observation is not sensitive enough, making it difficult to apply to the detection of trace analytes and unable to achieve quantitative detection. A catalytic colorimetric mode is used for result interpretation. A mixture of 0.5 mL pH 4.0 NaAC-HAC buffer, 300 μL 50 mM AEC, and 300 μL 5 M H2O2 is added to the sample pad and allowed to precipitate upwards. To further improve detection sensitivity, Raman detection mode is employed, utilizing the SERS activity of the nanoprobe. A 633 nm laser and a 50x telephoto objective lens are used, with a acquisition time of 2 s and one integration.

[0066] The results of a series of concentration gradients of p24 test strips, the catalytic colorimetric results, and the corresponding Raman spectra collected on the T line are shown in the following figures. Figure 7 As shown, the detection limit for p24 protein visualization is approximately 1 ng / mL, and the detection limit for catalytic colorimetric visualization is approximately 100 pg / mL, indicating further improved sensitivity. The sensitivity using SERS detection mode is further enhanced, with a detection limit of approximately 10 pg / mL. A series of concentration gradient CFP-10 test strip detection results, catalytic colorimetric results, and corresponding Raman spectra acquired on the T line are shown below. Figure 8 As shown, the limit of detection for CFP-10 protein visualization is approximately 500 pg / mL, the limit of detection for CFP-10 protein catalytic colorimetric visualization is approximately 50 pg / mL, and the limit of detection for CFP-10 protein SERS is approximately 5 pg / mL.

[0067] After testing, the multi-mode LFIA (colorimetric / catalytic colorimetric / surface-enhanced Raman scattering, CM / CL / SERS) has excellent signal output capability for the detection of TB-HIV, with good reproducibility and high specificity, and has good application prospects.

[0068] In summary, this invention provides a method for preparing a multifunctional nanozyme probe and its application in tuberculosis-AIDS comorbidity. The multifunctional nanozyme probe comprises a covalent organic framework (COF) material as a carrier substrate, and first and second noble metal nanoparticles loaded on the surface of the carrier substrate. The first noble metal nanoparticles enhance the Raman signal of the carrier substrate, and the second noble metal nanoparticles provide enzyme activity. The nanozyme probe provided by this invention uses a covalent organic framework (COF) material as a substrate and forms a composite nanomaterial by loading first and second noble metal nanoparticles. This material possesses excellent colorimetric response performance, peroxidase-like activity, and strong SERS (Surface-Enhanced Raman Spectroscopy) performance, and can serve as a multifunctional signal probe. Furthermore, applying this nanozyme probe to two-way lateral flow immunoassay (LFIA) can avoid potential interference between multiple targets. By modifying it with specific antibodies against tuberculosis (TB) and HIV biomarkers, simultaneous and bidirectional detection of TB and HIV biomarkers can be achieved. At the same time, the test strip supports three detection modes: visual colorimetry, catalytic amplification colorimetry, and SERS precise quantification, which significantly improves the sensitivity, dynamic range, and reliability of the detection. It is especially suitable for early screening, efficacy monitoring, and epidemic prevention and control of TB-HIV co-infection in resource-limited areas.

[0069] Specifically, this invention synthesizes star-shaped COFs@Au@PtNPs with plasma properties and excellent catalytic activity, which are assembled with bidirectional side-flow chromatography LFIA to form a multi-mode sensing platform, enabling highly specific, accurate, sensitive, rapid, and economical multi-modal detection of TB-HIV co-infection, and providing new ideas for the diagnosis of TB-HIV co-infection diseases.

[0070] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A multifunctional nanozyme probe, characterized in that, The invention includes a covalent organic framework material (COF) as a carrier substrate, and a first noble metal nanoparticle and a second noble metal nanoparticle loaded on the surface of the carrier substrate; the first noble metal nanoparticle is used to enhance the Raman signal of the carrier substrate; and the second noble metal nanoparticle is used to provide enzyme activity.

2. The multifunctional nanozyme probe according to claim 1, characterized in that, The first noble metal nanoparticles include one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles; the second noble metal nanoparticles include one or more of Pt nanoparticles, Pd nanoparticles, Au nanoparticles, Ag nanoparticles, Ir nanoparticles, Ru nanoparticles, and Rh nanoparticles.

3. The multifunctional nanozyme probe according to claim 1, characterized in that, The multifunctional nanozyme probe is also adsorbed with specific antibodies corresponding to CFP-10 and p24 proteins.

4. The multifunctional nanozyme probe according to claim 1, characterized in that, The nanozyme probe has a particle size of 350 nm-450 nm.

5. A method for preparing a multifunctional nanozyme probe as described in any one of claims 1-4, characterized in that, Including the following steps: 2,5-Dimethoxytetraphenylbenzene, 1,3,5-tris(4-aminophenyl)benzene, solvent, regulator and catalyst are mixed and reacted to obtain a COFs solution; The COFs solution is mixed with a first noble metal salt solution, a first reducing agent and a stabilizer to obtain a COFs nanoparticle solution loaded with the first noble metal. The COF nanoparticle solution loaded with the first noble metal is mixed with a surfactant and a second reducing agent, and then mixed with a second noble metal salt solution to obtain a multifunctional nanozyme probe.

6. The method for preparing the multifunctional nanozyme probe according to claim 5, characterized in that, The concentration of 2,5-dimethoxy-terephthalaldehyde is 25 mmol / L-35 mmol / L; the concentration of 1,3,5-tris(4-aminophenyl)benzene is 35 mmol / L-50 mmol / L; and the volume ratio of 2,5-dimethoxy-terephthalaldehyde to 1,3,5-tris(4-aminophenyl)benzene is (4-6):(5-7).

7. The method for preparing the multifunctional nanozyme probe according to claim 5, characterized in that, The concentration of the COFs solution is 25 mmol / L-35 mmol / L; the concentration of the first noble metal salt solution is 7 mmol / L-14 mmol / L; and the volume ratio of the COFs solution to the first noble metal salt solution is (1-25):

1.

8. The method for preparing the multifunctional nanozyme probe according to claim 5, characterized in that, The concentration of the COF nanoparticle solution loaded with the first noble metal is 25 mmol / L-35 mmol / L; the concentration of the second noble metal salt solution is 40 mmol / L-60 mmol / L; and the volume ratio of the COF nanoparticle solution loaded with the first noble metal to the second noble metal salt solution is (0.75-6):

1.

9. The application of a multifunctional nanozyme probe as described in any one of claims 1-4 in a two-way rapid test strip platform for tuberculosis and HIV comorbidities.

10. The application according to claim 9, characterized in that, The tuberculosis and HIV comorbidity two-way rapid test strip platform includes a test strip and a reaction system. The test strip includes a base plate, a first nitrocellulose membrane and a second nitrocellulose membrane located on one side of the base plate and spaced apart, and a sample pad located between the first nitrocellulose membrane and the second nitrocellulose membrane. The first nitrocellulose membrane has a first control line and a first detection line, and the second nitrocellulose membrane has a second control line and a second detection line. The first detection line and the second detection line are positioned close to the sample pad relative to the first control line and the second control line. The first detection line is loaded with anti-CFP-10 protein-specific monoclonal antibody Ab2, and the second detection line is loaded with anti-p24 protein-specific monoclonal antibody Ab2. The reaction system includes a nanozyme probe adsorbed with CFP-10 protein polyclonal antibody Ab1 and anti-p24 protein monoclonal antibody Ab1.

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