A method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection.

Abstract

This invention relates to the field of nanobiosensing technology, and particularly to a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection. The dual-mode test strip includes a multifunctional nanozyme probe, an anti-CFP-10 polyclonal detection antibody coupled to the surface of the nanozyme probe, and a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody embedded in the T and C lines. The nanozyme probe includes a flower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier. The nanozyme probe is coupled with the anti-CFP-10 polyclonal detection antibody Ab1 to form a SERS signal tag probe. This probe specifically binds to the capture antibody Ab2, recognizing the CFP-10 antigen and achieving specific, rapid, and sensitive detection of tuberculosis. Qualitative detection of CFP-10 protein can be achieved by visually interpreting the color development of the test lines. Utilizing the SERS performance of the multifunctional nanozyme probe, Raman signals are acquired from the test lines, and accurate quantitative detection can be achieved based on the correlation between signal intensity and CFP-10 concentration.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensing technology, and in particular to a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). It mainly affects the lungs (accounting for more than 85%), but can also invade other organs such as lymph nodes, bones, kidneys, and meninges, leading to extrapulmonary tuberculosis.

[0003] Currently, common methods for detecting tuberculosis include sputum smear microscopy, tuberculin skin test, nucleic acid amplification technology, and enzyme-linked immunosorbent assay (ELISA), but all have significant limitations: sputum smear microscopy has low sensitivity and is prone to missed diagnoses; the tuberculin skin test has poor specificity and is easily affected by BCG vaccination, resulting in false positives; nucleic acid amplification technology is complex to operate, relies on specialized instruments and personnel, and has high testing costs, making it difficult to popularize in primary healthcare institutions; and ELISA has a long testing cycle and cannot achieve rapid on-site testing.

[0004] In serological testing, Culture Filtrate Protein 10 (CFP-10), a low-molecular-weight protein secreted by Mycobacterium tuberculosis, can form a heterodimer with Early Secretory Target Antigen Protein 6 (ESAT-6), exhibiting good immunogenicity and stimulating a specific immune response. CFP-10 is encoded by the RD1 region gene Rv3874 and is specific to a few pathogenic mycobacteria, including Mycobacterium tuberculosis, but is absent in BCG and other non-pathogenic mycobacteria. Therefore, it can serve as a specific molecular target for tuberculosis diagnosis. After Mtb infection, antigenic proteins such as CFP-10 are secreted early, and direct detection of these proteins is of great value for early tuberculosis diagnosis. Furthermore, direct detection of the CFP-10 antigen provides direct evidence of Mtb infection, effectively avoiding false-negative results in immunological tests (such as the interferon-gamma release assay) caused by immune dysfunction or defects.

[0005] Lateral flow immunoassay (LFIA) strips are widely used for rapid on-site detection of various biomarkers due to their advantages such as ease of use, rapid detection, low cost, and no need for specialized instruments. However, traditional colloidal gold strips have low sensitivity and cannot meet the detection needs of low abundance CFP-10 in the early stages of tuberculosis.

[0006] In recent years, SERS technology has become a research hotspot in the field of biosensing due to its ultra-high sensitivity at the single-molecule level and its unique molecular "fingerprint" spectral characteristics. However, SERS-LFIA with single noble metal nanostructures suffers from problems such as limited stability, uniformity, selectivity, and enhancement effect, susceptibility to matrix interference, and susceptibility to oxidative denaturation.

[0007] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection, aiming to solve the problems of poor stability, uniformity, selectivity and enhancement effect of existing test strips.

[0009] The technical solution of the present invention is as follows: A colorimetric-SERS dual-mode test strip includes a multifunctional nanozyme probe and an anti-CFP-10 polyclonal detection antibody coupled to the surface of the multifunctional nanozyme probe, as well as a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody embedded in the detection line and control line, respectively; the multifunctional nanozyme probe includes a nanoflower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier.

[0010] The colorimetric-SERS dual-mode test strip, wherein the noble metal nanoparticles include one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles.

[0011] The colorimetric-SERS dual-mode test strip, wherein the multifunctional nanozyme probe has a particle size of 300 nm-480 nm.

[0012] The colorimetric-SERS dual-mode test strip further includes a base plate, a nitrocellulose membrane located on one side of the base plate, and a sample pad and an absorbent pad located at both ends of the nitrocellulose membrane; the detection line and the control line are located on the nitrocellulose membrane, with the detection line positioned closer to the sample pad and the control line positioned closer to the absorbent pad.

[0013] A method for preparing a colorimetric-SERS dual-mode test strip, comprising 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 was mixed with a noble metal salt solution, sodium citrate, and sodium borohydride solution, and after reaction, a multifunctional nanozyme probe solution was obtained. After adjusting the pH of the multifunctional nanozyme probe solution, anti-CFP-10 polyclonal detection antibody was added. After the reaction, bovine serum albumin solution was added for blocking to obtain a SERS signal tag probe coupled with anti-CFP-10 polyclonal detection antibody. Using CFP-10 monoclonal capture antibody and goat anti-rabbit secondary antibody as the detection line and control line, a colorimetric-SERS dual-mode test strip is formed with the SERS signal tag probe conjugated with anti-CFP-10 polyclonal detection antibody.

[0014] The method for preparing the colorimetric-SERS dual-mode test strip, wherein the concentration of 2,5-dimethoxy-terephthalaldehyde is 25 mmol / L-35 mmol / L; and the concentration of 1,3,5-tris(4-aminophenyl)benzene is 35 mmol / L-50 mmol / L. Preferably, the volume ratio of 2,5-dimethoxytetraphenylbenzene to 1,3,5-tris(4-aminophenyl)benzene is (4-6):(5-7).

[0015] The method for preparing the colorimetric-SERS dual-mode test strip, wherein the concentration of the COFs solution is 25 mmol / L-35 mmol / L; and the concentration of the noble metal salt solution is 4 mmol / L-15 mmol / L; Preferably, the volume ratio of the COFs solution to the noble metal salt solution is 25:(1-25).

[0016] The method for preparing the colorimetric-SERS dual-mode test strip, wherein the pH is between 6.5 and 8.0.

[0017] The method for preparing the colorimetric-SERS dual-mode test strip, wherein the concentration of the multifunctional nanozyme probe solution is 0.5 mg / mL-2 mg / mL; and the concentration of the anti-CFP-10 polyclonal detection antibody is 1.0 mg / mL-1.5 mg / mL; The volume ratio of the multifunctional nanozyme probe solution to the anti-CFP-10 polyclonal detection antibody is 1:(1-3).

[0018] Application of a colorimetric-SERS dual-mode test strip in rapid tuberculosis detection.

[0019] Beneficial Effects: This invention provides a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection. The colorimetric-SERS dual-mode test strip includes a multifunctional nanozyme probe and an anti-CFP-10 polyclonal detection antibody coupled to the surface of the multifunctional nanozyme probe, as well as a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody embedded in the detection line and control line, respectively. The multifunctional nanozyme probe includes a nanoflower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier. This invention utilizes the multifunctional nanozyme probe coupled with the anti-CFP-10 polyclonal detection antibody Ab1 to form a signal tag probe. This probe specifically binds to the capture antibody Ab2 immobilized on the nitrocellulose membrane (NC membrane) of the test strip, recognizing the CFP-10 antigen, and achieving specific, rapid, and sensitive detection of tuberculosis. Furthermore, qualitative detection of CFP-10 protein can be achieved by visually interpreting the color development of the test line on the test strip. Simultaneously, the SERS performance of the multifunctional nanozyme probe can be utilized to acquire Raman signals from the test line, and precise quantitative detection can be achieved based on the correlation between signal intensity and CFP-10 concentration. Therefore, this colorimetric-SERS dual-mode test strip integrates both visual colorimetric and SERS detection modes, offering outstanding advantages in speed, accuracy, and sensitivity. Moreover, the use of nanoflower-like COFs as a carrier ensures that the multifunctional nanozyme probe exhibits high stability, uniformity, selectivity, and enhanced performance. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the process flow for preparing a colorimetric-SERS dual-mode test strip according to the present invention; Figure 2 TEM images of COFs@Au NF with different Au loadings in Example 1; Figure 3 A graph showing the SERS performance comparison data for different Au loads; Figure 4 The color development results of the T line after different chromatography times for the test strip containing the target protein solution; Figure 5 The colorimetric detection results of test strips for different concentrations of CFP-10 protein and the corresponding SERS spectra of the T line; Figure 6 The results show the specificity of the test strip for CFP-10 and the corresponding SERS spectrum of the T line. Detailed Implementation

[0021] This invention provides a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection. 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.

[0022] 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.

[0023] Lateral Flow Immunoassay (LFIA) test strips are based on the principles of antigen-antibody specific binding, colloidal gold color development, and capillary chromatography. Using a nitrocellulose membrane as the reaction carrier, the chromatographic and immunoassay processes are achieved through the synergistic action of the sample pad, NC membrane, and absorbent pad. When the target analyte is present, the metal particles enriched on the detection line develop color, allowing for qualitative / semi-quantitative interpretation by visual inspection within minutes. However, traditional colloidal gold test strips have low sensitivity, making them unsuitable for detecting low-abundance CFP-10 in the early stages of tuberculosis.

[0024] The enhancement mechanism of SERS mainly relies on the local surface plasmon resonance effect generated by noble metal nanostructures such as gold and silver under specific wavelength light excitation: the collective oscillation of free electrons on the metal surface forms a locally enhanced electromagnetic field, which can increase the electromagnetic field strength by 10 on the nanoscale. 6 -10 14 Simultaneously, the chemical enhancement effect caused by charge transfer between molecules and the metal surface can further amplify the Raman scattering signal of adsorbed molecules, enabling precise quantitative detection of trace substances. However, SERS-LFIA with a single noble metal nanostructure suffers from limitations in stability, uniformity, selectivity, and enhancement effect, and is susceptible to matrix interference and oxidative degradation.

[0025] Based on this, the present invention provides a colorimetric-SERS dual-mode test strip, comprising a multifunctional nanozyme probe and an anti-CFP-10 polyclonal detection antibody coupled to the surface of the multifunctional nanozyme probe, as well as a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody respectively embedded in the detection line and the control line; the multifunctional nanozyme probe comprises a nanoflower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier.

[0026] In this embodiment, the multifunctional nanozyme probe is coupled with anti-CFP-10 polyclonal detection antibody Ab1 to form a signal tag probe. This probe specifically binds to capture antibody Ab2 immobilized on the nitrocellulose membrane (NC membrane) of the test strip, recognizing the CFP-10 antigen and achieving specific, rapid, and sensitive detection of tuberculosis. Furthermore, the qualitative detection of CFP-10 protein can be achieved by visually interpreting the color development of the test line (T line). Simultaneously, the SERS performance of the multifunctional nanozyme probe can be further utilized to acquire Raman signals from the test line. Based on the correlation between signal intensity and CFP-10 concentration, accurate quantitative detection can be achieved. Therefore, this colorimetric-SERS dual-mode test strip has the advantage of integrating both visual colorimetric and SERS detection modes, possessing outstanding advantages of speed, accuracy, and sensitivity. Moreover, the use of nanoflower-like COFs as a carrier gives the nanozyme probe high stability, uniformity, selectivity, and good enhancement effect.

[0027] Specifically, covalent organic frameworks (COFs) are a class of porous crystalline materials formed by organic monomers linked by covalent chains. They possess characteristics such as large specific surface area, abundant pore structure, tunable structure, and diverse functional groups. Their three-dimensional network structure can effectively load nanoparticles and prevent their aggregation. Flower-like COFs carriers have a larger specific surface area and more active sites. As a loading substrate for noble metal nanoparticles, they can significantly improve the dispersibility of noble metal nanoparticles, thereby enhancing the enhancement effect and stability of SERS signals. At the same time, the noble metal nanoparticles have good plasmon resonance effect, enabling visual detection of colorimetric signals. Furthermore, by coupling an anti-CFP-10 polyclonal detection antibody to the surface of a nanoprobe composed of noble metal nanoparticles and COFs carriers to form an SERS signal tag probe, and applying it to a test strip, a colorimetric-SERS dual-mode detection system can be constructed. This system can achieve dual detection of "visual qualitative analysis + instrument quantitative analysis," balancing the speed and accuracy of detection and addressing the pain points of traditional tuberculosis detection methods.

[0028] In this embodiment, the multifunctional nanozyme probe has the following advantages compared with the prior art: 1) Stable structure and good signal reproducibility: The flower-shaped COFs carrier has a porous structure, which can effectively prevent the aggregation of noble metal nanoparticles. The resulting nanoprobe structure is stable and has excellent dispersibility, ensuring the consistency and reliability of the detection signal and significantly improving the batch reproducibility of the test strip, making it suitable for clinical batch testing applications.

[0029] 2) Dual-mode detection, combining qualitative and quantitative analysis: SERS signal tag probes coupled with anti-CFP-10 polyclonal detection antibodies are applied to tuberculosis detection to construct colorimetric-SERS dual-mode test strips. This solves the technical pain points of low sensitivity or poor stability of traditional single-signal test strips, and realizes the integration of "on-site screening + precise quantification".

[0030] 3) High target specificity, avoiding false results: The SERS signal tag probe coupled with anti-CFP-10 polyclonal detection antibody targets the tuberculosis-specific marker CFP-10. Combined with the specific recognition function of CFP-10 monoclonal antibody, it only binds to CFP-10 secreted by the tuberculosis complex and does not react with BCG or non-tuberculous mycobacteria, effectively avoiding false positive and false negative results.

[0031] 4) Easy to operate and applicable to a wide range of scenarios: The SERS signal tag probe conjugated with anti-CFP-10 polyclonal detection antibody is applied to the test strip. The detection does not require professional instruments and technicians and can be quickly operated on site. It is suitable for various scenarios such as primary medical institutions, community screening, field epidemic prevention, and clinical diagnosis assistance. It is especially suitable for tuberculosis screening in resource-scarce areas.

[0032] In some embodiments, the noble metal nanoparticles include one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles. When the target analyte is present, the multifunctional nanozyme probe loaded with the aforementioned noble metal nanoparticles can be enriched and colorimetrically developed on the detection line, enabling qualitative / semi-quantitative interpretation by visual inspection. Furthermore, the aforementioned noble metal nanoparticles exhibit excellent plasmon resonance effects, enabling visual detection of colorimetric signals and achieving precise quantitative detection of trace substances.

[0033] In a preferred embodiment, the noble metal nanoparticles are Au nanoparticles.

[0034] In some embodiments, the multifunctional nanozyme probe has a particle size of 300 nm-480 nm. Controlling the particle size of the multifunctional nanozyme probe within this range exhibits excellent performance-enhanced Raman scattering (SERS) activity, allowing for the formation of abundant plasma "hot spots" on the probe surface. This avoids the reduced light scattering performance caused by excessively small particle sizes and the non-radiative mode excitation problems caused by excessively large particle sizes. A particle size between 300 nm and 480 nm provides the nanozyme probe with a larger specific surface area and more surface active sites. This not only enhances the binding ability of the nanozyme to Mycobacterium tuberculosis target molecules but also significantly improves its peroxidase-like or oxidase-like catalytic activity, resulting in a more pronounced color change signal in colorimetric detection mode. In SERS mode, probes in this particle size range can effectively couple the excitation light wavelength, generating a strong electromagnetic field enhancement effect, which significantly amplifies the Raman signal of tuberculosis markers, enabling trace detection.

[0035] In a preferred embodiment, the particle size of the multifunctional nanozyme probe is approximately 400 nm.

[0036] In some embodiments, the colorimetric-SERS dual-mode test strip further includes a base plate, a nitrocellulose membrane located on one side of the base plate, and a sample pad and an absorbent pad located at both ends of the nitrocellulose membrane; the detection line and the control line are located on the nitrocellulose membrane, with the detection line positioned closer to the sample pad and the control line positioned closer to the absorbent pad.

[0037] In addition, such as Figure 1 As shown, the present invention also provides a method for preparing a colorimetric-SERS dual-mode test strip, 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: The COFs solution is mixed with a noble metal salt solution, sodium citrate, and sodium borohydride solution. After reaction, a multifunctional nanozyme probe solution is obtained. Step S30: After adjusting the pH of the multifunctional nanozyme probe solution, add anti-CFP-10 polyclonal detection antibody. After the reaction, add bovine serum albumin solution for blocking to obtain a SERS signal tag probe coupled with anti-CFP-10 polyclonal detection antibody. Step S40: Using CFP-10 monoclonal capture antibody and goat anti-rabbit secondary antibody as detection line and control line, form a colorimetric-SERS dual-mode test strip with the SERS signal tag probe conjugated with anti-CFP-10 polyclonal detection antibody.

[0038] In this embodiment, COF nanoflowers with high specific surface area, tunable pore structure, and excellent stability are first synthesized as a carrier. Noble metal nanoparticles are then uniformly grown on their surface using an in-situ reduction method to form a composite nanoprobe material. This material possesses both intuitive colorimetric signals and excellent Raman enhancement performance. Next, a CFP-10 specific detection antibody (Ab1) is modified onto its surface to form a tag probe for a dual-mode test strip, specifically a SERS signal tag probe coupled with an anti-CFP-10 polyclonal detection antibody. This probe specifically binds to the capture antibody Ab2 immobilized on the nitrocellulose membrane (NC membrane) of the test strip, recognizing the CFP-10 antigen and achieving specific, rapid, and sensitive detection of tuberculosis. Furthermore, the qualitative detection of CFP-10 protein can be achieved by visually interpreting the color development of the test line (T line). Simultaneously, the SERS performance of the multifunctional nanozyme probe can be further utilized to acquire Raman signals from the test line. Based on the correlation between signal intensity and CFP-10 concentration, accurate quantitative detection can be achieved.

[0039] 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 reactants at the above concentration and volume ratio, the functional group ratio between aldehyde and amino monomers can be precisely controlled, ensuring that the Schiff base polycondensation reaction proceeds at a gentle and orderly rate. This concentration range can avoid the problems of incomplete polymerization and insufficient crystal nucleus generation caused by too low monomer concentration, while preventing the rapid aggregation of monomers and excessive local crosslinking caused by too high concentration, thus reducing the generation of amorphous impurity phases from the source. Combined with the above volume ratio, the actual molar ratio of the two monomers can be further optimized to match the anisotropic crystallization kinetics required for flower-like morphology growth, guiding COF crystals to grow oriented along specific crystal planes and stack layer by layer.

[0040] In a preferred embodiment, the concentration of 2,5-dimethoxy-terephthalaldehyde is 30 mmol / L; the concentration of 1,3,5-tris(4-aminophenyl)benzene is 40 mmol / L; and the volume ratio of 2,5-dimethoxy-terephthalaldehyde to 1,3,5-tris(4-aminophenyl)benzene is 5:6. Under these conditions, the nucleation rate and crystal growth rate reach a dynamic equilibrium, ensuring sufficient and uniformly dispersed nuclei in the reaction solvent while providing a stable monomer supply for subsequent crystal morphology evolution. This ultimately helps the material form a flower-like microstructure with regular contours, an expansive and uniformly distributed petal structure. Simultaneously, it improves the crystallinity and pore structure integrity of the COFs material, reduces defects such as particle adhesion and morphology collapse, and results in flower-like COFs with more uniform particle size and a stable skeletal structure.

[0041] In some embodiments, step S10 specifically includes: mixing 2,5-dimethoxytetraphenylbenzene (DMTP) and 1,3,5-tris(4-aminophenyl)benzene (TAPB), then adding acetonitrile and methanol as regulators and ultrasonically mixing until homogeneous, then adding ultrapure water as solvent and acetic acid as catalyst, thoroughly shaking and mixing, and allowing the reaction to stand at room temperature for 12 h to obtain a yellow product; washing twice by centrifugation with a 1:2 volume ratio ethanol / acetone mixture, then washing once by centrifugation with ultrapure water, and finally resuspending in ultrapure water to obtain a COFs solution, which is stored at 4°C for later use.

[0042] In some embodiments, the concentration of the COFs solution is 25 mmol / L-35 mmol / L; the concentration of the noble metal salt solution is 4 mmol / L-15 mmol / L; preferably, the volume ratio of the COFs solution to the noble metal salt solution is 25:(1-25). By controlling the concentration and volume ratio of the COFs solution and the noble metal salt solution within the above ranges, precise control of the loading ratio between the support and the precursor can be achieved. Relying on the porous framework, abundant functional groups, and surface adsorption sites of COFs, a uniform anchoring environment for noble metal ions can be provided.

[0043] Specifically, this COFs concentration range ensures sufficient dispersion of the support in the system, preventing agglomeration and sedimentation, maintaining the integrity of the framework structure and full exposure of active sites. It also inhibits particle stacking through a suitable solid-liquid environment, laying the foundation for uniform adsorption and in-situ reduction of noble metal ions. Pairing it with low to medium concentrations of noble metal salt solutions avoids the problems of autonomous nucleation and agglomeration caused by excessively high metal precursor concentrations, preventing a large number of free noble metal particles from detaching from the COFs support and forming impurity particles. Simultaneously, it avoids the drawbacks of insufficient loading and scarce active sites caused by excessively low concentrations. By controlling the volume ratio of the two solutions, it can flexibly adapt to experimental requirements with different target loadings, achieving controllable adjustment of the noble metal nanoparticles on the COFs surface from uniform distribution under low loading to dense modification under medium to high loading. This ensures uniform particle size, no obvious agglomeration, and close adhesion to the COFs framework for growth. Ultimately, it retains the original regular pore structure and high specific surface area advantages of COFs while enabling highly dispersed and firmly anchored noble metal nanoparticles, strengthening the interfacial synergy between the two.

[0044] In some embodiments, step S20 specifically includes the following steps: adding a noble metal salt solution and sodium citrate sequentially to the COFs solution at room temperature, stirring for 10 min, then quickly adding a freshly prepared sodium borohydride solution, and continuing to stir the reaction for 30 min to obtain a dark red product; washing twice by centrifugation with ultrapure water, and finally resuspending in 1 mL of ultrapure water to obtain a nanoprobe solution, which is stored at 4°C for later use.

[0045] In a preferred embodiment, the noble metal salt solution is chloroauric acid (HAuCl4).

[0046] In some embodiments, the pH is between 6.5 and 8.0. By adjusting the pH of the nanoprobe solution, the surface charge state of the material and the charged characteristics of the antibody protein can be precisely balanced. This weakly acidic to near-neutral environment avoids problems such as the hydrolysis and collapse of the COF backbone and the dissolution and aggregation of noble metal nanoparticles caused by strong acidic or strong alkaline conditions, thus fully preserving the original porous structure, high specific surface area, and dispersion stability of the metal particles in the composite material. Furthermore, within this pH range, the abundant oxygen- and nitrogen-containing functional groups on the COF surface can form appropriate electrostatic adsorption sites, while the surface of the noble metal nanoparticles can construct a mild interfacial binding environment. This avoids disordered antibody accumulation and a significant increase in non-specific adsorption due to excessive charge, while ensuring that antibody molecules are stably anchored to the surface of the composite material through hydrophobic interactions, hydrogen bonds, and electrostatic forces. At the same time, the pH range of 6.5 to 8.0 closely matches the stable range of the native spatial conformation of the antibody protein, effectively preventing antibody peptide chain denaturation and inactivation of antigen binding sites, maintaining its specificity and binding activity in immune recognition.

[0047] In some embodiments, the concentration of the multifunctional nanozyme probe solution is 0.5 mg / mL to 2 mg / mL; the concentration of the anti-CFP-10 polyclonal detection antibody is 1.0 mg / mL to 1.5 mg / mL; and the volume ratio of the nanoprobe solution to the anti-CFP-10 polyclonal detection antibody is 1:(1-3).

[0048] Specifically, step S30 includes the following steps: adding potassium carbonate (K2CO3) solution to the multifunctional nanozyme probe solution to adjust the pH; subsequently, adding anti-CFP-10 polyclonal detection antibody (Ab1), and gently shaking the reaction at 25°C and 220 rpm for 2 h; after the reaction, centrifuging at 4000 rpm for 15 min, discarding the supernatant, adding 1 mL of running buffer (0.01 M PBS containing 0.5% Tween 20 and 10% fetal bovine serum) to resuspend the precipitate, thus obtaining the SERS signal tag probe conjugated with anti-CFP-10 polyclonal detection antibody, which is stored at 4°C in the dark for later use. Preferably, before centrifugation, the probe is first blocked with bovine serum albumin (BSA) to block any vacancy sites that may exist on the surface of the multifunctional nanoprobe.

[0049] In addition, in view of the problems that existing tuberculosis detection methods are complicated to operate, have low sensitivity, poor specificity, and cannot achieve rapid and accurate on-site detection, this invention also provides an application of colorimetric-SERS dual-mode test strip in rapid tuberculosis detection.

[0050] In this embodiment, the colorimetric-SERS dual-mode test strip is applied to rapid tuberculosis detection. A tuberculosis dual-mode test strip platform is constructed. A SERS signal tag probe conjugated with an anti-CFP-10 polyclonal detection antibody is co-incubated with a test sample containing CFP-10 antigen, and then dropped onto the sample pad of the test strip. Under capillary action, the mixture migrates upwards along the test strip. If the target antigen is present in the sample, it is captured by the specific capture antibody (Ab2) wrapped in the test line (T line), forming a sandwich immune complex, causing the T line to appear as a deep red band, achieving rapid colorimetric interpretation. Excess multifunctional nanozyme probe conjugated with an anti-CFP-10 polyclonal detection antibody continues to chromatographically to the control line (C line), where it is captured by the secondary antibody, serving as a quality control marker for the effectiveness of the test strip.

[0051] Simultaneously, based on the excellent SERS performance of the multifunctional nanozyme probe, Raman signal acquisition of the T-line can be achieved for quantitative detection. When the target analyte is present, the immune complex labeled with the multifunctional nanozyme probe is enriched in the T-line. Raman laser focusing on the T-line region, based on the COFs at 1575 cm⁻¹, allows for quantitative detection. -1By analyzing the characteristic Raman signal intensity, a linear relationship between SERS intensity and CFP-10 concentration is established, thereby achieving qualitative and quantitative detection with higher sensitivity than visual colorimetry, effectively overcoming the shortcomings of traditional colorimetric methods in terms of sensitivity and accurate quantification. Therefore, the tuberculosis dual-mode test strip platform, constructed based on a SERS signal tag probe conjugated with an anti-CFP-10 polyclonal detection antibody, integrates both colorimetric and SERS dual detection modes, offering the advantages of speed, convenience, accuracy, and sensitivity, providing a novel solution for rapid tuberculosis detection.

[0052] In some embodiments, the colorimetric-SERS dual-mode test strip can be directly used as a tuberculosis dual-mode test strip platform, with a SERS signal tag probe coupled with an anti-CFP-10 polyclonal detection antibody as the reaction system, and a detection line and a control line respectively embedded with a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody as the detection system.

[0053] In this embodiment, the flow immunochromatographic test strip platform with colorimetric / SERS dual-mode output, constructed based on a SERS signal tag probe coupled with an anti-CFP-10 polyclonal detection antibody, can solve the limitations of existing technologies for early rapid screening of tuberculosis. 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 them. It breaks through the technical bottleneck of existing POCT platforms having a single signal output mode and being unable to balance screening convenience and result accuracy.

[0054] 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.

[0055] Example 1 This embodiment prepares nanoprobes (COFs@Au NF) with different Au loadings, specifically including the following steps: 250 µL of 30 mmol / L 2,5-dimethoxytetraphenylethylene (DMTP) and 300 µL of 40 mmol / L 1,3,5-tris(4-aminophenyl)benzene (TAPB) were added sequentially to a 10 mL brown sample vial. Then, 1 mL of acetonitrile and 3 mL of methanol were added, and the mixture was sonicated until homogeneous. Next, 2 mL of ultrapure water and 500 µL of acetic acid were added. The mixture was thoroughly shaken and allowed to stand at room temperature for 12 h to obtain a yellow product. The product was washed twice by centrifugation with a 1:2 ethanol / acetone mixture, followed by one washing with ultrapure water. Finally, the product was resuspended in 1 mL of ultrapure water to obtain a 35 mmol / L COFs solution, which was stored at 4 °C for later use.

[0056] Add 10 mL of ultrapure water to a 25 mL round-bottom flask, then add 200 µL of the COFs solution prepared above, and sonicate for 10 min. Place the flask on a magnetic stirrer, and add 100 µL of 10 mM chloroauric acid (HAuCl4) and 80 µL of 10 mM sodium citrate sequentially at room temperature. After stirring for 10 min, quickly add 100 µL of freshly prepared 1 mM sodium borohydride solution, and continue stirring for 30 min to obtain a deep red product. Wash twice with ultrapure water by centrifugation, and finally resuspend in 1 mL of ultrapure water to obtain a 15 mmol / L COFs@Au NF solution, which is stored at 4 °C for later use. To obtain composite materials with both excellent morphology and optimal Raman reinforcement properties, a series of samples were synthesized according to the above method, with only the volume of HAuCl4 added adjusted to 8 µL, 20 µL, 50 µL, 100 µL, 150 µL, and 200 µL. The morphology of the COFs@AuNFs with different Au loadings prepared above was characterized, and the results are as follows: Figure 2 As shown. Transmission electron microscope images show (e.g.) Figure 2 As shown in the figure, the obtained material is a composite flower-like structure with a particle size of about 400 nm. The core is a flower-like COFs, and Au NPs are uniformly adhered to the outer surface. The material has good dispersion, indicating that the COFs@Au NF nanomaterials were successfully prepared.

[0057] Example 2 The SERS performance of the COFs@Au NF nanoprobe prepared in Example 1 was verified as follows: The SERS performance of COFs@Au NF with different Au loadings was evaluated: 2.5 µL of each COFs@Au NF solution was dropped onto a clean glass slide, and Raman spectra were acquired using a microconfocal Raman spectrometer under the following conditions: 633 nm laser excitation, 50x telephoto objective, 5 mW laser power, 1 s acquisition time, and 1 integration. The comparison data of SERS performance for different Au loadings are shown in the figure below. Figure 3 As shown, the analysis results indicate that the Au loading level affects the Raman signal (1575 cm⁻¹) of COFs themselves. -1 The enhancement effect of the characteristic peak (at the location of the peak) has a significant impact. Based on a comprehensive evaluation of the colorimetric signal intensity and SERS enhancement performance of the material, subsequent experiments selected COFs@Au NF prepared with 100 µL of HAuCl4 as the optimal material, which exhibited the best colorimetric / SERS dual-mode signal response capability.

[0058] Example 3 This embodiment provides the assembly of a lateral flow immunochromatographic test strip and the preparation of a SERS nanotag, as detailed below: The lateral flow immunochromatographic test strip consists of four parts: a sample pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate. First, the NC membrane is adhered to the center of the PVC base plate and equilibrated in a 37°C drying oven for 2 hours. Using a membrane scrubbing instrument, 1 mg / mL of anti-CFP-10 protein-specific monoclonal capture antibody (Ab2) and 1 mg / mL of goat anti-rabbit secondary antibody are sprayed onto the NC membrane at a rate of 1 µL / cm, forming the T and C lines, respectively. Subsequently, the pretreated sample pad and absorbent pad are fixed to the top and bottom of the PVC base plate, respectively, ensuring an overlap of approximately 1 mm between each part to guarantee continuous and smooth capillary chromatography after sample solution addition. The assembled test strip is cut into 4 mm wide strips, placed in a custom-made plastic cartridge, sealed with desiccant, and stored for later use.

[0059] Anti-CFP-10 protein polyclonal detection antibody (Ab1) was coupled to the surface of COFs@Au NF with an Au loading of 100 μL via physical adsorption. First, 2 μL of 0.2 M K₂CO₃ solution was added to an equal volume of COFs@Au NF solution to adjust the pH. Then, 3 µL of 1.0 mg / mL Ab1 antibody was added to each tube, and the reaction was carried out with gentle shaking at 25 °C and 220 rpm for 2 h. Next, 100 µL of 10% bovine serum albumin (BSA) solution was added for blocking, and the reaction was continued for another 2 h. After the reaction, the sample was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of running buffer (0.01 M PBS containing 0.5% Tween 20 and 10% fetal bovine serum) to obtain the COFs@Au NF-Ab1 signal tag probe, which was stored at 4 °C protected from light for later use.

[0060] Take 10 µL of the tag probe resuspended in 0.01 M running buffer, mix it with 10 µL of 100 ng / mL CFP-10 protein standard, and add it to the assembled test strip sample pad. After chromatography for 15 min, observe the color development of the test strip. The color development results of the T line of the test strip containing the target protein solution after different chromatography times are shown in the figure. Figure 4 As shown in the figure; the results showed that after about 9 minutes of chromatography, a clearly visible band appeared at the T line, indicating that the prepared test strip can achieve rapid colorimetric detection of CFP-10 protein.

[0061] Example 4 COFs@Au NF combined with lateral flow immunochromatography for the ultrasensitive and rapid detection of the Mycobacterium tuberculosis marker CFP-10 is as follows: 10 µL of running buffer containing the COFs@AuNF-Ab1 tag probe (from Example 3) was mixed with 10 µL of CFP-10 protein standard solutions of different concentrations. If the CFP-10 antigen is present in the sample, it specifically binds to Ab1 on the tag probe to form an immune complex. The mixture was then added to the sample pad of the test strip for chromatographic reaction. For high-concentration CFP-10 samples, the T-line was clearly visible, allowing for qualitative or semi-quantitative interpretation by direct visual observation. However, for low-abundance targets, the sensitivity of visual colorimetry is limited, making trace detection and accurate quantification difficult. To improve detection sensitivity, the SERS activity of the tag probe can be utilized for quantitative analysis using Raman detection mode. A 633 nm laser, a 50x telephoto objective lens, a laser power of 5 mW, an acquisition time of 1 s, and one integration were used to acquire the Raman spectrum of the T-line region. The colorimetric detection results of test strips for different concentrations of CFP-10 protein and the corresponding SERS spectra of the T-line are shown below. Figure 5 As shown, the test results indicate that the visually perceptible limit of detection (LOD) for CFP-10 protein on this test strip is approximately 500 pg / mL. Using the SERS detection mode, the detection sensitivity is significantly improved, with a LOD for CFP-10 protein reaching approximately 5 pg / mL.

[0062] To evaluate the specificity of the developed colorimetric / SERS dual-mode test strip (CM / SERS-LFIA) for CFP-10 detection, five interfering antigens associated with lung diseases (such as N protein, ConA, FluA, FluB, and p24) were detected under the same conditions. The specificity detection results of the test strip for CFP-10 and the corresponding SERS spectra of the T line are shown below. Figure 6 As shown, the results indicate that the test strips using interfering antigens as detection samples showed no visible color development on the T-line after chromatography, and almost no SERS signal was detected. However, samples containing CFP-10 antigen showed clear and visible color development on the T-line, and a strong SERS signal was obtained. These results demonstrate that the constructed SERS-LFIA exhibits excellent specificity for CFP-10 detection, and also confirm that the specific antibody used has a strong affinity for the CFP-10 antigen.

[0063] Clearly, the constructed colorimetric / SERS dual-mode test strip (CM / SERS-LFIA) exhibits excellent signal output capability, good detection reproducibility, and high specificity for the detection of the Mycobacterium tuberculosis marker CFP-10, demonstrating broad application prospects.

[0064] In summary, this invention provides a method for preparing a colorimetric-SERS dual-mode test strip and its application in rapid tuberculosis detection. The colorimetric-SERS dual-mode test strip comprises a multifunctional nanozyme probe and an anti-CFP-10 polyclonal detection antibody coupled to the surface of the multifunctional nanozyme probe, as well as a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody embedded in the detection line and control line, respectively. The multifunctional nanozyme probe comprises a nanoflower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier. This invention utilizes the multifunctional nanozyme probe coupled with the anti-CFP-10 polyclonal detection antibody Ab1 to form a signal tag probe. This probe specifically binds to the capture antibody Ab2 immobilized on the nitrocellulose membrane (NC membrane) of the test strip, recognizing the CFP-10 antigen, and achieving specific, rapid, and sensitive detection of tuberculosis. Furthermore, qualitative detection of CFP-10 protein can be achieved by visually interpreting the color development of the test line on the test strip. Simultaneously, the SERS performance of the multifunctional nanozyme probe can be utilized to acquire Raman signals from the test line, and precise quantitative detection can be achieved based on the correlation between signal intensity and CFP-10 concentration. Therefore, this colorimetric-SERS dual-mode test strip integrates both visual colorimetric and SERS detection modes, offering outstanding advantages in speed, accuracy, and sensitivity. Moreover, the use of nanoflower-like COFs as a carrier ensures that the multifunctional nanozyme probe exhibits high stability, uniformity, selectivity, and enhanced performance.

[0065] 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 colorimetric-SERS dual-mode test strip, characterized in that, The invention includes a multifunctional nanozyme probe and an anti-CFP-10 polyclonal detection antibody coupled to the surface of the multifunctional nanozyme probe, as well as a CFP-10 monoclonal capture antibody and a goat anti-rabbit secondary antibody embedded in the detection line and control line, respectively; the multifunctional nanozyme probe includes a nanoflower-shaped COFs carrier and noble metal nanoparticles loaded on the surface of the COFs carrier.

2. The colorimetric-SERS dual-mode test strip according to claim 1, characterized in that, The noble metal nanoparticles include one or more of Au nanoparticles, Ag nanoparticles, Pt nanoparticles, Pd nanoparticles, Ru nanoparticles, and Cu nanoparticles.

3. The colorimetric-SERS dual-mode test strip according to claim 1, characterized in that, The particle size of the multifunctional nanozyme probe is 300 nm-480 nm.

4. The colorimetric-SERS dual-mode test strip according to claim 1, characterized in that, The colorimetric-SERS dual-mode test strip also includes a base plate, a nitrocellulose membrane located on one side of the base plate, and a sample pad and an absorbent pad located at both ends of the nitrocellulose membrane; the detection line and the control line are located on the nitrocellulose membrane, with the detection line positioned closer to the sample pad and the control line positioned closer to the absorbent pad.

5. A method for preparing a colorimetric-SERS dual-mode test strip 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 was mixed with a noble metal salt solution, sodium citrate, and sodium borohydride solution, and after reaction, a multifunctional nanozyme probe solution was obtained. After adjusting the pH of the multifunctional nanozyme probe solution, anti-CFP-10 polyclonal detection antibody was added. After the reaction, bovine serum albumin solution was added for blocking to obtain a SERS signal tag probe coupled with anti-CFP-10 polyclonal detection antibody. Using CFP-10 monoclonal capture antibody and goat anti-rabbit secondary antibody as the detection line and control line, a colorimetric-SERS dual-mode test strip is formed with the SERS signal tag probe conjugated with anti-CFP-10 polyclonal detection antibody.

6. The method for preparing the colorimetric-SERS dual-mode test strip according to claim 5, characterized in that, The concentration of 2,5-dimethoxytetraphenylaldehyde is 25 mmol / L-35 mmol / L; the concentration of 1,3,5-tris(4-aminophenyl)benzene is 35 mmol / L-50 mmol / L. Preferably, the volume ratio of 2,5-dimethoxytetraphenylbenzene to 1,3,5-tris(4-aminophenyl)benzene is (4-6):(5-7).

7. The method for preparing the colorimetric-SERS dual-mode test strip according to claim 5, characterized in that, The concentration of the COFs solution is 25 mmol / L-35 mmol / L; the concentration of the noble metal salt solution is 4 mmol / L-15 mmol / L. Preferably, the volume ratio of the COFs solution to the noble metal salt solution is 25:(1-25).

8. The method for preparing the colorimetric-SERS dual-mode test strip according to claim 5, characterized in that, The pH is between 6.5 and 8.

0.

9. The method for preparing the colorimetric-SERS dual-mode test strip according to claim 5, characterized in that, The concentration of the multifunctional nanozyme probe solution is 0.5 mg / mL-2 mg / mL; the concentration of the anti-CFP-10 polyclonal detection antibody is 1.0 mg / mL-1.5 mg / mL. The volume ratio of the multifunctional nanozyme probe solution to the anti-CFP-10 polyclonal detection antibody is 1:(1-3).

10. The application of a colorimetric-SERS dual-mode test strip as described in any one of claims 1-4 in the rapid detection of tuberculosis.

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