A mercaptobenzoic acid-mediated nano-enzyme immunochromatography reagent and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a thiol phenylboronic acid mediated nano-enzyme immunochromatography reagent and application thereof. The present application utilizes the broad-spectrum binding ability of MPBA to the glycosylated molecules on the surface of pathogens, and combines the colorimetric catalytic double enhancement effect of the virus-like biomimetic magnetic nano-enzyme (FeAu@AuIr), to improve the detection sensitivity. The probe takes Fe3O4 magnetic nanoparticles as the core, and sequentially loads large-diameter gold nanoparticles (Au) and gold-iridium alloy (AuIr) with high catalytic activity, to enhance the pathogen capture capacity and signal amplification effect. Combined with the antibody modified immunochromatography strip, Streptococcus pneumoniae, Pseudomonas aeruginosa and SARS-CoV-2 can be simultaneously detected, and the detection sensitivity is greatly improved. Through verification of 170 clinical samples, the present application has good application prospect in on-site rapid detection, and provides an efficient and low-cost solution for precise screening of respiratory tract infectious pathogens.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mercaptophenylboronic acid-mediated nanoenzyme immunochromatographic reagent and its application. Background Technology

[0002] Immunochromatographic analysis (ICA) is a rapid, low-cost (<$2), and portable immunoassay technique widely used in food safety, environmental monitoring, and home testing. It has great potential for rapid diagnosis of respiratory infections, directly detecting pathogen structural proteins and integrating multiple antibodies to achieve simultaneous detection of viruses and bacteria. However, current ICA faces two major technical bottlenecks in pathogen detection: (1) Insufficient signal strength and stability: Traditional nanotags (such as colloidal gold and latex beads) have weak colorimetric signals and poor stability in complex matrices, affecting sensitivity. Although fluorescent microspheres, quantum dots, and SERS tags can improve signal strength, they require specialized instruments for reading, reducing the convenience of ICA. (2) Reliance on antibody detection strategies, affecting cost and stability: Existing ICA relies on a double-antibody sandwich strategy, which is time-consuming and expensive to screen for matching antibody pairs. Furthermore, the antibodies on the surface of nanoprobes are easily affected by complex samples, leading to decreased activity and affecting detection accuracy and stability. Currently, there is no ICA technology that simultaneously possesses high sensitivity and ease of use for efficient detection of bacteria and viruses.

[0003] Therefore, it is necessary to develop an efficient and sensitive ICA method for detecting bacteria and viruses. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention develops a universal colorimetric ICA platform that combines the broad-spectrum recognition of MPBA with the colorimetric-catalytic dual-signal enhancement of nanozymes, achieving highly sensitive simultaneous detection of respiratory bacteria and viruses. This invention designs a virus-like biomimetic magnetic nanozyme (FeAu@AuIr): a core Fe3O4 (160nm) for magnetic enrichment; Au nanoparticles (15nm) to enhance the colorimetric signal and provide additional loading space; and a multi-tentacle AuIr (5nm) shell to provide high catalytic activity and MPBA binding sites.

[0005] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides a nanoenzyme comprising a magnetic core, gold nanoparticles, and gold-iridium alloy nanoparticles.

[0007] In some embodiments of the present invention, the gold nanoparticles are loaded on the surface of a magnetic core to form gold-shelled magnetic beads.

[0008] In some embodiments of the present invention, the gold-iridium alloy nanoparticles are loaded on the surface of the gold nanoparticles and the surface of the magnetic core.

[0009] In some embodiments of the present invention, the magnetic core and the surface of the gold-shelled magnetic beads are modified with a cationic polymer layer.

[0010] In some embodiments of the present invention, the magnetic core includes at least one of Fe3O4, Fe2O3, Co-Ni alloy, Fe-Co alloy, and Fe-Pt alloy.

[0011] In some embodiments of the present invention, the magnetic core is Fe3O4.

[0012] In some embodiments of the present invention, the particle size of the Fe3O4 is 80-500 nm; preferably 100-300 nm.

[0013] In some embodiments of the present invention, the particle size of the gold nanoparticles is 10-100 nm; preferably 10-50 nm.

[0014] In some embodiments of the present invention, the particle size of the gold-iridium alloy nanoparticles is 1–30 nm.

[0015] In some embodiments of the present invention, the cationic polymer layer is PEI.

[0016] In some embodiments of the present invention, the molecular weight of the PEI is 5000-80000 Da; preferably 10000-50000 Da.

[0017] A second aspect of the present invention provides a method for preparing nanozymes according to the first aspect of the present invention, comprising the following steps:

[0018] 1) Modify the magnetic core with a cationic polymer layer;

[0019] 2) Gold nanoparticles are loaded onto the magnetic core of the modified cationic polymer layer to obtain gold-shelled magnetic beads;

[0020] 3) Modify the gold-shelled magnetic beads with a cationic polymer layer;

[0021] 4) Gold-iridium alloy nanoparticles were loaded onto gold-shelled magnetic beads modified with cationic polymer layers to obtain nanozymes.

[0022] In some embodiments of the present invention, step 1) is: dispersing Fe3O4 nanoparticles in water (to obtain Fe3O4 dispersion), mixing it with PEI aqueous solution, and using ultrasound to uniformly coat the Fe3O4 surface with PEI to form a positively charged Fe3O4-PEI complex.

[0023] In some embodiments of the present invention, the particle size of the Fe3O4 nanoparticles ranges from 80 to 500 nm; preferably from 100 to 300 nm.

[0024] In some embodiments of the present invention, 0.1 to 5 g of Fe3O4 nanoparticles are dispersed in 100 mL of deionized water; preferably 0.5 to 1.5 g.

[0025] In some embodiments of the present invention, the molecular weight of the PEI is 5000-80000 Da; preferably 10000-50000 Da.

[0026] In some embodiments of the present invention, the mass concentration of the PEI aqueous solution is 0.1 to 5 mg / mL.

[0027] In some embodiments of the present invention, the volume ratio of the Fe3O4 dispersion to the PEI aqueous solution is 1 to 50:1.

[0028] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 10-60min.

[0029] In some embodiments of the present invention, step 2) is: Fe3O4-PEI is enriched, collected, and dispersed in a negatively charged Au aqueous solution, and Au is uniformly deposited on the surface of Fe3O4-PEI under intense ultrasonic treatment to form gold-shelled magnetic beads (FeAu).

[0030] In some embodiments of the present invention, the Au particle size is 10 to 100 nm.

[0031] In some embodiments of the present invention, the concentration of the Au aqueous solution is 1 to 10 mg / mL.

[0032] In some embodiments of the present invention, the amount of Fe3O4-PEI nanospheres added per 100 mL of Au aqueous solution is 0.5–5 g.

[0033] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 10-60min.

[0034] In some embodiments of the present invention, step 3) is: collecting and dispersing the FeAu in an aqueous solution, and mixing it again with the PEI aqueous solution, and then using intense ultrasonic treatment to make the PEI uniformly coat the FeAu surface again, forming a positively charged FeAu-PEI complex.

[0035] In some embodiments of the present invention, the molecular weight of the PEI is 5000-80000 Da; preferably 10000-50000 Da.

[0036] In some embodiments of the present invention, the mass concentration of the PEI aqueous solution is 0.1 to 5 mg / mL.

[0037] In some embodiments of the present invention, 0.1 to 5 g of FeAu is dispersed in every 100 mL of deionized water.

[0038] In some embodiments of the present invention, the volume ratio of FeAu dispersion to PEI aqueous solution is 1-50:1.

[0039] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 10-60min.

[0040] In some embodiments of the present invention, step 4) is: dispersing the FeAu-PEI in an aqueous solution of AuIr alloy nanoparticles, mixing them evenly, and then promoting the uniform self-assembly of AuIr nanoparticles onto the FeAu-PEI surface under ultrasonic action, ultimately forming a virus-like biomimetic magnetic nanozyme (FeAu@AuIr).

[0041] In some embodiments of the present invention, the AuIr alloy nanoparticles have a particle size of 1 to 30 nm.

[0042] In some embodiments of the present invention, the concentration of the aqueous solution of AuIr alloy nanoparticles is 1-30 mg / mL.

[0043] In some embodiments of the present invention, the amount of FeAu-PEI nanospheres added per 100 mL of AuIr aqueous solution is 0.1–5 g.

[0044] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 10-60min.

[0045] A third aspect of the present invention provides a nanozyme probe comprising a virus-like biomimetic magnetic nanozyme (FeAu@AuIr) from the first aspect of the present invention, wherein the nanozyme is surface-modified with 4-mercaptophenylboronic acid (MPBA).

[0046] A fourth aspect of the present invention provides a method for preparing the nanozyme probe of the third aspect of the present invention, comprising the following steps:

[0047] The virus-like biomimetic magnetic nanozyme (FeAu@AuIr) of the first aspect of the present invention was dispersed in water, mixed with an ethanol solution of mercaptophenylboronic acid (MPBA), and ultrasonically treated to obtain a nanozyme probe (FeAu@AuIr-MPBA).

[0048] In some embodiments of the present invention, the mass concentration of the MPBA ethanol solution is 0.1–5 mg / mL; preferably 0.5–2 mg / mL.

[0049] In some embodiments of the present invention, 0.1 to 5 g of FeAu@AuIr is dispersed in every 100 mL of deionized water; preferably 0.5 to 1.5 g.

[0050] In some embodiments of the present invention, the volume ratio of FeAu@AuIr dispersion to MPBA ethanol solution is preferably 10 to 2000:1.

[0051] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 30-500min.

[0052] A fifth aspect of the present invention provides a quality control line probe comprising a virus-like biomimetic magnetic nanozyme (FeAu@AuIr) from the first aspect of the present invention, wherein the nanozyme is surface-modified with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0053] A sixth aspect of the present invention provides a method for preparing the quality control line probe of the fifth aspect of the present invention, comprising the following steps:

[0054] 1) The virus-like biomimetic magnetic nanozyme (FeAu@AuIr) of the first aspect of the present invention is dispersed in water and mixed with a 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) ethanol solution and ultrasonically treated to obtain a virus-like biomimetic magnetic nanozyme with carboxyl groups on its surface.

[0055] 2) After activating the carboxyl-containing biomimetic magnetic nanozyme, it was coupled with biotin-BSA and incubated at room temperature for 2 hours. Bovine serum albumin (BSA) solution was added to block the unreacted carboxyl groups to prepare a quality control line probe.

[0056] In some embodiments of the present invention, the mass concentration of the DTNB ethanol solution is 0.1–5 mg / mL; preferably 0.5–2 mg / mL.

[0057] In some embodiments of the present invention, 0.1 to 5 g of FeAu@AuIr is dispersed in every 100 mL of deionized water; preferably 0.5 to 1.5 g.

[0058] In some embodiments of the present invention, the volume ratio of FeAu@AuIr dispersion to DTNB ethanol solution is preferably 10 to 2000:1.

[0059] In some embodiments of the present invention, the ultrasonic frequency is 50-100Hz, the temperature is 10-40℃, and the duration is 30-500min.

[0060] In some embodiments of the present invention, the activation treatment includes mixing a virus-like biomimetic magnetic nanozyme with an outer surface modified with DTNB with an activation solution and then subjecting it to ultrasonic treatment.

[0061] In some embodiments of the present invention, the activation solution is prepared by adding 0.002-0.2 g of N-hydroxysuccinimide (NHS), 0.002-0.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 0.002-0.2 g of morpholine ethanesulfonic acid (MES) to 100 mL of deionized water.

[0062] In some embodiments of the present invention, the amount of biotin-BSA added to each 1g of the DTNB-modified viral biomimetic magnetic nanoenzyme is 0.1-2mg, preferably 0.1-0.5mg.

[0063] In some embodiments of the present invention, the incubation treatment is carried out at a temperature of 20-40°C for a time of 20-180 min.

[0064] A seventh aspect of the present invention provides the use of the nanozyme of the first aspect, the nanoprobe of the third aspect, and the quality control probe of the fifth aspect in the preparation of products for detecting bacteria and / or viruses.

[0065] In some embodiments of the present invention, the bacteria include Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, and Salmonella typhimurium.

[0066] In some embodiments of the present invention, the virus includes SARS-CoV-2, monkeypox virus (MPXV), Ebola virus (EBOV), etc.

[0067] According to relevant research (https: / / doi.org / 10.1002 / smll.202310014, https: / / doi.org / 10.1021 / acsnano.4c01824), MPBA has a broad-spectrum ability to bind to bacteria and viruses, including but not limited to Staphylococcus aureus, Salmonella typhimurium, monkeypox virus (MPXV), and Ebola virus (EBOV). Therefore, those skilled in the art can reasonably expect that replacing the bacteria and viruses specifically detected in the embodiments of this invention with other bacteria and viruses known in the art that MPBA can bind to can also achieve the corresponding technical effects.

[0068] An eighth aspect of the present invention provides a kit comprising a nanoprobe from the third aspect of the present invention and a quality control line probe from the fifth aspect of the present invention.

[0069] In some embodiments of the present invention, the kit includes test strips.

[0070] In some embodiments of the present invention, the test strip includes a sample pad for loading a sample solution, a nitrocellulose membrane, and an absorbent pad, wherein the nitrocellulose membrane is provided with a detection line and a control line.

[0071] In some embodiments of the present invention, the detection lines include 1 to 9 lines, preferably 3 lines.

[0072] In some embodiments of the present invention, the detection line is modified with specific substances for detecting different pathogens.

[0073] In some embodiments of the present invention, there are three detection limits, which are respectively modified with anti-SARS-CoV-2 spike protein antibody, anti-Pseudomonas aeruginosa antibody and anti-Streptococcus pneumoniae antibody, and the order of the three control lines is not limited.

[0074] In some embodiments of the present invention, the concentrations of the anti-SARS-CoV-2 antigen spike protein antibody, anti-Pseudomonas aeruginosa antibody, and anti-Streptococcus pneumoniae antibody sprayed (saturated) on the detection line are all 0.1-2.5 mg / mL.

[0075] In some embodiments of the present invention, the kit further includes a running buffer and a catalytic buffer.

[0076] In some embodiments of the present invention, the running buffer comprises Tween 20, milk, and PBS buffer; the mass fraction of milk is 10%; and the component ratio of the running buffer is Tween 20:Milk:PBS = (1-10):(0.001-10):(10-1000).

[0077] In some embodiments of the present invention, the catalyst solution consists of sodium acetate buffer, 3-amino-9-ethylcarbazole (AEC) and hydrogen peroxide (H2O2) solution, wherein the pH of the sodium acetate buffer is 2-6, the concentration of AEC is 1-100 mmol / L, and the concentration of H2O2 is 0.1-50 mol / L.

[0078] In some embodiments of the present invention, the composition ratio of the catalyst solution is sodium acetate buffer: AEC: H2O2 = (1-100): (1-100): (1-100).

[0079] A ninth aspect of the present invention provides a method for detecting bacteria and / or viruses, said method for non-disease treatment purposes, comprising the following steps:

[0080] The test sample was mixed with nanozyme probe (FeAu@AuIr-MPBA) and control line probe (FeAu@AuIr-DTNB), incubated, magnetically separated, resuspended in running buffer, loaded onto the sample pad of the immunochromatographic test strip, and brought into contact with the detection line on the immunochromatographic test strip to carry out the chromatographic reaction. Catalytic solution was added, and the signal was read.

[0081] In some embodiments of the present invention, the concentration of the nanozyme probe is 0.01–5 mg / mL.

[0082] In some embodiments of the present invention, the volume ratio of the sample to the nanozyme probe is 1000:(1-5).

[0083] In some embodiments of the present invention, the incubation time is 1 to 15 minutes.

[0084] In some embodiments of the present invention, the chromatography reaction time is 5 to 20 minutes.

[0085] In some embodiments of the present invention, the catalytic time is 10 to 200 s.

[0086] The beneficial effects of this invention are:

[0087] (1) This invention proposes a virus-like biomimetic magnetic nanozyme (FeAu@AuIr): the core Fe3O4 (160nm) is used for magnetic enrichment; Au nanoparticles (15nm) enhance the colorimetric signal and provide additional loading space; the multi-tentacle AuIr (5nm) shell provides high catalytic activity and MPBA binding sites. This nanoprobe can efficiently capture pathogens within 5 minutes and perform dual-mode colorimetric catalytic detection on antibody-modified test strips.

[0088] (2) This invention proposes a method for preparing a virus-like biomimetic magnetic nanozyme probe. The prepared nanozyme has high hydrophilicity, magnetic responsiveness and high efficiency peroxidase-like activity.

[0089] (3) Modify MPBA on the surface of the prepared virus-like biomimetic magnetic nanozyme probe to give it the ability to capture bacterial and viral glycoproteins simultaneously on a broad spectrum, thereby achieving universal capture of a variety of bacteria and viruses and avoiding the limitations of antibody dependence.

[0090] (4) This invention proposes an immunochromatographic method based on a virus-like biomimetic magnetic nanozyme probe. This method can simultaneously detect two respiratory bacteria and one respiratory virus in a complex sample matrix within 15 minutes, thereby improving the detection throughput.

[0091] (5) The immunochromatographic method based on virus-like biomimetic magnetic nanozyme probe proposed in this invention can significantly amplify the signal after catalysis by the catalytic solution, thereby improving the detection sensitivity.

[0092] In summary, an immunochromatographic method based on virus-like biomimetic magnetic nanozyme probes was proposed. Using broad-spectrum virus-like biomimetic magnetic nanozyme probes, bacteria and viruses in complex samples can be rapidly enriched simultaneously, and rapid and highly sensitive quantitative detection of bacteria and viruses can be achieved on immunochromatographic test strips. Attached Figure Description

[0093] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0094] Figure 1 The diagrams are schematic diagrams of the preparation methods for synthesizing the virus-like biomimetic magnetic nanozyme probes and control line probes in Examples 1 and 2 of the present invention, where i is a schematic diagram of nanozyme preparation and ii is a schematic diagram of nanozyme probe and control line probe preparation.

[0095] Figure 2High-resolution transmission electron microscopy (TEM) images, scanning electron microscopy (SEM) images, and elemental mappings of the virus-like biomimetic magnetic nanozyme probes at each stage are shown. Among them: (a) typical TEM images of Fe3O4 MNPs, (e) FeAu MNPs, and (i) FeAu@AuIr nanozymes, and magnified TEM images of the particles in (b), (f), and (j); (c) locally magnified TEM images of the surface structures of Fe3O4, (g) FeAu, and (k) FeAu@AuIr nanocomposites; (d) representative SEM images of Fe3O4, (h) FeAu, and (l) FeAu@AuIr nanozymes; (m) EDS elemental line scan of a single multitentacle and (n) elemental mapping results for FeAu@AuIr.

[0096] Figure 3 To verify the peroxidase-like activity of the virus-inspired biomimetic magnetic nanozyme probe, including: (a) different nanomaterials (Au) 15 (a) Photographs and UV-Vis absorption spectra of TMB catalyzed by FeAu, Fe3O4, AuIr5, Fe@AuIr and multi-tendril FeAu@AuIr; (b) Photographs of TMB (1 mM) solution catalyzed by Fe3O4, FeAu, Fe@AuIr and FeAu@AuIr in the presence of H2O2 at 0.49–1000 fM; (c) the corresponding absorbance values ​​at 652 nm after catalytic oxidation; (d) Steady-state kinetic analysis of FeAu@AuIr: (I) Relationship between reaction rate and H2O2 concentration with fixed TMB concentration; (II) Relationship between reaction rate and TMB concentration with fixed H2O2 concentration; Inset: Lineweaver-Burk plot; (e) Free energy diagrams of FeAu and FeAu@AuIr during catalysis; (f) Calculated near-field electromagnetic field distributions of (I) FeAu and (II) FeAu@AuIr nanozymes.

[0097] Figure 4 To verify the ability of this type of viral biomimetic magnetic nanozyme probe to simultaneously capture two bacteria and one virus, the following were performed: (a) fluorescence microscopy analysis to verify the binding ability of FeAu@AuIr-MPBA to SARS-CoV-2SP; (b) TEM images of Pseudomonas aeruginosa (I) and Streptococcus pneumoniae (II) captured by FeAu@AuIr-MPBA; (c) BCA assay and plate culture results showing the concentrations of residual viral proteins and bacteria in the supernatant before and after FeAu@AuIr-MPBA capture; (d) calculation of the capture efficiency of FeAu@AuIr-MPBA for the three target pathogens; and (e) the relationship between the capture efficiency of FeAu@AuIr-MPBA and incubation time.

[0098] Figure 5This diagram illustrates the working principle of immunochromatography based on a broad-spectrum virus-inspired biomimetic magnetic nanozyme probe for SARS-CoV-2 antigen spike protein, Pseudomonas aeruginosa, and Streptococcus pneumoniae.

[0099] Figure 6 The results of the immunochromatographic verification of the broad-spectrum viral biomimetic magnetic nanozyme probe are as follows: (a) Photographs of the T lines of the corresponding test strips for different pathogens after catalytic activation in (I) and (II) and (bc) measured colorimetric signals: (1) SARS-CoV-2SP / Pseudomonas aeruginosa / Streptococcus pneumoniae, (2) SARS-CoV-2SP, (3) Pseudomonas aeruginosa, (4) Streptococcus pneumoniae, ( 5) Influenza A, (6) Influenza B, (7) Respiratory syncytial virus, (8) Adenovirus, (9) Staphylococcus aureus, (10) Mycobacterium pneumoniae, (11) Klebsiella pneumoniae, (12) Legionella pneumophila, (13) Blank control; (d) Typical SEM images of the T-line inner region of SARS-CoV-2, Pseudomonas aeruginosa, Streptococcus pneumoniae and negative samples; (I-IV) and (V-VIII) are test strip images before and after catalysis, respectively.

[0100] Figure 7 The results of the optimization of the tag amount (a), run buffer composition (b), and incubation time (c) for the immunochromatographic assay of a broad-spectrum virus-inspired magnetic nanozyme probe.

[0101] Figure 8 The results show the optimized reaction time (a), catalytic time (b), T-line concentration (c), and catalytic system (d) of the immunochromatographic assay for a broad-spectrum virus-inspired biomimetic magnetic nanozyme probe.

[0102] Figure 9 Photographs (I), thermograms (II), and detailed colorimetric intensities (III) of FeAu@AuIr-ICA T-line for simultaneous detection of Streptococcus pneumoniae, Pseudomonas aeruginosa, and SARS-CoV-2SP before (a) and after (b) catalytic activation; calibration curves of FeAu@AuIr-ICA against Streptococcus pneumoniae (c), Pseudomonas aeruginosa (d), and SARS-CoV-2SP (e) before and after catalysis.

[0103] Figure 10 Immunochromatographic images and signal diagrams of a broad-spectrum viral biomimetic magnetic nanozyme probe before (a) and after (b) catalysis at high, medium, and low concentrations.

[0104] Figure 11This study presents the application of immunochromatography based on a broad-spectrum virus-inspired biomimetic magnetic nanozyme probe in the detection of clinical samples. (a) Results of FeAu@AuIr-ICA on swab samples containing pathogens before (I) and after (II) catalysis; (b) T-line colorimetric signal (I) on the FeAu@AuIr-ICA strip (catalysis mode), OD value (II) of ELISA, and Ct value (III) of qPCR in detecting clinical positive specimens; (c) qPCR standard curves for detecting Streptococcus pneumoniae (I) and Pseudomonas aeruginosa (II); (d) Deming regression analysis results of SARS-CoV-2 in positive pharyngeal swab specimens detected by FeAu@AuIr-ICA and ELISA. Deming regression analysis results of Pseudomonas aeruginosa (e) and Streptococcus pneumoniae (f) in bronchoalveolar lavage fluid positive specimens detected by FeAu@AuIr-ICA and qPCR; ROC curves of infection prediction models for (g) SARS-CoV-2, (h) Pseudomonas aeruginosa and (i) Streptococcus pneumoniae based on FeAu@AuIr-ICA. Detailed Implementation

[0105] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0106] Example 1: Preparation of Virus-like Biomimetic Magnetic Nanozyme Probes with Broad-Spectrum Capture Capabilities

[0107] This invention provides a method for preparing a virus-like biomimetic magnetic nanozyme probe with broad-spectrum capture capability. The method is characterized by first loading a layer of gold nanoparticles (Au NPs) providing a strong colorimetric signal to increase the surface area using electrostatic self-assembly technology, with a magnetic bead (Fe3O4) core. Then, a layer of gold-iridium alloy particles (AuIr NPs) with excellent peroxidase catalytic activity is loaded to prepare a virus-like biomimetic magnetic nanozyme (FeAu@AuIr). Finally, the surface is modified with mercaptophenylboronic acid (MPBA) to endow it with the ability to simultaneously capture multiple bacterial and viral glycoproteins. The preparation process is as follows: Figure 1 As shown.

[0108] The specific steps are as follows:

[0109] (1) 0.5 g of Fe3O4 nanoparticles (particle size 160 nm) were dispersed in 50 mL of aqueous solution, and 1 mL of PEI aqueous solution (25000 Da, 5 mg / mL) was added. The PEI was uniformly coated on the Fe3O4 surface under ultrasonic treatment (100 Hz, 25 °C, 30 min) to form a positively charged Fe3O4-PEI complex.

[0110] (2) 0.5g of Fe3O4-PEI was enriched, collected and dispersed in 150mL of negatively charged Au aqueous solution (5mg / mL). Under intense ultrasonic treatment (100Hz, 25℃, 30min), Au was uniformly deposited on the surface of Fe3O4-PEI to form gold-shelled magnetic beads (FeAu).

[0111] (3) Collect 0.5g of gold-shelled magnetic beads (FeAu) and redisperse them in 50mL of PEI aqueous solution (25000Da, 2mg / mL). Through vigorous ultrasonic treatment (100Hz, 25℃, 20min), PEI is uniformly coated on the FeAu surface again to form a positively charged FeAu-PEI complex.

[0112] (4) 0.5g FeAu-PEI was dispersed in 100mL of AuIr alloy nanoparticle aqueous solution (15mg / mL). After mixing evenly, AuIr nanoparticles were uniformly self-assembled onto the FeAu-PEI surface under ultrasonic treatment (100Hz, 25℃, 20min) to finally form a virus-like biomimetic magnetic nanozyme (FeAu@AuIr).

[0113] (5) Disperse 0.5g FeAu@AuIr in 100mL of aqueous solution, add 100μL of MPBA ethanol solution (1mg / mL), mix and sonicate (100Hz, 25℃, 120min) to obtain a virus-like biomimetic magnetic nanozyme probe (FeAu@AuIr-MPBA) with broad-spectrum capture capability.

[0114] Example 2: Preparation of Virus-Inspired Biomimetic Magnetic Nanoenzyme Control Line Probe

[0115] This embodiment provides a method for preparing a virus-like biomimetic magnetic nanozyme control line probe. The method is characterized by first loading a layer of gold nanoparticles (AuNPs) providing a strong colorimetric signal to increase the surface area using electrostatic self-assembly technology, with a magnetic bead (Fe3O4) core. Then, a layer of gold-iridium alloy particles (AuIrNPs) with excellent peroxidase catalytic activity is loaded to prepare a virus-like biomimetic magnetic nanozyme (FeAu@AuIr). Finally, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is modified on its surface. The preparation process is as follows: Figure 1 As shown.

[0116] The specific steps are as follows:

[0117] (1) A virus-like biomimetic magnetic nanozyme (FeAu@AuIr) was prepared according to Example 1.

[0118] (2) 1.5g FeAu@AuIr was mixed with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) ethanol solution and ultrasonically treated (100HZ, 25℃, 120min) to obtain a virus-like biomimetic magnetic nanozyme with carboxyl groups on the surface.

[0119] (3) Mix 2g of DTNB-modified viral biomimetic magnetic nanozyme with 100mL of activation solution and sonicate (100HZ, 25℃, 120min).

[0120] The activation solution formula is as follows: each 100 mL of deionized water contains 0.2 g N-hydroxysuccinimide (NHS), 0.2 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 0.2 g morpholine ethanesulfonic acid (MES).

[0121] (4) The activated DTNB-modified viral biomimetic magnetic nanozyme was coupled with biotin-BSA (0.2 mg biotin-BSA was added to every 1 g of DTNB-modified viral biomimetic magnetic nanozyme), incubated at room temperature for 2 h, and bovine serum albumin (BSA) solution was added to block the unreacted carboxyl groups to prepare the viral biomimetic magnetic nanozyme control probe (FeAu@AuIr-DTNB).

[0122] Example 3 Structural Characterization Results

[0123] The products of each stage in Examples 1 and 2 were detected by high-resolution transmission electron microscopy, scanning electron microscopy, and energy-dispersive X-ray spectroscopy.

[0124] Figure 2 Transmission electron microscopy (TEM) images a, e, and i show the typical morphologies of the prepared Fe3O4, FeAu, and FeAu@AuIr nanozymes, respectively. Figure 2 Images b, f, and g show magnified views of Fe3O4, FeAu, and FeAu@AuIr, respectively. It can be seen that Au is densely and uniformly loaded on the Fe3O4 surface. Figure 2 (f), magnified TEM image of FeAu@AuIr ( Figure 2 In the image (j), AuIr can be clearly seen on the FeAu surface and in the Au gaps. High-resolution TEM image (j) Figure 2The c, g, and k sequences further revealed the single crystals of the assembled Fe3O4, FeAu, and FeAu@AuIr particles, with lattice spacings of 0.261 nm, 0.231 nm, and 0.227 nm, respectively, corresponding to the (311) plane of the cubic Fe3O4 structure, the (111) plane of Au, and the (111) plane of Ir. Energy-scattered X-ray spectroscopy (EDS) elemental scanning... Figure 2 (m) and element mapping ( Figure 2 The n) can clearly show the composition and distribution of each element in FeAu@AuIr.

[0125] Example 4: Detection of the hydrogen peroxide catalytic ability of virus-inspired biomimetic magnetic nanozymes

[0126] FeAu@AuIr, Fe@AuIr (prepared using the same method as in Example 1, without an Au shell), AuIr5 (5nm), Fe3O4, FeAu (prepared using the same method as in Example 1, without an AuIr shell), and Au were selected. 15 Using 15nm as the analyte, the peroxidase-like activity of nanozymes was indirectly quantified using the conventional 3,3',5,5'-tetramethylbenzidine (TMB) oxidation colorimetric method. (200 μL of sodium acetate buffer (pH=4), 10 μL of TMB, 10 μL of H2O2, and 1 μL of nanozyme (2 mg / mL) were mixed and allowed to stand for 2 min.) The absorbance of oxidized TMB was measured using a microplate reader to determine the catalytic activity of the corresponding nanozymes. DFT and FDTD simulation models were constructed to simulate the peroxidase-like activities of FeAu@AuIr and Fe@AuIr. The catalytic ability was verified using the Michaelis-Menten equation (the rate equation relating the initial rate of an enzyme-catalyzed reaction to the substrate concentration).

[0127] The results are as follows Figure 3 As shown, FeAu@AuIr exhibits the strongest peroxidase-like activity.

[0128] The results are as follows Figure 3 As shown in Figure a, the presence of the Au shell significantly increases the colorimetric signal of the nanozyme, while the presence of AuIr greatly increases its catalytic activity. Furthermore, since Au simultaneously provides a larger surface area for AuIr deposition, FeAu@AuIr not only exhibits a higher colorimetric signal but also possesses higher peroxidase-like activity. Figure 3As shown in Figure bc, the concentrations of the four magnetic nanozymes were sequentially diluted (1000-0.49 fM). 200 μL of sodium acetate buffer (pH=4), 10 μL of TMB, 10 μL of H₂O₂, and 1 μL of nanozyme (2 mg / mL) were mixed, and the absorbance of the mixed solution at 652 nm was measured after 2 min. As the concentrations of the four magnetic nanozymes decreased, the solution color gradually changed from blue to colorless. Figure 3 In part b), the absorbance of the reaction solution at 652 nm decreased accordingly. Figure 3 (c) Visual detection limits for TMB by Fe3O4, FeAu, Fe@AuIr, and FeAu@AuIr were 125, 250, 7.81, and 1.95 fM, respectively, for the blue signal observed with the naked eye. Steady-state kinetics experiments were used to quantify the peroxidase-like activity and substrate binding affinity of the nanozyme. The Michelis constant (Km) represents the affinity for substrate binding, while the maximum reaction rate (Vmax) represents the catalytic activity. Figure 3 As shown in Figure d, the Vmax and Km of FeAu@AuIr for H2O2 are 85.27 M / s (I) and 1.85 mM, respectively, while the Vmax and Km of FeAu@AuIr for TMB are 32.89 M / s and 2.48 mM (II), respectively, further demonstrating that the proposed multi-tentacle magnetic nanozyme possesses the highest biocatalytic activity. Figure 3 As shown in Figure f, the strong electric field is mainly distributed in the gaps between the particles. The maximum enhancement factors for FeAu and FeAu@AuIr are 284 times and 340 times, respectively. These results indicate that the presence of AuIr enhances the electronic properties of the nanozyme. Figure 3 As shown in Figure e, the energy barrier of FeAu@AuIr is significantly lower than that of FeAu in the key step (step four), indicating that FeAu@AuIr has less resistance when generating free radicals. That is, the large amount of AuIr loaded on the surface of the nanomaterial has a significant effect on enhancing its peroxidase-like catalytic activity.

[0129] Example 5: Detection of FeAu@AuIr-MPBA capture results

[0130] The SARS-CoV-2 antigen spike protein, Pseudomonas aeruginosa, and Streptococcus pneumoniae were detected using FeAu@AuIr-MPBA. FeAu@AuIr-MPBA (5 μL, 2 mg / mL) and naked FeAu@AuIr (1 μL, 2 mg / mL) were incubated with biotin-conjugated SARS-CoV-2 SP (2 μL, 1 mg / mL) for 5 min. After incubation, magnetic separation and washing were performed, followed by reaction with a streptavidin-FITC fluorescent probe. The success of SARS-CoV-2 SP capture by FeAu@AuIr-MPBA was determined by observing the fluorescent molecules. FeAu@AuIr-MPBA (5 μL, 2 mg / mL) was also incubated with Pseudomonas aeruginosa (1 μL, 10 mg / mL) for 5 min. 5 cells / mL) and Streptococcus pneumoniae (1μL,10 5 After mixing (cells / mL) and vortexing for 5 min, the capture efficiency of FeAu@AuIr-MPBA against viral proteins and the two target bacteria was observed using transmission electron microscopy. Next, the classic BCA protein quantification method and plate counting method were used to determine the capture efficiency of FeAu@AuIr-MPBA against viral proteins and the two target bacteria, respectively.

[0131] Bacterial capture efficiency (%) = (initial bacterial concentration - bacterial concentration after capture) / initial bacterial concentration × 100%.

[0132] Viral protein capture efficiency (%) = (initial absorbance - absorbance after capture) / initial absorbance × 100%.

[0133] Figure 4 As shown in Figure a, after reaction with SARS-CoV-2 SP-biotin, a large number of green fluorescent molecules specifically bound to FeAu@AuIr-MPBA, while no obvious green fluorescent signal appeared on the surface of unmodified FeAu@AuIr under the same conditions. These findings indicate that only MPBA-modified FeAu@AuIr has the ability to capture target viral proteins. Simultaneously, TEM observation... Figure 4 As shown in b), MPBA-modified FeAu@AuIr can rapidly bind to the surfaces of Pseudomonas aeruginosa (I) and Streptococcus pneumoniae (II), indicating its ability to simultaneously recognize Gram-positive and Gram-negative bacteria. Results based on the BCA detection kit and plate counting method are as follows... Figure 4As shown in Figure c, the capture efficiencies of FeAu@AuIr-MPBA against SARS-CoV-2SP, Pseudomonas aeruginosa, and Streptococcus pneumoniae were 87.83%, 93.42%, and 93.33%, respectively. Figure 4 (d). Furthermore, FeAu@AuIr-MPBA exhibited a clear time-dependent effect in the efficient enrichment of bacteria / viruses; a 5-minute incubation time was sufficient to achieve saturation capture of viral glycoproteins and bacteria. Figure 4 (e).

[0134] Example 6 Immunochromatography based on broad-spectrum virus-like biomimetic magnetic nanozyme probes

[0135] This invention provides an immunochromatographic assay based on a broad-spectrum virus-inspired biomimetic magnetic nanozyme probe. Figure 5 The kit includes a sample pad for loading sample solutions, a nitrocellulose membrane, an absorbent pad, a broad-spectrum virus-inspired magnetic nanozyme probe and a control line probe for detection, and a run buffer.

[0136] The running buffer composition ratio is Tween 20:Milk (10wt%):PBS = 1:10:1000.

[0137] The sample pad is used to load the sample solution to be tested; the absorbent pad is used to provide capillary force; the nitrocellulose membrane is loaded with several test lines and one control line. There are three test lines, which are modified with antibodies against the SARS-CoV-2 antigen spike protein, Pseudomonas aeruginosa, and Streptococcus pneumoniae, respectively. The control line is modified with streptavidin.

[0138] The specific testing process is as follows:

[0139] 1 mL of the sample to be tested was mixed with 4 μL of virus-like biomimetic magnetic nanozyme probe (FeAu@AuIr-MPBA, 2 mg / mL) and 2 μL of quality control line probe (FeAu@AuIr-DTNB, 2 mg / mL), and then incubated together on a shaker for 5 minutes. The bacterial-probe immune complex was then recovered by magnetic enrichment of the magnetic nanozyme probe.

[0140] The sample was resuspended in 80 μL of running buffer and loaded onto the sample pad of the immunochromatographic test strip. The sample was then brought into contact with the detection line on the immunochromatographic test strip to initiate the chromatographic reaction. After 13 min, the sample was catalyzed for 2 min with a catalyst. The colorimetric signal on the detection line of the immunochromatographic test strip was then visually read and read using an immunochromatographic analyzer.

[0141] Example 7 Specificity Detection

[0142] The experimental groups included mixed samples of Streptococcus pneumoniae / Aeruginosa / SARS-CoV-2 (1), SARS-CoV-2SP (2), Pseudomonas aeruginosa (3), Streptococcus pneumoniae (4), and non-target pathogen samples, including influenza A virus (5), influenza B virus (6), respiratory syncytial virus (7), adenovirus (8), Staphylococcus aureus (9), Mycoplasma pneumoniae (10), Klebsiella pneumoniae (11) and Legionella pneumophila (12), as well as a blank control group (13).

[0143] The concentration of SARS-CoV-2SP protein was 10 ng / mL; the concentration of bacteria and viruses was 10 ng / mL. 5 cells / mL.

[0144] Referring to the detection process in Example 6, the above-mentioned pathogens were subjected to immunochromatographic testing.

[0145] The results are as follows Figure 6 As shown in a, b, and c, the photographs of the immunochromatographic test strips and the colorimetric signals on the detection lines clearly demonstrate that the broad-spectrum viral biomimetic magnetic nanozyme probe has good selectivity for the target pathogen.

[0146] Furthermore, scanning electron microscopy (SEM) was used to observe the T-line of positive and negative samples after the reaction. The target-virus-viral biomimetic magnetic nanozyme probe complex was clearly observed at the T-line of the positive sample. Figure 6 (d).

[0147] Example 8: Optimization of Reaction Conditions

[0148] This embodiment optimizes the amount of broad-spectrum virus-like biomimetic magnetic nanozyme probe added. Figure 7 a) Running buffer components ( Figure 7 (b) Incubation time ( Figure 7 c), reaction time ( Figure 7 a), catalytic time ( Figure 8 (b) Different target antibody concentrations on the NC membrane ( Figure 8 c) Proportion of effective components in the catalyst solution ( Figure 8 (d).

[0149] The specific process is as follows: 1 / 2 / 3 / 4 / 5 μL of virus-like biomimetic magnetic nanozyme probe and 2 μL of quality control line probe (both 2 mg / mL) are added to 1 mL of the sample to be tested. After mixing and incubating for 3 / 4 / 5 / 6 / 7 min, the bacteria-probe immune complex is recovered by magnetic enrichment of the magnetic nanozyme probe. The sample was resuspended in 80 μL of running buffer (1% PBST, 1% PBST + 5% FBS, 1% PBST + 10% FBS, 1% PBST + 5% Milk, 1% PBST + 10% Milk) and loaded onto the sample pad of an immunochromatographic test strip loaded with different coating concentrations (0.6 / 0.8 / 1.0 / 1.2 / 1.5 mg / mL). The sample pad was then brought into contact with the detection line on the immunochromatographic test strip to initiate the chromatographic reaction. After 10 / 11 / 12 / 13 / 14 / 15 minutes, a catalytic solution (H2O2:AEC = 2:8 / 3:7 / 4:6 / 5:5 / 6:4 / 7:3 / 8:2) was applied to initiate catalysis. After 40 / 60 / 80 / 100 / 120 / 140 / 160 seconds, the colorimetric signal on the detection line of the immunochromatographic test strip was visually read, and the colorimetric signal was also read using an immunochromatographic analyzer.

[0150] The above experiment is a horizontal comparison, with only one variable changing each time, and the remaining variables being the first condition mentioned above.

[0151] Based on the signal-to-noise ratio, the optimal conditions were selected as follows: 4 μL probe volume, incubation for 5 min, running buffer composition of 1% PBST + 5% Milk, chromatography time of 13 min, catalysis time of 2 min, and the membrane scratching concentrations of SARS-CoV-2SP, Pseudomonas aeruginosa, and Streptococcus pneumoniae were 1.0, 1.2, and 1.2 mg / mL, respectively. The effective component ratio of the catalytic solution was H2O2:AEC = 4:6.

[0152] Example 9 Sensitivity Test

[0153] This example selects different concentrations (0-10 ng / mL; 0-10 5 To evaluate the sensitivity of simultaneous detection of respiratory bacteria and viruses using a mixed sample of SARS-CoV-2, Pseudomonas aeruginosa, and Streptococcus pneumoniae (cells / mL), a virus-like biomimetic magnetic nanozyme immunochromatography method was employed. Detection was performed using the final optimized conditions described in Example 8.

[0154] Figure 9Figure 'a' shows a photograph of the test strip and a colorimetric signal thermogram after direct detection of the mixed sample solution. The visual limits of detection (vLOD) for SARS-CoV-2, Pseudomonas aeruginosa, and Streptococcus pneumoniae in the pre-catalytic colorimetric mode were 0.1 ng / mL, 5000 cells / mL, and 5000 cells / mL, respectively.

[0155] Photographs of the test strip enhanced with catalytic substrate and the intensity of the T-line signal are as follows: Figure 9 As shown in Figure b, at concentrations of 0.005 ng / mL, 100 cells / mL, and 100 cells / mL in the post-catalysis colorimetric mode, the vLOD of the post-catalysis colorimetric mode was 50 times higher than that of the pre-catalysis colorimetric mode. An S-shaped calibration curve was plotted to show the relationship between the colorimetric signal and the target concentration on the T-line. Figure 9 The results showed that the magnetic nanozyme probe immunochromatographic assay had high analytical performance against Helicobacter pylori, a wide detection dynamic range (5 orders of magnitude), and a good correlation coefficient (Rc). 2 >0.98), and LODs were 0.0012 ng / mL, 17 cells / mL, and 21 cells / mL, respectively.

[0156] Example 10 Repeatability Test

[0157] This embodiment uses three different concentrations (10, 1, 0.1 ng / mL, 10... 5 10 4 10 3 A mixed sample of SARS-CoV-2, Pseudomonas aeruginosa, and Streptococcus pneumoniae (cells / mL) was used to verify the reproducibility of immunochromatography based on virus-like biomimetic magnetic nanozyme probes. The detection method used the optimal parameters obtained in Example 8.

[0158] like Figure 10 As shown, the colorimetric signal on the detection line changed little, and the RSD values ​​of each test group were ≤7.5%, indicating that the established immunochromatographic assay based on virus-like biomimetic magnetic nanozyme probes has good reproducibility.

[0159] Example 11 Spike Recovery and Real Sample Detection

[0160] This example uses different concentrations (10, 1, 0.1 and 0.01 ng / mL, 10 5 10 4 10 3 and 10 2The SARS-CoV-2 antigen spike protein (cells / mL), Pseudomonas aeruginosa, and Streptococcus pneumoniae were added to approximately 5 mL of throat swab samples from healthy volunteers to evaluate the practicality of immunochromatography based on broad-spectrum virus-like biomimetic magnetic nanozyme probes. For example... Figure 11 As shown, the recoveries of the three targets before and after catalysis were calculated based on the gray values ​​of the detection lines. The recoveries before catalysis were 86.19%-115.94%, and the recoveries after catalysis were 87.08%-111.00%, indicating that this method has good accuracy and reliability for the detection of real saliva samples. It is worth noting that the detection range for lower concentrations of bacteria before catalysis could not be calculated using the corresponding standard curve, while the detection range was significantly improved after catalysis.

[0161] In addition, this embodiment collected 59 SARS-CoV-2 positive pharyngeal swab samples, 59 P. aeruginosa positive bronchoalveolar lavage fluid samples, 52 S. pneumoniae positive bronchoalveolar lavage fluid samples, 15 negative pharyngeal swab samples, and 20 negative bronchoalveolar lavage fluid samples from the Department of Laboratory Medicine of Guangdong Provincial People's Hospital. This further validated the immunochromatography based on a broad-spectrum virus-like biomimetic magnetic nanozyme probe (the collection of the above samples was approved by the Ethics Review Committee of Guangdong Provincial People's Hospital, and informed consent was obtained from the patients). The detection results of SARS-CoV-2 were compared with those of ELISA, and the detection results of the two bacteria were compared with those of qPCR. Deming regression analysis showed that the slopes for SARS-CoV-2, Pseudomonas aeruginosa, and Streptococcus pneumoniae were all close to 1 (SARS-CoV-2: 1.019, Pseudomonas aeruginosa: 0.849, Streptococcus pneumoniae: 0.842), with 95% confidence intervals of 0.9690–0.9893, 0.9498–0.9821, and 0.9570–0.9858, respectively. The ROC curves further demonstrated that the reagents of this invention have 100% sensitivity and specificity for real clinical specimens. Furthermore, they offer significant advantages in terms of ease of operation and analysis time, and are expected to become a powerful tool for the detection of respiratory pathogens in clinical practice.

Claims

1. A nanozyme probe, characterized in that: Including magnetic cores, gold nanoparticles, and gold-iridium alloy nanoparticles; The gold nanoparticles are loaded on the surface of the magnetic core; The gold-iridium alloy nanoparticles are loaded on the surface of the gold nanoparticles and the surface of the magnetic core. The magnetic core and the surface of the gold nanoparticles are modified with a cationic polymer layer; The cationic polymer layer includes PEI, PLL, PAAs, or PAEs; The surface of the nanozyme probe is modified with 4-mercaptophenylboronic acid.

2. The nanozyme probe according to claim 1, characterized in that: The magnetic core includes at least one of Fe3O4, Fe2O3, Co-Ni alloy, Fe-Co alloy, and Fe-Pt alloy.

3. The method for preparing the nanozyme probe according to any one of claims 1 to 2, comprising the following steps: 1) Modify the magnetic core with a cationic polymer layer; 2) Gold nanoparticles are loaded onto the magnetic core of the modified cationic polymer layer to obtain gold-shelled magnetic beads; 3) Modify the gold-shelled magnetic beads with a cationic polymer layer; 4) Gold-iridium alloy nanoparticles were loaded onto gold-shelled magnetic beads modified with cationic polymer layers to obtain nanozymes; 5) Disperse the nanozyme in water, mix it with a mercaptophenylboronic acid ethanol solution, and sonicate to obtain a nanozyme probe.

4. The use of the nanozyme probe according to any one of claims 1 to 2 in the preparation of products for detecting bacteria or viruses.

5. A reagent kit, characterized in that: The kit includes a control line probe and a nanozyme probe as described in any one of claims 1 to 2.

6. The reagent kit according to claim 5, characterized in that: The quality control line probe comprises a magnetic core, gold nanoparticles, and gold-iridium alloy nanoparticles. The gold nanoparticles are loaded on the surface of the magnetic core; The gold-iridium alloy nanoparticles are loaded on the surface of the gold nanoparticles and the surface of the magnetic core. The magnetic core and the surface of the gold nanoparticles are modified with a cationic polymer layer; The cationic polymer layer includes PEI, PLL, PAAs, or PAEs; The surface of the quality control line probe is modified with 5,5'-dithiobis(2-nitrobenzoic acid).

7. The kit according to claim 5 or 6, characterized in that: The kit includes test strips; The test strip includes a sample pad for loading the sample solution, a nitrocellulose membrane, and an absorbent pad, wherein the nitrocellulose membrane is provided with a detection line and a control line.

8. The reagent kit according to claim 7, characterized in that: The kit includes a running buffer and a catalytic buffer.

Images