Method for constructing label-free immune platform through enzyme / antibody non-covalent modification of ZnNFs and ZnNPs

Abstract

The invention relates to a method for constructing a label-free immune platform through enzyme / antibody non-covalent modification of ZnNFs and ZnNPs, and belongs to the technical field of nano biosensing. The method comprises the following steps: respectively synthesizing ZnNFs and ZnNPs under the conditions of 25 DEG C and 4 DEG C based on a zinc nitrate and 3-methyl-1H-1, 2, 4-triazole (Hmtz) one-pot synthesis method, and constructing a completely non-labeled enzyme-linked immunosorbent assay platform to realize the detection of cardiac troponin I. The ZnNFs and the ZnNPs have the advantages of simple synthesis, uniform size, strong adsorption capacity and the like, the newly constructed magnetic separation complete non-labeled enzyme-linked immunosorbent assay platform successfully realizes the detection of cardiac troponin I and new coronavirus N protein in a complex environment, and the standard recovery rate experiment is within the standard error range. The sensing platform has huge potential in the aspect of disease diagnosis.

Description

Technical Field

[0001] The invention relates to a method for constructing a label-free immune platform by non-covalently modifying ZnNFs and ZnNPs through enzyme / antibody, and belongs to the field of nanotechnology. Background Art

[0002] ELISA is a very efficient in vitro diagnostic method that can quickly and accurately detect compounds such as antibodies, antigens, proteins and hormones, and is widely used in environmental monitoring, food safety and medical clinical testing. The advantage of ELISA is that it does not require complicated sample pretreatment, nor does it require the use of radioactive substances and most organic solvents, and it has strong specificity, high sensitivity and extremely strong safety. However, traditional ELISA technology has the disadvantages of cumbersome operation steps, long time consumption, and the need for enzyme labeling of antibodies. Therefore, it is urgent to further improve the traditional ELISA technology to meet the needs of simpler, faster and more efficient in vitro diagnosis.

[0003] So far, some strategies have been proposed to improve the analytical performance of traditional ELISA, such as developing more sensitive enzyme colorimetric and fluorescent substrates, using blockers and coatings that can reduce background noise, improving antibody affinity, introducing new antibody fragments and nanobodies, using biotin-streptavidin system, coupling ELISA with polymerase chain reaction or using microfluidics, and introducing nanomaterials as carriers. Among them, the immobilization of multiple enzymes and antibodies by introducing various nanomaterials as carriers has always been the focus of research. This method can immobilize multiple enzymes and antibodies on a single nanoparticle, thereby significantly improving the sensitivity of ELISA and expanding the detection range. However, these nanoparticles used for immunoassays are covalently linked to antibodies and enzymes. The covalent method is not only expensive and complicated, but also inevitably affects the catalytic performance of the enzyme and the biological activity of the antibody. In recent years, many label-free biosensors have been developed, such as quartz crystal-microbalance biosensors, surface plasmon resonance biosensors, optical resonance biosensors, field effect transistor biosensors, etc. The development of these new sensors enables us to quickly detect analytes without the need for culture or washing processes. However, there are relatively few reports on the construction of ELISA using label-free modification methods. Biswas et al. reported a label-free electrochemical immunosensor based on biocompatible MOF-808 / CNT immobilized antibody for the detection of carbohydrate antigen 125. However, the immobilization of the capture antibody in this immunoassay is mainly based on the biotin-avidin system, which is easily interfered by free biotin, thus affecting the detection results, and the immunoassay only achieves partial label-free in the sensing system. Therefore, it is necessary to introduce a new method to develop a completely label-free ELISA method.

[0004] In order to overcome these shortcomings, we used zinc nitrate hexahydrate and Hmtz as raw materials to synthesize a particle-wire-flower directional self-assembled ZnNFs and a nanoparticle ZnNPs, respectively, and proposed a new strategy for the efficient combination of ZnNFs and ZnNPs with proteins or enzymes (capture antibody @ ZnNFs as solid phase carrier, enzyme / labeled antibody @ ZnNPs as signal reporting unit), and constructed a completely label-free enzyme-linked immunosorbent sensing platform. The successful implementation of this work provides a revolutionary method for traditional enzyme-linked immunosorbent assay detection. Summary of the invention

[0005] The technical problem solved by the present invention is: in order to overcome the shortcomings of the prior art, a construction strategy of a completely label-free enzyme-linked immunosorbent sensing platform based on ZnNFs and ZnNPs is proposed, which is used for sensitive and selective detection of cardiac troponin and new coronavirus N protein. With ZnNFs and ZnNPs as anchors, ZnNFs replace 96-well plates and combine with monoclonal antibodies to capture disease markers, ZnNPs adsorb HRP and primary antibodies to be responsible for signal output, and the experimental signal is then output as a fluorescent or colorimetric signal through a DA-DHA-H2O2 or TMB system. In addition, in view of the co-synthesis of magnetic iron oxide nanoparticles, zinc nitrate and 3-methyl-1H-1,2,4-triazole to produce magnetic zinc-based complex nanoflowers, we designed a dual-signal completely label-free magnetic separation enzyme-linked immunosorbent platform for specific colorimetric and fluorescent detection of cTnI and new coronavirus N protein.

[0006] In order to solve the technical problem of the present invention, the proposed technical solution is: a method for constructing a completely label-free enzyme-linked immunosorbent assay platform for detecting cardiac troponin I by non-covalent modification of ZnNFs and ZnNPs by enzyme / antibody.

[0007] The particle-wire-flower self-assembled ZnNFs successively adsorbed the capture antibody Capture ab-mouse anti-cTnI monoclonal antibody and bovine serum albumin BSA to form a BSA / Capture ab@ZnNFs complex. The spherical ZnNPs were sequentially combined with horseradish peroxidase HRP, labeled with antibody Ab1-sheep anti-cTnI primary antibody and BSA to generate a BSA / Ab1 / HRP@ZnNPs complex. In PBS buffer solution, different concentrations of cTnI were incubated with the BSA / Capture ab@ZnNFs complex, and the BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution. After further incubation and washing, 300 μL of 400 μM DA, 30 μM DHA and 60 μM H2O2 in 10 mM, pH = 7.4 Tris-HCl buffer solution generate a yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer. The excitation wavelength and emission wavelength of AFC are 490 nm and 550 nm, respectively.

[0008] Preferably, the synthesis conditions of ZnNFs are as follows: 1 mL of zinc nitrate (1.6 mM) solution is poured into 1 mL of Hmtz (8.0 mM) solution (the solvents are all ultrapure water), and the mixture is gently mixed. The mixed solution is kept at a constant temperature of 25° C. for 6 hours to obtain a mixed solution containing white precipitated ZnNFs, which is then washed and used for later use.

[0009] The synthesis conditions of ZnNPs are as follows: 2 mL of zinc nitrate solution (0.4 mM) was added to 2 mL of Hmtz solution (2.0 mM) in batches of 200 μL (all solvents were ultrapure water, vortexed while adding), and placed in a constant temperature environment of 4°C for 24 hours to obtain a mixed solution of white precipitated ZnNPs.

[0010] Preferably, the sensing platform conditions are as follows: the concentration of the capture antibody is 1 μg / mL, the concentration of BSA bound to ZnNFs is 20 mg / mL, the concentration of HRP is 1 U / mL, the concentration of the labeled antibody is 2 μg / mL, the concentration of BSA bound to ZnNPs is 10 mg / mL, and the two complexes of BSA / Ab1 / HRP@ZnNPs and BSA / Capture ab@ZnNFs are prepared and placed at 4°C for use.

[0011] In order to solve the technical problem of the present invention, another technical solution is proposed: a method for detecting cTnI based on a fluorescent colorimetric dual-channel non-labeled enzyme-linked immunosorbent sensor constructed based on ZnNFs, a series of cTnI with different concentrations and BSA / Capture ab@ZnNFs complexes are incubated at 37°C, and then Ab1 and HRP-secondary antibody (HRP-labeled rabbit anti-sheep polyclonal antibody) are incubated in the system in turn, and DA, DHA and H2O2 or TMB colorimetric solution are added to the final complex Tris-HCl buffer solution. After reacting at room temperature for several minutes, the fluorescence spectrum and absorption intensity analysis are performed, and the absorption of the oxidized TMB is fixed at 652nm.

[0012] The synthesis conditions of ZnNFs are as follows: 1 mL of zinc nitrate (1.6 mM) solution is poured into 1 mL of Hmtz (8.0 mM) solution (all solvents are ultrapure water), and the mixture is gently mixed. The mixed solution is kept at a constant temperature of 25° C. for 6 hours to obtain a mixed solution containing white precipitated ZnNFs, which is washed for later use; the particle-wire-flower self-assembled ZnNFs successively adsorb capture antibodies (Capture ab) and bovine serum albumin (BSA, blocking agent) to form a BSA / Capture ab@ZnNFs complex. According to claim 5, the method for detecting cTnI based on ZnNFs using a fluorescent colorimetric dual-channel non-labeled enzyme-linked immunosorbent sensor is characterized in that: the sensing platform conditions are as follows: the concentration of the labeled antibody is Ab1 2 μg / mL, the concentration of the HRP-secondary antibody is 1 μg / mL, and the system is subjected to fluorescence and absorption dual signal analysis and detection by a fluorescence spectrometer and an ultraviolet spectrophotometer.

[0013] In order to solve the technical problem of the present invention, another technical solution is proposed: a method for detecting cTnI based on a non-labeled enzyme-linked immunosorbent assay constructed by ZnNPs, wherein the capture antibody is incubated with a 96-well plate overnight and then blocked with BSA; a series of cTnI with different concentrations are added to each well for incubation and washing; finally, a BSA / Ab1 / HRP@ZnNPs complex is added, incubated and washed again, and a Tris-HCl buffer solution containing DA, DHA and H2O2 is added, and the fluorescence spectrum and absorption intensity are analyzed after a few minutes of reaction.

[0014] The synthesis conditions of ZnNPs are as follows: 2 mL of zinc nitrate solution (0.4 mM) was added to 2 mL of Hmtz solution (2.0 mM) in batches of 200 μL (the solvents were all ultrapure water, and vortexing was performed while adding), and the mixture was placed in a constant temperature environment of 4 °C for 24 hours to obtain a mixed solution of white precipitated ZnNPs. The spherical ZnNPs were sequentially combined with horseradish peroxidase (HRP), labeled antibody (Ab1) and BSA to generate BSA / Ab1 / HRP@ZnNPs complexes.

[0015] Preferably, the sensing platform conditions are as follows: the concentration of the capture antibody is 1 μg / mL, the concentration of BSA is 1 mg / mL, and the fluorescence signal analysis and detection of the system is performed by a fluorescence spectrometer.

[0016] In order to solve the technical problem of the present invention, another technical solution is proposed: a completely label-free magnetic separation enzyme-linked immunosorbent sensor method for detecting cTnI or N protein based on magnetic ZnNFs and ZnNPs, wherein the magnetic ZnNFs successively adsorb the capture antibody and BSA to form a BSA / Capture ab@magnetic ZnNFs complex, and the spherical ZnNPs are successively combined with HRP, the labeled antibody (Ab1) and BSA to form a BSA / Ab1 / HRP@ZnNPs complex; in a PBS buffer solution, different concentrations of cTnI and BSA / Capture ab@magnetic ZnNFs are mixed. After the ab@ZnNFs complex was incubated for reaction, BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution; after further incubation and washing, Tris-HCl (10mM, pH=7.4) buffer solution or TMB colorimetric solution containing dopamine (DA, 400μM), 1,5-naphthalenediol (DHA, 30μM) and hydrogen peroxide (H2O2, 60μM) was added. The former was used to detect the fluorescence intensity of the synthesized AFC by a fluorescence spectrometer, and the latter was used to detect the absorption intensity of the synthesized oxTMB by a UV spectrophotometer.

[0017] Magnetic ZnNFs successively adsorbed capture antibody (mouse anti-N protein monoclonal antibody, Capture ab) and BSA to form BSA / Capture ab@magnetic ZnNFs complex. Spherical ZnNPs successively combined with HRP, labeled antibody (rabbit anti-N protein primary antibody, Ab1) and BSA to form BSA / Ab1 / HRP@ZnNPs complex. In PBS buffer solution, different concentrations of N protein were mixed with BSA / Capture ab. After the ab@ZnNFs complex was incubated, BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution; after further incubation and washing, Tris-HCl (10mM, pH=7.4) buffer solution or TMB colorimetric solution containing dopamine (DA, 400μM), 1,5-naphthalenediol (DHA, 30μM) and hydrogen peroxide (H2O2, 60μM) was added, and the fluorescence intensity and absorption intensity of the synthesized AFC and oxTMB were detected by fluorescence spectrometer and UV spectrophotometer, respectively.

[0018] Preferably, the synthesis conditions of magnetic ZnNFs are as follows: 0.2703g of ferric chloride hexahydrate (0.2mM) and 0.0663g of ferrous chloride tetrahydrate (0.0667mM) are dissolved in 5mL of ultrapure water, stirred evenly at room temperature, and 1.5mL of ammonia water is quickly added to the mixed solution (the color of the solution immediately changes from orange to black), the mixed solution is ultrasonicated for 10 minutes, and then allowed to stand at room temperature for 10 minutes to obtain a black Fe3O4 NPs suspension; then, 1mL of zinc nitrate (1.6mM) solution is poured into 1mL of Hmtz (8.0mM) solution (the solvents are all ultrapure water), and gently mixed; then, 0.0160g of magnetic iron oxide nanoparticles are weighed and poured into the former mixed solution, and slowly mixed. Then, the mixed solution is placed in a 25°C water bath for 6 hours to obtain a solution containing brown magnetic ZnNFs precipitate, and the synthesized magnetic ZnNFs are characterized by an X-ray diffractometer and a hysteresis loop.

[0019] Zn(NO3)2 and Hmtz were reacted at room temperature for 6 hours to synthesize ZnNFs. The directional self-assembly process of ZnNFs was characterized and analyzed in detail by scanning electron microscopy (SEM), X-ray diffractometer (XRD), energy spectrum element analyzer (EDS), etc. It was inferred that ZnNFs are zinc-based coordination compounds formed by directional self-assembly of particles-wires-flowers.

[0020] Furthermore, in order to explore the formation mechanism of ZnNFs nanoflower materials, the products at different stages of the ZnNFs formation process were characterized by SEM. As the substrate reaction time increases, SEM results show that ZnNFs exist in three states: particles, wires, and flowers.

[0021] Furthermore, in order to further explore the influence of other factors on the morphology of ZnNFs, the products of the overall substrate concentration were characterized by SEM. 2+ :Hmtz=1:5,Zn 2+ :0.1, 0.2, 0.4, 0.8, 1.6 mM) on ZnNFs. The SEM results showed that only these results showed that the overall concentration of the substrate played a key role in the formation of ZnNFs. 2+ Only when Hmtz is 1.6mM and Hmtz is 8mM can nanoflowers with uniform size and symmetrical morphology be formed.

[0022] Furthermore, in order to verify the feasibility of protein adsorption on ZnNFs nanoflowers, green fluorescent protein (GFP), rhodamine B-labeled BSA (RB-BSA) and Cy5-labeled antibody (Cy5-ab) were incubated with ZnNF nanoflowers and centrifuged and washed to obtain GFP / Ab / BSA@ZnNFs. We characterized GFP / Ab / BSA@ZnNFs using fluorescence confocal microscopy and proved that the three proteins (GFP, BSA and antibody) effectively bound to ZnNFs. It can be seen that ZnNFs can not only be used as its carrier to bind to proteins through bioconjugation, but also its binding ability is universal.

[0023] Furthermore, the prepared BSA / Capture ab@ZnNFs can replace the traditional ELISA 96-well plate to construct a ZnNFs-based cTnI immunosensor. The immunosensor uses mouse anti-cTnI monoclonal antibody (Capture ab), goat anti-cTnI antibody (Ab1) and enzyme-labeled rabbit anti-goat IgG (HRP-ab2) for cTnI detection. The blocking protein BSA blocks the redundant sites. Previous work reported that HRP can induce a rapid in situ fluorescent reaction between DA and DHA in the presence of H2O2. Similarly, HRP can in situ catalyze TMB to generate blue oxTMB. Therefore, DA-DHA and TMB were selected as substrates of HRP-H2O2 for the fluorescence and colorimetric signal output of the immunosensor, respectively.

[0024] ZnNPs can be prepared by reacting Zn(NO3)2 with Hmtz at 4°C for 24 hours. SEM characterization shows that the obtained ZnNPs are spherical structures with an average size of about 100nm. Furthermore, in order to verify whether the prepared ZnNPs have good protein fixation ability, three proteins with different fluorescence wavelengths (GFP, RB-BSA and Cy5-ab) were incubated and combined with ZnNPs. The product was imaged by fluorescence confocal microscopy, and it can be observed that the three fluorescent proteins can be adsorbed to the surface of ZnNPs. This shows that ZnNPs can fix proteins well.

[0025] Furthermore, ZnNPs can be used to construct a non-labeled enzyme-linked immunosorbent sensing platform for detecting cTnI. The non-labeled sensor incubates and adsorbs three proteins, BSA (blocking part), Ab1 (recognition part) and HRP (catalytic part) onto ZnNPs to obtain a BSA / Ab1 / HRP@ZnNPs complex, which can be used to replace the expensive enzyme label ab2 in traditional ELISA. Capture ab is fixed on a 96-well plate. Through the specific binding of antigen and antibody, the Capture ab on the 96-well plate successively recognizes and captures cTnI and BSA@Ab1@HRP@ZnNPs complex. In the non-labeled platform system constructed based on ZnNPs, the relative fluorescence intensity shows a good linear relationship with the cTnI concentration.

[0026] Furthermore, the stability of the non-labeled platform system constructed based on ZnNPs was also explored. After the reaction solution system was added for the first time to detect the fluorescence spectrum for catalytic reaction, the supernatant was removed by centrifugation, and a new reaction solution was added to continuously carry out the catalytic reaction. After 6 cycles of reaction, the immunosensor system still maintained a catalytic activity of more than 90%. Alpha-fetoprotein (AFP), lysozyme (Lyso), trypsin (Try) and BSA were added to the system as interfering proteins to study the selectivity of the immunosensor. Only cTnI can cause a significant enhancement of the fluorescence signal. Therefore, a highly sensitive, highly selective and highly stable enzyme-linked immunosorbent sensing platform can be constructed based on the BSA / Ab1 / HRP@ZnNPs complex.

[0027] Furthermore, based on the excellent performance of ZnNFs and ZnNPs in constructing the immunosensing platform, a completely label-free fluorescent immunosensor was constructed by centrifugation. ZnNFs and ZnNPs acted as label-free carriers of proteins and combined with Capture ab, BSA, Ab1 and HRP accordingly. Then we used SEM to characterize the complexes assembled by ZnNFs and ZnNPs complexes with the corresponding proteins. With the gradual modification of proteins (Capture ab, BSA and cTnI), the rod-like structures on the ZnNPs nanoflowers gradually became thicker and larger. At the same time, SEM can also observe that the BSA / Capture ab@ZnNFs complex and the BSA / Ab1 / HRP@ZnNPs complex are anchored together by the antigen (cTnI). The combination of ZnNPs and ZnNFs also shows that the fluorescent immunosensor based on ZnNFs&ZnNPs was successfully constructed. Subsequently, we applied the label-free fluorescent immunosensor to detect cTnI in the dual channels of fluorescence and colorimetry.

[0028] Furthermore, the selectivity and stability of the fluorescent immunosensor system based on ZnNFs&ZnNPs were also studied. Only when cTnI was present in the reaction system could the system respond better. The constructed ZnNFs&ZnNPs complex was placed in an ultrasonic cleaner for different ultrasonic treatment times (0-120s), the last incubation solution was removed and cleaned as usual, and then the reaction solution was added to trigger the fluorescent reaction. The external ultrasonic force could not destroy the effective combination of ZnNFs and ZnNPs in the system within 2 minutes of treating the ZnNFs&ZnNPs complex, and it also had little effect on the adsorption force between the enzyme and ZnNPs.

[0029] Furthermore, the fluorescent colorimetric dual-channel sensing system based on magnetic ZnNFs & ZnNPs uses the magnetic ZnNFs complex with adsorbed capture antibody and BSA as the solid phase carrier, and the ZnNPs complex with fixed specific primary antibody and HRP as the signal reporting unit. The two are connected together through antigen-antibody specific binding, with cTnI as the intermediate medium. The large complex formed by the immune response can be enriched by the magnetic response performance carried by the magnetic ZnNFs. The ZnNPs in the complex show that the adsorbed HRP can catalyze the fluorescent colorimetric dual-channel reaction to quantify cTnI. Compared with the sensing platform constructed by magnetic iron oxide nanoparticles and ZnNFs alone, the newly constructed sensing platform has the advantages of convenient operation, low cost, wide detection range and high sensitivity, that is, it achieves the result of synergistic enhancement. Furthermore, in order to verify the versatility of the developed completely non-labeled magnetic separation immunosensor, the new coronavirus N protein was used to replace cTnI as a model antigen, and the detection linear relationship was still good. This is of great value to the development and application of composite materials, and also provides a new method for the widespread application of nanomaterials in clinical testing.

[0030] The present invention is further described below.

[0031] For the ZnNFs-based cardiac troponin I immunoassay, 100 μL of BSA / Captureab@ZnNFs complex solution was added to a centrifuge tube. Then, 100 μL of cTnI solution of different concentrations (0-300 ng / mL) was added and incubated at 37°C for 1 hour. The precipitate in the centrifuge tube was then washed 3 times with 200 μL TBST at 6000 rpm for 1 min, and then washed once with 100 μL TBST. 100 μL Ab1 (2 μg / mL) was added to the tube and incubated on a shaker at 37°C for 1 hour. After centrifugation, the supernatant in the tube was aspirated, washed 3 times with 200 μL TBST, washed once with 100 μL TBST, and then 100 μL of HRP-labeled rabbit anti-goat antibody (HRP-ab2, 1 μg / mL) diluted with PBS buffer was added to the tube and incubated at 37°C for 1 hour. Then the solution was aspirated and washed 4 times with TBST centrifugal washing solution. Finally, 300 μL of Tris-HCl buffer (10 mM, pH = 7.4) reaction solution containing DA (400 μM), DHA (30 μM) and H2O2 (60 μM) was added to each tube. Finally, the solution was placed at room temperature for reaction for 3 minutes and then the fluorescence spectrum (Ex = 490 nm) was measured. The experiment can also use a total volume of 300 μL of a mixed solution of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution A and B in equal proportions to replace the final reaction solution and react at room temperature for 15 minutes to measure the absorption spectrum.

[0032] In the method of cardiac troponin I immunoassay based on ZnNPs, 100 μL of Capture ab (1 μg / mL, prepared with PBS buffer) solution was added to a 96-well plate, and the well plate was incubated at 4°C overnight. After the incubation solution in the well was aspirated, it was washed 3 times with 200 μL TBST, and then washed once with 100 μL TBST, and then 200 μL of 20% BSA solution was added to each well and incubated at 37°C for 1 hour. After the solution was aspirated and washed 4 times with TBST, 100 μL of cTnI solution of different concentrations (0-200 ng / mL) was added to each well and incubated at 37°C for 1 hour. After the incubation solution in the well was aspirated and washed 4 times with TBST, 100 μL of BSA / Ab1 / HRP@ZnNP solution was added to each well and incubated at 37°C for 1 hour, and then the solution was aspirated and washed 4 times with TBST. Finally, 300 μL of the mixed solution containing DA-DHA-H2O2 was added to each well of the above plate and kept at room temperature for 3 minutes before measuring the fluorescence spectrum.

[0033] Method for cardiac troponin I immunoassay based on ZnNFs & ZnNPs: 100 μL BSA / Capture ab@ZnNFs complex solution was added to a 1.5 mL centrifuge tube. Then, 100 μL of cTnI solution (0-250 ng / mL) of different concentrations was added to the tube and placed on a shaker at 37°C for 1 hour. After washing 4 times with TBST at 6000 rpm for 1 minute, 100 μL BSA / Ab1 / HRP@ZnNPs complex solution was injected and incubated at 37°C for 1 hour. Finally, the supernatant was removed by centrifugation and washed again 4 times with TBST. After that, 300 μL of a mixed solution containing DA-DHA-H2O2 was added to each centrifuge tube, and the fluorescence spectrum was measured after the solution was allowed to stand for 3 minutes. Similarly, the reaction solution can also be replaced with TMB colorimetric solution, and the absorption spectrum is measured after reacting at room temperature for 15 minutes. In order to test the selectivity of the sensing platform for cTnI, some interfering substances were added to the system to replace cTnI to study their effects on the sensing system. These substances included AFP, Lyso, Try and BSA.

[0034] The method of constructing a sensing platform based on magnetic ZnNFs & ZnNPs to detect cTnI and SARS-CoV-2 N protein, 100μL BSA / Capture ab@magnetic ZnNFs complex solution was added to multiple 1.5mL centrifuge tubes. Then, 100μL cTnI solution (0-200ng / mL) of different concentrations was injected into each tube, and then incubated at 37°C for 1 hour. After washing 4 times with TBST under magnetic separation, 100μL BSA / Ab1 / HRP@ZnNPs complex solution was added and incubated at 37°C for 1 hour. Finally, the supernatant was removed after external magnet adsorption and washed 4 times with TBST by centrifugation. After that, 300μL of a mixture containing DA-DHA-H2O2 was added to each centrifuge tube, and the fluorescence spectrum was measured after being reacted at room temperature for 3 minutes. Similarly, the reaction solution can also be replaced with TMB colorimetric solution, and the absorption spectrum can be measured after reacting at room temperature for 15 minutes. The above platform can also be used to analyze and detect SARS-CoV-2N protein by replacing the detection protein and the corresponding antibody.

[0035] Beneficial effects:

[0036] We used zinc nitrate hexahydrate and Hmtz as raw materials to synthesize a particle-wire-flower directed self-assembled ZnNFs and a nanoparticle ZnNPs, respectively, and proposed a new strategy for the efficient combination of ZnNFs and ZnNPs with proteins or enzymes (capture antibody@ZnNFs as solid phase carrier, enzyme / labeled antibody@ZnNPs as signal reporting unit), and constructed a completely label-free enzyme-linked immunosorbent sensing platform for detecting cTnI. In addition, the developed sensing platform has been successfully applied to the detection of cTnI in complex environments. This work is of great value for the detection of disease markers.

[0037] Method 1 of the present invention uses zinc nitrate and Hmtz as raw materials, synthesizes ZnNFs and ZnNPs with strong adsorption characteristics at 25°C and 4°C respectively, proposes a new strategy of using the two as anchors, ZnNFs replaces 96-well plates and combines with monoclonal antibodies to capture disease markers, and ZnNPs adsorbs HRP and primary antibodies to output signals, and constructs a non-labeled immunosensor platform for detecting cTnI. ZnNFs and ZnNPs can be prepared in large quantities in advance, the material preparation operation is simple, the reaction conditions are mild, the raw materials are cheap, and two enzyme-linked complexes based on ZnNFs and ZnNPs can be prepared simultaneously. The enzyme-linked operation time is half a day, while the traditional enzyme-linked operation takes one day and requires expensive enzyme secondary antibodies.

[0038] The invention also carries out detailed characterization and analysis on the directional self-assembly process of ZnNFs by means of SEM, XRD, EDS and the like.

[0039] In addition, magnetic ZnNFs composite materials were synthesized in one step at 25°C using magnetic iron oxide nanoparticles, zinc nitrate and triazole as raw materials. A new completely non-labeled magnetic separation enzyme-linked immunosorbent sensing platform was constructed based on magnetic ZnNFs and ZnNPs, which achieved quantitative detection of cTnI and N proteins. The present invention has important value for the development and application of composite materials, and also provides a new method for the application of nanomaterials in clinical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

[0041] Figure 1 This is a scanning electron microscope image of the zinc-based complex nanoflower prepared in Example 1;

[0042] Figure 2 Elemental analysis diagram of the zinc-based complex nanoflower prepared in Example 1;

[0043] Figure 3 is the X-ray diffraction pattern of the zinc-based complex nanoflower prepared in Example 1;

[0044] Figure 4is a nitrogen adsorption-desorption isotherm diagram of the zinc-based complex nanoflower prepared in Example 1;

[0045] Figure 5 The schematic diagram of the directional self-assembly of the zinc-based complex nanoflowers prepared in Example 2 from particles-wires-flowers, the corresponding scanning electron microscope and the particle size statistics;

[0046] Figure 6 The SEM characterization and particle size statistics of the zinc-based complex nanoflower substrates of different molar concentrations prepared in Example 2;

[0047] Figure 7 This is a fluorescence microscope characterization image of the zinc-based complex nanoflower prepared in Example 3 adsorbing fluorescent protein;

[0048] Figure 8 Schematic diagram of the fluorescent colorimetric dual-channel enzyme-linked immunosorbent sensor based on nanoflowers in Example 4;

[0049] Fig. 9 This is a feasibility fluorescence image of the nanoflower-based fluorescent immunosensor in Example 4;

[0050] Fig.10 This is the feasibility absorption diagram of the nanoflower-based colorimetric immunosensor in Example 4;

[0051] Fig.11 This is the fluorescence emission spectrum of cTnI detected based on the nanoflower fluorescence method in Example 4;

[0052] Fig.12 This is a quantitative relationship diagram for detecting cTnI based on the nanoflower fluorescence method in Example 4;

[0053] Fig.13 This is the absorption spectrum of cTnI detected based on the nanoflower colorimetry in Example 4;

[0054] Fig.14 This is a quantitative relationship diagram for detecting cTnI based on the nanoflower colorimetric method in Example 4;

[0055] Fig.15 is a scanning electron microscope image of the zinc-based complex nanoparticles prepared in Example 5;

[0056] Fig.16 This is a fluorescence microscope characterization image of the zinc-based complex nanoparticles prepared in Example 5 adsorbing fluorescent protein;

[0057] Fig.17 is a schematic diagram of the nanoparticle-based fluorescent enzyme-linked immunosorbent assay sensor in Example 6;

[0058] Fig.18 The fluorescence emission spectrum of cTnI detected based on the nanoparticle fluorescence method in Example 6;

[0059] Fig.19 This is a quantitative relationship diagram of cTnI detection based on nanoparticle fluorescence method in Example 6;

[0060] Fig. 20 This is a repeated catalytic diagram of detecting cTnI based on nanoparticle fluorescence method in Example 6;

[0061] Fig.21 This is the selectivity spectrum of cTnI detection based on nanoparticle fluorescence method in Example 6;

[0062] Fig. 22 This is a scanning electron microscope image of the construction process of the nanoflower and nanoparticle sensor in Example 7;

[0063] Fig.23 The fluorescence emission spectrum of cTnI detected by the fluorescence method based on nanoflowers and nanoparticles in Example 7;

[0064] Fig.24 This is a quantitative relationship diagram for detecting cTnI based on the fluorescence method of nanoflowers and nanoparticles in Example 7;

[0065] Fig.25 This is the selectivity spectrum of cTnI detection based on nanoflower and nanoparticle fluorescence method in Example 7;

[0066] Fig.26 This is the ultrasonic stability diagram based on the fluorescence method of nanoflowers and nanoparticles in Example 7;

[0067] Fig. 27 Schematic diagram of the fluorescent enzyme-linked immunosorbent sensor based on magnetic nanoflowers and nanoparticles in Example 8;

[0068] Fig.28 This is a diagram showing the optimization of the amount of magnetic nanoparticles used in preparing magnetic nanoflowers in Example 8;

[0069] Fig.29 is the X-ray diffraction pattern of the nanoflowers, magnetic nanoflowers and magnetic nanoparticles in Example 8;

[0070] Fig.30 hysteresis loop diagram of magnetic nanoparticles and magnetic nanoflowers in Example 8;

[0071] Fig.31 This is the absorption spectrum of cTnI detected based on the magnetic nanoflower and nanoparticle colorimetric method in Example 8;

[0072] Fig.32 This is a quantitative relationship diagram for detecting cTnI based on the magnetic nanoflower and nanoparticle colorimetric method in Example 8;

[0073] Fig.33The fluorescence emission spectrum of cTnI detected by the magnetic nanoflower and nanoparticle fluorescence method in Example 8;

[0074] Fig.34 This is a quantitative relationship diagram for detecting cTnI based on magnetic nanoflowers and nanoparticle fluorescence in Example 8;

[0075] Fig.35 This is the absorption spectrum of the N protein detected by the colorimetric method based on magnetic nanoflowers and nanoparticles in Example 9;

[0076] Fig.36 This is a quantitative relationship diagram for detecting N protein based on magnetic nanoflowers and nanoparticle colorimetry in Example 9;

[0077] Fig.37 This is the fluorescence emission spectrum of the N protein detected by the magnetic nanoflower and nanoparticle fluorescence method in Example 9;

[0078] Fig.38 This is a quantitative relationship diagram for detecting cTnI based on magnetic nanoflowers and nanoparticle fluorescence in Example 9;

[0079] Fig.39 The table shows the recovery rate of cTnI detected in clinical samples using the developed method in Example 10.

[0080] Fig.40 This is a table showing the recovery rates of N protein detected in clinical samples using the developed method in Example 10.

[0081] Fig.41 This is a schematic diagram of the fluorescent enzyme-linked immunosorbent assay based on nanoflowers and nanoparticles in Example 7. DETAILED DESCRIPTION

[0082] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0083] Example 1: The directed self-assembly synthesis process of ZnNFs was characterized and analyzed in detail by SEM, XRD, EDS and other methods. Pour 1 mL of zinc nitrate (1.6 mM) solution into 1 mL of Hmtz (8.0 mM) solution (the solvents are all ultrapure water) and mix gently. The mixed solution is then allowed to stand at a constant temperature of 25°C for 6 hours to obtain a mixed solution containing a white precipitate. After the reaction solution was centrifuged at 6000 rpm for 1 min, the precipitate was collected and washed three times with ultrapure water. The white precipitate was placed in an oven at 50°C and dried to obtain a solid powder of nanoflower ZnNFs. Figure 1As shown in the figure, the macroscopic and microscopic morphology of ZnNFs were characterized by scanning electron microscopy, and it was observed that the shape of ZnNFs was similar to aster flowers, and the size was between 8-10μm. The "petals" of ZnNFs are composed of many rod-like structures (4.41μm long and 52.88nm wide), and the rods are assembled from many nanoparticles (~50nm). The constituent elements and element distribution states of ZnNFs were characterized by energy spectrum element analysis, and the element composition diagram and element resolution diagram were drawn, as shown in the figure. Figure 2 The presence and uniform distribution of elements (Zn, C, N, O) in ZnNFs are shown in Figure 2. The crystal structures of ZnNFs and the known metal organic framework material MAF-7 (the ligands and metal elements of the two are the same) were analyzed by X-ray diffractometer, as shown in Figure 2. Figure 3 As shown in Figure 2, the diffraction peaks of ZnNFs are different from those of MAF-7 reported in the literature. The specific surface area of ​​ZnNFs was analyzed by nitrogen adsorption desorption instrument, as shown in Figure 2. Figure 4 As shown in Figure 2, the specific surface area of ​​ZnNFs is 16.08 m 2 / g, which is much smaller than the specific surface area of ​​MAF-7. These characteristics indicate that ZnNFs are not organic metal framework materials, but are more likely to be composed of ligands and Zn 2+ Coordination copolymers synthesized by coupling.

[0084] Example 2: 1 mL of zinc nitrate (1.6 mM) solution was poured into 1 mL of Hmtz (8.0 mM) solution (the solvents were all ultrapure water) and gently mixed. The mixed solution was then kept at a constant temperature of 25°C. The products of different reaction times (0 min, 10 min, 30 min, 1 hour, and 6 hours) during the synthesis of ZnNFs were selected and characterized by scanning electron microscopy to study the particle-wire-flower self-assembly synthesis mechanism of ZnNFs, and a size statistical diagram was drawn, such as Figure 5 As shown in the figure, in the early stage, Zn-Hmtz primary crystals are rapidly generated and appear in the form of nano-sized particles. At this stage, we speculate that Hmtz and Zn 2+ Mainly through the N and Zn in the Hmtzs main chain 2+ The coordination of Zn-Hmtz crystals forms complexes, which provide locations for the nucleation of primary crystals and then aggregate into small particles. In the second growth step, a large number of irregular nanowires gradually appear in the reaction solution. The growth of Zn-Hmtz crystals may be controlled to a certain extent by the single Zn on the surface of the aggregates. 2+ / Hmtz, thus directing self-assembly to form individual nanowires. In the final stage, the "construction" of the nanoflower scaffold is basically completed, and the nanowires assemble and grow to form a flower-like structure with independent branches. As time goes on, the size of the nanoflower gradually increases. We speculate that in this growth process, after the Zn-Hmtz crystals nucleate and then form the nanowire scaffold, one end of the nanorod grows, a section agglomerates, and finally assembles three-dimensional ZnNFs with uniform size and stable morphology. Fixing other conditions, different concentrations of substrate molar concentration (Zn 2+ :Hmtz=1:5,Zn 2+ :0.1, 0.2, 0.4, 0.8, 1.6mM) on the effects of ZnNFs Figure 6 As shown. 2+ When the concentration is 0.1 mM, the product morphology is nanoparticles of 26.01 nm. 2+ When the concentration increased to 0.2 mM, the product morphology was a nanoparticle of 100 nm assembled from many small nanoparticles. 2+ concentration (0.4 mM), the newly generated self-assembled nanoparticles merged into more complete and smoother nanoparticles. 2+ When the concentration reaches 0.8mM, the nanoparticles aggregate in a certain order to form nanowires, and then aggregate to form three-dimensional nanoflowers of about 12μm. At this time, the nanowires assembled into nanoflowers are in a disordered state. 2+ In the presence of Zn, the nanowires are orderly aggregated into three-dimensional nanoflowers of ~9 μm. 2+ As the concentration increases further, we find that the morphology and size of ZnNFs remain unchanged. These results indicate that the overall concentration of the substrate plays a key role in the formation of ZnNFs, and that only within a specific concentration range can the substrate form nanoflowers with uniform size and symmetrical morphology.

[0085] Example 3: Green fluorescent protein (GFP), rhodamine B-labeled BSA (RB-BSA) and Cy5-labeled antibody (Cy5-ab) were incubated with ZnNF nanoflowers and centrifuged and washed to obtain GFP / Ab / BSA@ZnNFs complex. The fluorescence characteristics of the complex under three different wavelengths of excitation light were characterized by fluorescence microscopy, bright field images, and the fluorescence characteristics under three different wavelengths of excitation light. Figure 7As shown in the figure, the fluorescence confocal microscope characterization images show the fluorescence characteristics of GFP / Ab / BSA@ZnNFs under three different wavelengths of excitation light, the bright field image of ZnNFs, and the fluorescence characteristics of GFP / Ab / BSA@ZnNFs under three different wavelengths of excitation light. When ZnNFs are excited at 488nm, green fluorescent protein can be observed to be evenly distributed on the surface of ZnNFs in the field of view of fluorescence microscope. When GFP / Ab / BSA@ZnNFs are excited at 600nm, the specific red fluorescence of Cy5-ab can be observed in the field of view. When GFP / Ab / BSA@ZnNFs are excited at 543nm, we can observe the characteristic yellow fluorescence of RB-BSA. The merged image shows that the three proteins are successfully adsorbed on the surface of ZnNFs. The characterization results show that ZnNFs can not only be used as its carrier to bind to proteins through bioconjugation, but also its binding ability is universal. Furthermore, the prepared ZnNFs replaced the traditional ELISA 96-well plate to construct a cTnI immunosensor based on ZnNFs. The immunosensor uses mouse anti-cTnI monoclonal antibody (Capture ab), goat anti-cTnI antibody (Ab1) and enzyme-labeled rabbit anti-goat IgG (HRP-ab2) for cTnI detection. Figure 8 DA-DHA and TMB were selected as substrates of HRP-H2O2 for the fluorescence and colorimetric signal output of the immunosensor, respectively. Compared with the control group without cTnI (0 ng / mL), the experimental group with cTnI (150 ng / mL) showed a significant increase in fluorescence at 550 nm and a significant increase in absorbance at 652 nm, as shown in Figure 2. Fig. 9 , Fig.10 cTnI was further quantified by fluorescence colorimetry dual channels, such as Fig.11 , Fig.12 As shown, with the increase of cTnI concentration, the fluorescence intensity gradually increased. The relative fluorescence intensity showed a good linear relationship with the cTnI concentration in 0-50ng / mL and 50-150ng / mL. The fitting linear equations corresponding to the two concentration ranges were Y1=1.1366+0.0375CcTnI (R12=0.9725) and Y2=2.6616+0.0045CcTnI (R22=0.9846), and the detection limit (LOD) was 0.33ng / mL. Fig.13 , Fig.14As shown in the colorimetric channel of the sensor, a linear relationship between relative absorbance and cTnI concentration (2-20 and 20-300 ng / mL) can be found, and the regression equations are Y1=1.2926+0.0281CcTnI (R12=0.9673) and Y2=1.7996+0.0015CcTnI (R22=0.9822), and the LOD is 0.67 ng / mL. The experimental results show that the developed fluorescence colorimetric dual-channel immunosensor is feasible and has excellent performance for cTnI analysis.

[0086] Example 4: 2 mL of zinc nitrate solution (0.4 mM) was added to 2 mL of Hmtz solution (2.0 mM) in batches of 200 μL (the solvents were all ultrapure water, vortexed while adding), and placed in a constant temperature environment of 4°C. After standing for 24 hours, a mixed solution containing a white precipitate was obtained. The reaction solution was centrifuged at 12000 rpm for 1 min to collect the sample, which was then washed three times with ultrapure water. Finally, the precipitate was placed in an oven at 50°C and dried to obtain a solid powder of nanoparticles ZnNPs. ZnNPs were characterized by scanning electron microscopy, such as Fig.15 Green fluorescent protein (GFP), rhodamine B-labeled BSA (RB-BSA) and Cy5-labeled antibody (Cy5-ab) were incubated with ZnNF nanoflowers and centrifuged and washed to obtain GFP / Ab / BSA@ZnNPs complex. The fluorescence characteristics of the complex under three different wavelengths of excitation light were characterized by fluorescence microscopy, bright field images and co-excitation of the three different wavelengths of excitation light. Fig.16 . ZnNPs were used to construct a non-labeled enzyme-linked immunosorbent sensor platform for detecting cTnI. The non-labeled sensor incubated and adsorbed three proteins, BSA (blocking part), Ab1 (recognition part) and HRP (catalytic part) onto ZnNPs to obtain a BSA / Ab1 / HRP@ZnNPs complex, which can be used to replace the expensive enzyme label ab2 in traditional ELISA. Capture ab was fixed on a 96-well plate. Through the specific binding of antigen and antibody, the Capture ab on the 96-well plate successively recognized and captured cTnI and BSA@Ab1@HRP@ZnCPs complex, such as Fig.17 . Further draw fluorescence spectrum and linear relationship analysis diagram, such as Fig.18 , Fig.19The relative fluorescence intensity of the system is well linearly correlated with the cTnI concentration (0-200 ng / mL) (Y=1.142+0.0134CcTnI, R2=0.9830), and the LOD is 0.33 ng / mL. After adding the reaction solution system to catalyze the reaction and detect the fluorescence spectrum, the supernatant is removed by centrifugation, and new reaction solution is added to continue the catalytic reaction. After 6 cycles of reaction, the immunosensor system still maintains a catalytic activity of more than 90%, as shown in FIG. Fig. 20 The selectivity of the immunosensor was studied using AFP, Try, Lys and BSA as interfering proteins. Only cTnI could cause a significant fluorescence signal enhancement, such as Fig.21 The experimental results show that a high-sensitivity, high-selectivity and strong-stability enzyme-linked immunosorbent sensing platform can be constructed based on the BSA / ab1 / HRP@ZnNPs complex.

[0087] Example 5: Based on the excellent performance of ZnNFs and ZnNPs in constructing an immunosensor platform, a completely label-free fluorescent immunosensor was constructed using the centrifugation method. Fig.41 . The complexes at each stage of the enzyme linkage were characterized by scanning electron microscopy. With the gradual modification of proteins (Capture ab, BSA and cTnI), the rod-like structure on the ZnNPs nanoflower gradually became thicker and larger. At the same time, SEM can also observe that the BSA / Capture ab@ZnNFs complex and the BSA / Ab1 / HRP@ZnNPs complex are anchored together by the antigen (cTnI). The combination of ZnNPs and ZnNFs also shows that the fluorescent immunosensor based on ZnNFs&ZnNPs has been successfully constructed, such as Fig. 22 . The completely label-free fluorescent immunosensor was further applied to the detection of cTnI, such as Fig.23 , Fig.24 The linear data of the fluorescence method shown is Y1 = 1.3691 + 0.026C cTnI (R1 2 =0.9845)(1-75ng / mL), Y2=2.9353+0.0049C cTnI (R2 2 =0.9976)(75-250ng / mL), LOD was 0.33ng / mL. The selectivity and stability of the detection system were studied. Other interfering proteins were added separately. Only when cTnI was present in the reaction system, the response was better. Fig.25The constructed ZnNFs&ZnNPs complex was placed in an ultrasonic cleaning machine for different ultrasonic treatment times (0-120s), the last incubation solution was removed and the cleaning was continued, and then the reaction solution was added to trigger the fluorescence reaction. The external ultrasonic force could not destroy the effective combination of ZnNFs and ZnNPs in the system within 2 minutes of treating the ZnNFs&ZnNPs complex. Fig.26 .

[0088] Example 6: Take 0.2703g of ferric chloride hexahydrate (0.2mM) and 0.0663g of ferrous chloride tetrahydrate (0.0667mM) and dissolve them in 5mL of ultrapure water. After stirring evenly at room temperature, quickly add 1.5mL of ammonia water to the mixed solution (the solution color immediately changes from orange to black). Ultrasonicate the mixed solution for 10 minutes and then let it stand at room temperature for 10 minutes to obtain a black Fe3O4 NPs suspension. Freeze-dry the suspension for later use. At the beginning of the reaction of the ZnNFs reaction solution, add different concentrations of Fe3O4 NPs (0, 0.2, 0.4, 0.8, 1.6, 3.2mg / mL) to prepare magnetic ZnNFs complexes. Fig.28 As shown in the figure, when 0.8 mg / mL magnetic particles were added, the magnetic ZnNFs complex could be quickly separated within 10 seconds, and the fluorescence signal ratio between the experimental group with cTnI and the control group without cTnI was the highest. According to the experimental results, it is speculated that the low concentration of Fe3O4 NPs is insufficient to form a magnetic ZnNFs composite material with strong magnetic responsiveness, and too much Fe3O4 NPs will interfere with the ZnNFs crystal assembly process and reduce the overall product. At the same time, in order to verify the successful synthesis of the magnetic ZnNFs complex, XRD characterization of Fe3O4 NPs, ZnNFs and magnetic ZnNFs was performed respectively ( Fig.29 ). The differences in crystal structure and the corresponding peak positions of the three crystals provide evidence for the successful formation of the magnetic ZnNFs complex. Then, the hysteresis loops of Fe3O4 NPs and Fe3O4 NPs@ZnNFs were compared using VSM. Fig.30 As shown in the figure, the saturation magnetization (Ms) of Fe3O4 NPs is 49.9emu / g, and the saturation magnetization (Ms) of Fe3O4 NPs@ZnNFs is 27.2emu / g. Although the Ms value of Fe3O4 NPs@ZnNFs is reduced due to the presence of ZnNFs, Fe3O4 NPs@ZnNFs is still suitable for simple magnetic separation in an external magnetic field. Fig.30The inset photo shows that the magnet can easily separate Fe3O4 NPs@ZnNFs from the liquid suspension within 10 seconds. In addition, the prepared magnetic composites have no remanence and coercivity, and no hysteresis, showing superparamagnetic behavior at room temperature, indicating that these composites can be redispersed immediately after the magnet is removed. The above results indicate that Fe3O4 NPs@ZnNFs are suitable for cTnI detection and have good recyclability and reusability in practical applications.

[0089] Example 8: First, add 100 μL of BSA / Capture ab@magnetic ZnNFs complex solution to multiple 1.5 mL centrifuge tubes. Then, inject 100 μL of cTnI solution of different concentrations (0-200 ng / mL) into each tube, and then incubate on a shaker at 37°C for 1 hour. After washing 4 times with TBST under magnetic separation, add 100 μL of BSA / Ab1 / HRP@ZnNPs complex solution and incubate at 37°C for 1 hour. Finally, remove the supernatant after external magnetic adsorption and wash 4 times with TBST by centrifugation. Afterwards, add 300 μL of TMB colorimetric solution to each centrifuge tube, react at room temperature for 15 minutes, and measure the absorption spectrum and linear relationship as shown in the figure. Fig.31 , the colorimetric linear fitting data shown in 32 is Y = 1.1463 + 0.0046C cTnI (R 2 =0.9876)(5-200ng / mL), the LOD of the colorimetric channel is 1.67ng / mL. Similarly, 300μL of a mixed solution containing DA (400μM), DHA (30μM) and H2O2 (60μM) can be replaced and reacted at room temperature for 3min to obtain the fluorescence spectrum and linear relationship as shown in the figure. Fig.33 , 34 shows that the linear data of the fluorescence method is Y = 0.9999 + 0.0092C cTnI (R 2 =0.9939) (0-200 ng / mL), and the LOD of the fluorescence channel was 0.33 ng / mL.

[0090] Example 9: In order to verify the versatility of the developed completely label-free magnetic separation immunosensor, the novel coronavirus N protein was used to replace cTnI as a model antigen, and the capture antibody and labeling antibody were replaced by mouse anti-cTnI monoclonal antibody and sheep anti-cTnI primary antibody to mouse anti-N protein monoclonal antibody and rabbit anti-N protein primary antibody, and a label-free magnetic separation immunosensor for N protein was constructed. Fig.35 As shown in Figure 36, with the increase of N protein concentration, the fluorescence intensity at 550nm and the absorbance at 652nm gradually increased. The fluorescence intensity ratio or absorption intensity ratio of the fluorescence / colorimetric immunosensor was linearly related to the N protein concentration (0-250ng / mL). Fig.37, 38, for the absorbance method, the fitted linear data is Y1 = 1.0063 + 0.0260C N蛋白 (R1 2 =0.9814)(0-20ng / mL) and Y2=1.5003+0.0015C N蛋白 (R2 2 =0.9783) (20-250ng / mL), LOD is 1ng / mL. The linear data of the fluorescence method is Y=0.9958+0.0075C N蛋白 (R 2 =0.9972), the range was 0-250 ng / mL, and the LOD was 0.33 ng / mL.

[0091] Example 10: Serum recovery rate of cTnI determined by magnetic ZnNFs & ZnNPs based sensing platform Fig.39 As shown in the figure, the detection results are not much different from the spiked cTnI concentration, the recovery rate of the fluorescence channel is 98.7-103.6%, and the recovery rate of the colorimetric channel is 96.4-104.1%. The sensing platform based on magnetic ZnNFs & ZnNPs also has excellent results for the recovery rate of N protein in 1% serum by both fluorescence and colorimetric methods. Fig.40 As shown, the results were consistent with those of spiked N protein, with the recovery rates of the fluorescence channel ranging from 95.1% to 104.6% and the colorimetric channel ranging from 96.5% to 104.8%. In summary, this detection strategy has good practical application potential.

[0092] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A method for constructing an immune platform for detecting cardiac troponin I by non-covalently modifying ZnNFs and ZnNPs with enzymes / antibodies, characterized in that: The particle-wire-flower self-assembled ZnNFs successively adsorbed the capture antibody mouse anti-cTnI monoclonal antibody Capture ab and the bovine serum albumin BSA as a blocking agent to form a BSA / Capture ab@ZnNFs complex. The spherical ZnNPs were sequentially combined with horseradish peroxidase HRP, labeled antibody Ab1-sheep anti-cTnI primary antibody, and BSA to generate a BSA / Ab1 / HRP@ZnNPs complex. In PBS buffer solution, different concentrations of cTnI were incubated with the BSA / Capture ab@ZnNFs complex, and the BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution. After further incubation and washing, 400μM dopamine DA, 30μM 1,5-Naphthalene diol DHA and 60 μM hydrogen peroxide H2O2 in 10 mM, pH = 7.4 Tris-HCl buffer solution generate a yellow fluorescent compound AFC. The fluorescence intensity of the synthesized AFC is detected by a fluorescence spectrometer. The excitation wavelength and emission wavelength of AFC are 490 nm and 550 nm, respectively.

2. The method for constructing an immune platform for detecting cardiac troponin I using ZnNFs and ZnNPs according to claim 1, characterized in that: The synthesis conditions of ZnNFs are as follows: 1 mL of 1.6 mM zinc nitrate solution is poured into 1 mL of 8.0 mM Hmtz solution, and the solvents are all ultrapure water. The mixture is gently mixed, and the mixed solution is kept at a constant temperature of 25°C for 6 hours to obtain a mixed solution containing white precipitated ZnNFs, which is then washed and used for later use. The synthesis conditions of ZnNPs are as follows: 2 mL of 0.4 mM zinc nitrate solution is added to 2 mL of 2.0 mM Hmtz solution in batches of 200 μL. The solvents are all ultrapure water. The mixture is vortexed while being added and placed in a constant temperature environment of 4°C for 24 hours to obtain a mixed solution of white precipitated ZnNPs.

3. The method for constructing an immune platform for detecting cardiac troponin I using ZnNFs and ZnNPs according to claim 1, characterized in that: The sensing platform conditions are as follows: the concentration of capture antibody is 1 μg / mL, the concentration of BSA bound to ZnNFs is 20 mg / mL, the concentration of HRP is 1 U / mL, the concentration of labeled antibody is 2 μg / mL, the concentration of BSA bound to ZnNPs is 10 mg / mL, and the two complexes BSA / Ab1 / HRP@ZnNPs and BSA / Capture ab@ZnNFs are prepared and placed at 4°C for use.

4. A method for detecting cTnI based on a fluorescent colorimetric dual-channel non-labeled enzyme-linked immunosorbent assay sensor constructed using ZnNFs, characterized in that: A series of cTnI with different concentrations and BSA / Capture ab@ZnNFs complexes were incubated at 37°C, and then Ab1 and HRP-labeled rabbit anti-sheep polyclonal antibody HRP-ab2 were incubated in the system in turn. DA, DHA and H2O2 or TMB colorimetric solution were added to the final complex Tris-HCl buffer solution. After reacting at room temperature for several minutes, the fluorescence spectrum and absorption intensity were analyzed. The absorption of oxidized TMB was fixed at 652nm. The synthesis conditions of ZnNFs are as follows: 1 mL of 1.6 mM zinc nitrate solution is poured into 1 mL of 8.0 mM Hmtz solution, and the solvents are all ultrapure water. The mixture is gently mixed and the mixed solution is kept at a constant temperature of 25°C for 6 hours to obtain a mixed solution containing white precipitated ZnNFs, which is then washed for use; the particle-wire-flower self-assembled ZnNFs successively adsorb the capture antibody Capture ab and bovine serum albumin BSA to form a BSA / Captureab@ZnNFs complex.

5. The method of detecting cTnI based on ZnNFs using a fluorescent colorimetric dual-channel non-labeled enzyme-linked immunosorbent assay according to claim 4, characterized in that: The sensing platform conditions are as follows: the concentration of the labeled antibody Ab1 is 2 μg / mL, the concentration of HRP-ab2 is 1 μg / mL, and the system is subjected to fluorescence and absorption dual signal analysis and detection by a fluorescence spectrometer and an ultraviolet spectrophotometer.

6. A method for detecting cTnI using a non-labeled enzyme-linked immunosorbent assay based on ZnNPs, characterized in that: The capture antibody was incubated with the 96-well plate overnight and then blocked with BSA; a series of different concentrations of cTnI were added to each well for incubation and washing; finally, the BSA / Ab1 / HRP@ZnNPs complex was added and incubated again for washing, and then a Tris-HCl buffer solution containing DA, DHA and H2O2 was added. After reacting for several minutes, the fluorescence spectrum and absorption intensity were analyzed; The synthesis conditions of ZnNPs are as follows: 2mL of 0.4mM zinc nitrate solution was added to 2mL of 2.0mM Hmtz solution in batches of 200μL. The solvents were all ultrapure water. The mixture was vortexed while added and placed in a constant temperature environment of 4℃ for 24 hours to obtain a mixed solution of white precipitated ZnNPs. The spherical ZnNPs were sequentially combined with horseradish peroxidase HRP, labeled antibody Ab1 and BSA to generate BSA / Ab1 / HRP@ZnNPs complex.

7. The method for detecting cTnI using a non-labeled enzyme-linked immunosorbent assay based on ZnNPs according to claim 6, characterized in that: The sensing platform conditions are as follows: the concentration of the capture antibody is 1 μg / mL, the concentration of the BSA blocking the 96-well plate is 20 mg / mL, and the fluorescence signal analysis and detection of the system is performed by a fluorescence spectrometer.

8. A completely label-free magnetic separation enzyme-linked immunosorbent assay method for detecting cTnI or SARS-CoV-2 N protein based on magnetic ZnNFs and ZnNPs, characterized in that: Magnetic ZnNFs adsorbed capture antibody and BSA successively to form BSA / Capture ab@magnetic ZnNFs complex, and spherical ZnNPs bound HRP in turn, labeled antibody Ab1 and BSA to generate BSA / Ab1 / HRP@ZnNPs complex; in PBS buffer solution, cTnI with different concentrations was incubated with BSA / Capture ab@ZnNFs complex, and then BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution; after further incubation and washing, 300 μL of 10 mM, pH=7.4 Tris-HCl buffer solution or TMB colorimetric solution containing 400 μM dopamine DA, 30 μM 1,5-naphthalene diol DHA and 60 μM hydrogen peroxide H2O2 was added, and the fluorescence intensity of the synthesized AFC was detected by fluorescence spectrometer in the former, and the absorption intensity of the synthesized oxTMB was detected by UV spectrophotometer in the latter; Magnetic ZnNFs successively adsorbed capture antibody Capture ab-mouse anti-N protein monoclonal antibody and BSA to form BSA / Capture ab@magnetic ZnNFs complex, and spherical ZnNPs successively bound HRP, labeled antibody Ab1-rabbit anti-N protein primary antibody and BSA to generate BSA / Ab1 / HRP@ZnNPs complex; in PBS buffer solution, different concentrations of N protein were incubated with BSA / Capture ab@ZnNFs complex, and BSA / Ab1 / HRP@ZnNPs complex was added to the mixed solution; after further incubation and washing, 300 μL of 10 mM, pH=7.4 Tris-HCl buffer solution or TMB colorimetric solution containing 400 μM dopamine DA, 30 μM 1,5-naphthalene diol DHA and 60 μM hydrogen peroxide H2O2 was added, and the fluorescence intensity and absorption intensity of the synthesized AFC and oxTMB were detected by fluorescence spectrometer and UV spectrophotometer, respectively.

9. The completely label-free magnetic separation enzyme-linked immunosorbent assay method for detecting cTnI or N protein based on magnetic ZnNFs and ZnNPs according to claim 8 is characterized in that: The synthesis conditions of magnetic ZnNFs are as follows: 0.2703g of ferric chloride hexahydrate and 0.0663g of ferrous chloride tetrahydrate are dissolved in 5mL of ultrapure water, and after stirring evenly at room temperature, 1.5mL of ammonia water is quickly added to the mixed solution, and the color of the solution immediately changes from orange to black. The mixed solution is ultrasonicated for 10 minutes and then allowed to stand at room temperature for 10 minutes to obtain a black Fe3O4 NPs suspension; then, 1mL of 1.6mM zinc nitrate solution is poured into 1mL of 8.0mM Hmtz solution and gently mixed; then, 0.0160g of magnetic iron oxide nanoparticles is weighed and poured into the former mixed solution, and slowly mixed. Then, the mixed solution is placed in a 25°C water bath and allowed to stand for 6 hours to obtain a solution containing a brown magnetic ZnNFs precipitate. The synthesized magnetic ZnNFs are characterized by an X-ray diffractometer and a hysteresis loop.

Images