Magnetic nanoscale enzyme and application of enzyme catalysis and multi-channel microfluidic chip immune detection method

Abstract

The application discloses a magnetic nano-enzyme, and an enzyme catalysis and multi-channel micro-fluidic chip immunodetection method and application thereof. The application provides a manganese-iron oxide magnetic nano-enzyme with magnetic separation and catalysis functions, which is a manganese-iron oxide solid microsphere with a dopamine group on the surface, and has an average particle size of 200-400 nm, and has the functions of magnetic separation, good enzyme catalysis and solid phase carrier, realizes immunodetection based on manganese-iron oxide magnetic nano-enzyme catalysis fluorescence, and expands the application method of nano-enzyme in immunodetection. The application provides a multi-channel micro-fluidic rapid fluorescence immunodetection method based on the manganese-iron oxide magnetic nano-enzyme, and introduces a nano-enzyme catalysis fluorescence detection system with low cost, and the detection speed can be much higher than that of traditional immunodetection based on a micro-hole plate, the method realizes rapid immunodetection of color development and fluorescence, and fills the vacancy of the nano-enzyme catalysis fluorescence system in immunodetection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay. More particularly, it relates to a magnetic nanoscale enzyme, and an immunoassay method and application of enzyme catalysis and multi-channel microfluidic chip. BACKGROUND

[0002] The process of immunoassay generally includes sample processing, capture and separation of target molecules, and signal amplification, among which the capture and separation of target sample molecules and the amplification of detection signal are the key steps and important research contents in the process of immunoassay. The capture of target molecules relies on the specific binding of antigen and antibody, and the result of capture directly determines the specificity and accuracy of immunoassay; and the accurate separation of captured target molecules from the environment containing a large amount of non-target substances is the key to ensure low background and high sensitivity of the detection result. The signal amplification process can convert the number of target molecules obtained by capture and separation into color, fluorescence, luminescence and other signals that are easier to read, and the amplification and reading of weak signals are conducive to further improving the sensitivity of immunoassay to meet the detection needs of some low-concentration target substances.

[0003] In the traditional immunoassay method, the amplification of detection signal is usually realized by the catalysis of biological enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP). Biological enzyme catalysis technology is a mature and efficient method, which has been widely used in catalytic color development, fluorescence, chemiluminescence and other detection, especially in immunoassay; and enzyme-labeled antibody has become one of the most commonly used signal amplification materials. However, biological enzymes are easy to denature at inappropriate pH and temperature, and are easy to be degraded by proteases, and the cost of preparation and preservation is high. Nanoscale enzymes have similar catalytic activity to biological enzymes, and also have magnetism and higher stability, which can solve the problems existing in biological enzymes to a certain extent. Therefore, nanoscale enzymes have attracted widespread attention in various fields requiring catalytic reaction.

[0004] Materials capable of realizing sample capture, separation function and catalytic reaction at the same time are very important in the field of immunoassay. In order to realize multiple functions at the same time, researchers usually use double labeling method to couple magnetic beads, antibodies and enzymes together, such as coupling antibodies and enzymes to the surface of magnetic beads at the same time, or using multiple avidin sites of streptavidin to simultaneously connect antibodies and enzymes (Methods in Enzymology, 2016, 571, 135-150). Among them, ferrite nanoparticles are a kind of potential magnetic material that can be applied in immunoassay, which can not only capture target molecules, but also catalyze peroxide substrates to amplify signals. The latest research shows that ferrite nanoparticles with manganese ferrite as the core have excellent performance in strong magnetism and high catalytic efficiency. In various detections that require signal amplification, magnetic nanoscale enzymes not only participate in sample capture and separation processes as carriers while catalyzing, but also show excellent performance in immunoassay. However, there are still some problems in the application of magnetic nanoscale enzyme immunoassay: although the catalytic performance of small-sized and porous nanoscale enzyme is stronger, the magnetic nanoscale enzyme with too small size cannot be used as a reaction solid-phase carrier for coupling antibodies, and it cannot couple enough antibodies for immunoassay. Therefore, the size of the magnetic nanoparticle used as an immunoassay carrier should be large enough to couple enough antibodies, but at this time it is difficult to ensure the catalytic activity of the magnetic nanoscale enzyme. The method for detecting biomolecules by using the new nanoscale enzyme immunoassay sandwich technology of patent CN108982834A solves the above problems by providing two different sizes of magnetic nanoscale enzymes, but it still cannot realize the functions of reaction solid-phase carrier, magnetic separation and catalysis based on one kind of magnetic nanoscale enzyme.

[0005] At present, researchers pay much attention to the application of nanoscale enzymes in various detections. In catalytic fluorescence detection, nanoscale enzymes have been widely used in the detection of small molecules, such as uric acid, glucose, ascorbic acid, lactic acid, etc. However, in the application of immunoassay of proteins, researchers pay more attention to the catalytic color development and chemiluminescence detection of nanoscale enzymes, and there is no report on the immunoassay based on the catalytic fluorescence of nanoscale enzymes. On the one hand, there are few types of substrates that can be used for nanoscale enzyme catalytic fluorescence detection, and on the other hand, the nanoscale enzyme catalytic quenching type fluorescence detection technology widely used in the detection of small molecules cannot work well in immunoassay. Therefore, providing a magnetic nanoscale enzyme with the functions of magnetic separation, catalytic activity and reaction solid-phase carrier, and developing an immunoassay method based on the catalytic fluorescence of nanoscale enzymes will help to expand the application occasions of nanoscale enzymes and promote the development of nanoscale enzyme catalytic fluorescence immunoassay. SUMMARY

[0006] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a manganese ferrite magnetic nanoscale enzyme.

[0007] The second object of the present application is to provide an immune detection method based on the manganese ferrite magnetic nanoscale enzyme catalysis and a multi-channel microfluidic chip.

[0008] The third object of the present application is to provide an application of the immune detection method.

[0009] The above objects of the present application are achieved by the following technical solutions.

[0010] The present application first provides a manganese ferrite magnetic nanoscale enzyme with both magnetic separation and catalytic functions, which is a manganese ferrite oxide solid microsphere with a dopamine PDA group on the surface, and the average particle size is 200-400 nm.

[0011] The manganese ferrite nanoscale enzyme provided by the present application has magnetic characteristics, and the average particle size is in the range of 200-400 nm. It can be controlled by magnetic force to move in the solution and magnetically fixed for rapid separation, and at the same time has good enzyme catalytic fluorescence or color development signal amplification function, i.e. it can catalyze H2O2 and TMB or HPA substrate to cause color development or fluorescence reaction, thereby realizing enzyme catalytic signal amplification. That is, the manganese ferrite magnetic nanoscale enzyme has the functions of magnetic carrier and catalytic color development and catalytic fluorescence substrate, and can be used as a magnetic separation carrier at the same time, and instead of HRP enzyme to be applied to immune detection. On this basis, the size of the manganese ferrite magnetic nanoscale enzyme provided by the present application can be coupled with enough specific antibodies, thereby serving as a reaction solid-phase carrier. The manganese ferrite magnetic nanoscale enzyme can be used to realize a microfluidic immune detection method based on magnetic nanoscale enzyme catalytic fluorescence / color development, specifically by controlling the movement of the magnetic nanoscale enzyme in the solution by magnetic force and capturing sample molecules, and separating them from other interference components, using the catalytic fluorescence or color development ability of the magnetic nanoscale enzyme, further realizing signal amplification in the microfluidic chip, thereby completing the detection of sample molecules.

[0012] The application also provides a preparation method of the manganese ferrite magnetic nanoscale enzyme, which is a method for preparing a magnetic nanoscale enzyme based on a double-solvent method and surface modification of dopamine, and can simply realize synthesis of a multifunctional magnetic carrier; the double solvents are ethylene glycol and diethylene glycol, and particles with different sizes (average particle diameter) can be prepared when the volume ratio of ethylene glycol and diethylene glycol is different, the average particle diameter is larger when the ratio of diethylene glycol is larger, and the manganese ferrite magnetic nanoscale enzyme with a suitable average particle diameter (200-400 nm) can be prepared when the volume ratio of the double solvents ethylene glycol and diethylene glycol is 1:2-4; and the ratio of the material and the solvent does not affect the size of the synthesized particles, because the particle size is determined by the nucleation and growth process of the material, and in the double-solvent method, the process depends on the ratio of different solvents. For example, when pure ethylene glycol is used as the solvent, the material quickly nucleates and agglomerates to obtain large particles, and the average particle diameter becomes smaller after mixing with a certain proportion of diethylene glycol, and increasing the amount of the solvent does not affect the size. Meanwhile, the synthesis is hydrothermal synthesis, and a relatively narrow average particle diameter distribution of the nanoscale enzyme can be obtained by adjusting the reaction temperature in the hydrothermal synthesis. Specifically, the method comprises the following steps:

[0013] S1. Dissolving iron salt and manganese salt by a double-solvent method to obtain a reaction solution; the double solvents are ethylene glycol and diethylene glycol;

[0014] S2. Performing hydrothermal reaction on the reaction solution obtained in S1 to obtain a synthetic product MnFe2O4NPs with an average particle diameter of 100-200 nm;

[0015] S3. Mixing the synthetic product MnFe2O4NPs with dopamine hydrochloride, magnetically separating, washing, and drying to obtain manganese ferrite oxide solid microspheres MnFe2O4NPs-PDA with dopamine groups on the surface.

[0016] Further, the molar ratio of the iron salt and the manganese salt in step S1 is 1-2:1.

[0017] Preferably, the molar ratio of the iron salt and the manganese salt in step S1 is 2:1.

[0018] Further, the iron salt and the manganese salt in step S1 are FeCl3·6H2O and MnCl2·xH2O, respectively.

[0019] Further, the volume ratio of the ethylene glycol and the diethylene glycol in step S1 is 1:2-4.

[0020] Preferably, the volume ratio of the ethylene glycol and the diethylene glycol is 1:4.

[0021] Further, the temperature of the hydrothermal reaction in step S2 is 100-200℃.

[0022] Further, the method comprises the following specific steps:

[0023] S1. Dissolve the iron salt and manganese salt in a mixed solution of ethylene glycol and diethylene glycol, mix, add polyvinylpyrrolidone (PVP) to obtain a suspension, heat to obtain a transparent solution; the volume ratio of ethylene glycol and diethylene glycol in the mixed solution of ethylene glycol and diethylene glycol is 1:2-4.

[0024] S2. Add sodium acetate to the transparent solution obtained in S1, stop heating and mix, carry out hydrothermal reaction at 100-200°C, cool to room temperature to obtain a synthesis product MnFe2O4NPs;

[0025] S3. Mix and react the synthesis product MnFe2O4NPs with dopamine hydrochloride, magnetically separate, wash, dry to obtain manganese iron oxide solid microspheres MnFe2O4NPs-PDA with surface modified dopamine PDA groups.

[0026] Further, the mixing in step S1 is stirring for 30-60 minutes.

[0027] Preferably, the mixing in step S1 is stirring for 30 minutes.

[0028] Further, the mass fraction of polyvinylpyrrolidone in the suspension obtained in step S1 is 1-3%.

[0029] Preferably, the mass fraction of polyvinylpyrrolidone in the suspension obtained in step S1 is 1%.

[0030] Further, the heating in step S1 is heating at 80-120°C.

[0031] Preferably, the heating in step S1 is heating at 120°C.

[0032] Further, the concentration of sodium acetate in the transparent solution in step S2 is 0.01-0.1 mol / L.

[0033] Preferably, the optional amount of sodium acetate in step S2 is 1.5 g.

[0034] Further, the mixing in step S2 is stirring for 30-60 minutes.

[0035] Further, the hydrothermal reaction time in step S2 is 6-12 h.

[0036] Preferably, the hydrothermal reaction time in step S2 is 12 h.

[0037] Preferably, the step S2 is adding sodium acetate to the transparent solution obtained in S1, stopping heating and stirring for 30 minutes, carrying out hydrothermal reaction at 200°C for 12 h, cooling to room temperature to obtain a synthesis product.

[0038] Further, the synthetic product obtained in step S2 is further centrifuged at 10000-2000 rpm for 10-30 min, washed with water / ethanol for several times, dispersed in deionized water, and stored at 4°C for standby use.

[0039] Preferably, the synthetic product obtained in step S2 is further centrifuged at 10000 rpm for 10 min, washed with water / ethanol for several times, dispersed in deionized water, and stored at 4°C for standby use.

[0040] Further, the dosage of dopamine hydrochloride in step S3 is such that the final concentration after addition is between 1-10 mM.

[0041] Further, in step S3, after ultrasonic dispersion, the mixture is stirred for 60 min, and then Tris-HCl is added to adjust the pH to 8.5-9.0, and mechanical stirring is performed for 2 hours.

[0042] Preferably, the optional concentration and dosage of dopamine hydrochloride is 10 mg / mL, 2 mL; and the optional concentration and dosage of Tris-HCl is 40 mg / mL, 20 mL.

[0043] Further, in step S3, the mixing reaction of the synthetic product and dopamine hydrochloride is that the synthetic product is dispersed in deionized water, dopamine hydrochloride is added to the solution, ultrasonic dispersion is performed, the mixture is stirred for 60 min, and then Tris-HCl is added, and mechanical stirring is performed for 2 hours.

[0044] Further, in step S3, the washing is performed by using ethanol for three times.

[0045] As a preferred embodiment, the manganese-iron oxide magnetic nanoscale enzyme and the preparation method thereof. The preparation method is as follows:

[0046] S1. FeCl3.6H2O (4 / 3 mmol) and MnCl2.xH2O (2 / 3 mmol) are dissolved in 20 mL of a mixed solution of ethylene glycol and diethylene glycol (the volume ratio of ethylene glycol to diethylene glycol is 1:4), and stirred for 30 minutes.

[0047] S2. 2 g of PVP is added to the above solution, and the suspension is heated to solution transparency at 120°C.

[0048] S3. After 1 h, 1.5 g of sodium acetate is added to the above solution and heating is stopped, and further stirring is performed for 30 minutes.

[0049] S4. The above mixed solution is transferred to an autoclave, and reacted at 200°C for 12 h, and then the autoclave is cooled to room temperature.

[0050] S5. The obtained product was centrifuged at 10000 rpm for 10 min, washed with water / ethanol several times, dispersed in deionized water, and stored at 4°C for standby.

[0051] S6. The synthesized product was dispersed into deionized water, 2 mL of 10 mg / mL dopamine hydrochloride was added to the solution, and after ultrasonic dispersion, stirring was performed for 60 min, then 20 mL of 40 mg / mL Tris-HCl was added, mechanical stirring was performed for 2 hours, magnetic separation was performed, ethanol washing was performed three times, and drying was performed.

[0052] The application provides application of the manganese ferrimagnetic nanoscale enzyme in immunodetection of nanoscale enzyme catalytic fluorescence.

[0053] The application provides an immunodetection method based on manganese ferrimagnetic nanoscale enzyme catalysis and a multi-channel microfluidic chip, and the method comprises the following steps:

[0054] S1. Coupling of manganese ferrimagnetic nanoscale enzyme and first antibody;

[0055] S2. Mixing of manganese ferrimagnetic nanoscale enzyme coupled with the first antibody and antigen solution to be detected, incubation, and magnetic adsorption separation to obtain a magnetic immunocomplex;

[0056] S3. Loading of the magnetic immunocomplex solution into a microfluidic chip coated with a second antibody in advance, and reciprocating flow to capture the magnetic immunocomplex;

[0057] S4. Addition of a color developing / fluorescent substrate solution, reciprocating flow for catalytic signal amplification, reading of a signal, and completion of detection.

[0058] Further, the step S1 is mixing of a manganese ferrimagnetic nanoscale enzyme solution and an antibody, incubation at room temperature with vibration, magnetic adsorption of the manganese ferrimagnetic nanoscale enzyme, removal of supernatant, addition of a blocking solution, incubation at room temperature with vibration, and magnetic adsorption separation to obtain manganese ferrimagnetic nanoscale enzyme coupled with the first antibody.

[0059] Preferably, the first antibody in the step S1 is goat anti-human IgG.

[0060] Preferably, the mass ratio of the manganese ferrimagnetic nanoscale enzyme and the antibody in the step S1 is 10:1-3.

[0061] Further preferably, the mass ratio of the manganese ferrimagnetic nanoscale enzyme and the antibody in the step S1 is 10:1.

[0062] Further preferably, the optional concentration and amount of the manganese ferrimagnetic nanoscale enzyme and the antibody are 1 mg / mL of nanoscale enzyme 1 mL and 1 mg / mL of goat anti-human IgG 100 μL.

[0063] Preferably, the blocking solution in the step S1 is BSA.

[0064] Preferably, the optional concentration and amount of the blocking solution in step S1 is 1-2 mL of PBS containing 1-3% BSA.

[0065] Further preferably, the optional concentration and amount of the blocking solution in step S1 is 1 mL of PBS containing 1% BSA.

[0066] Further, the magnetic adsorption in step S2 is magnet adsorption.

[0067] Preferably, step S2 is mixing the manganese-iron oxygen magnetic nanoscale enzyme coupled with the first antibody with the antigen solution to be detected, incubating for 15 min, adsorbing the magnetic immune complex with a magnet, removing the supernatant, adding a cleaning solution to repeat cleaning three times, adding PBS to disperse the immune complex, and obtaining the magnetic immune complex.

[0068] Preferably, the second antibody in step S3 is a rabbit anti-human IgG antibody.

[0069] Preferably, the microfluidic chip structure in step S3 is composed of a substrate, a channel, and a top layer, the channel has a length of 15-30 mm and a height of 0.3-2 mm; the material of the microfluidic chip can be one or more of PMMA, PC, and PVC plastic. Preferably, the chip can have multiple channels at the same time, and multiple indexes can be detected at the same time.

[0070] Preferably, the length of the channel is 20 mm.

[0071] Preferably, the control device for reciprocating flow in steps S3 and S4 is any one of a gas pump, a water bottle, or a magnetic control device.

[0072] Preferably, the control device for reciprocating flow in steps S3 and S4 is a magnetic control device.

[0073] Preferably, the substrate in step S4 can be selected from H2O2 and TMB or H2O2 and HPA.

[0074] Preferably, the signal in step S4 can be selected from a colorimetric signal or a fluorescent signal.

[0075] Preferably, the signal in step S4 is a fluorescent signal.

[0076] The application also provides the use of any of the above-mentioned immune detection methods in the detection of macromolecular biomarkers.

[0077] Compared with the prior art, the application has the following beneficial effects:

[0078] (1) The present application provides a manganese ferrite magnetic nanoscale enzyme with appropriate size, which has the functions of magnetic separation, good enzyme catalysis and solid carrier, realizes the immunoprotein detection based on the magnetic nanoscale enzyme catalytic fluorescence, and can be applied to the detection of macromolecular biomarkers, and expands the application method of nanoscale enzyme in immunodetection.

[0079] (2) The present application provides a multi-channel microfluidic rapid fluorescence immunoassay method based on the manganese ferrite magnetic nanoscale enzyme, and introduces a low-cost nanoscale enzyme catalytic fluorescence detection system, which can detect at a speed far exceeding the traditional immunodetection based on a microplate, and expands the application of magnetic nanoscale enzyme in multi-channel immunodetection. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 It is a picture of the morphology of the magnetic nanoscale enzyme.

[0081] Figure 2 It is a picture of the particle size change and magnetic separation effect of the magnetic nanoscale enzyme after coupling with the antibody.

[0082] Figure 3 It is a schematic diagram of the immunodetection method based on the magnetic nanoscale enzyme and the multi-channel microfluidic chip.

[0083] Figure 4 It is the result of the colorimetric detection of the human IgG by the microfluidic immunodetection method based on the magnetic nanoscale enzyme.

[0084] Figure 5 It is the result of the fluorescence detection by the microfluidic immunodetection method based on the magnetic nanoscale enzyme. DETAILED DESCRIPTION

[0085] The present application will be further described below in combination with the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0086] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0087] Example 1 Preparation of manganese ferrite magnetic nanoscale enzyme

[0088] A manganese ferrite magnetic nanoscale enzyme and a preparation method thereof, and the preparation process is as follows:

[0089] S1, dissolve FeCl3.6H2O (4 / 3 mmol) and MnCl2.xH2O (2 / 3 mmol) in 20 mL of a mixed solution of ethylene glycol and diethylene glycol (the volume ratio of ethylene glycol to diethylene glycol is 1:4), and stir for 30 minutes.

[0090] S2, 2 g PVP was added into the above solution, and the suspension was heated at 120 °C until the solution was transparent.

[0091] S3, 1.5 g sodium acetate was added into the above solution after 1 h and the heating was stopped, and the further stirring was performed for 30 min.

[0092] S4, the above mixed solution was transferred into a hydrothermal kettle, and reacted at 200 °C for 12 h, and then the hydrothermal kettle was cooled to room temperature.

[0093] S5, the obtained product was centrifuged at 10000 rpm for 10 min, washed with water / ethanol several times, dispersed in deionized water, and stored at 4 °C for standby.

[0094] S6, the synthesized product was dispersed into 130 mL deionized water, 2 mL of 10 mg / mL dopamine hydrochloride was added into the solution, after ultrasonic dispersion, stirring was performed for 60 min, then 20 mL of 40 mg / mL Tris-HCl was added, mechanical stirring was performed for 2 hours, magnetic separation was performed, ethanol washing was performed for three times, and drying was performed.

[0095] The prepared magnetic nanoszyme was as shown in Figure 1 The results showed that the magnetic nanoszyme was a solid spherical particle, the average particle size was 132 nm, and the average particle size became 225 nm after the surface was modified by PDA. The larger the particle size, the more the number of modified antibodies on the surface, but the catalytic performance would be reduced. In addition, when the particle size was reduced, the magnetic moment of the particle was reduced, which led to a significant increase in the number of orders of the magnetic separation time. Therefore, to ensure sufficient coupling of the antibody and the catalytic performance, the suitable average particle size range was 200-400 nm. Figure 2 The figure for the characterization of the antibody coupled to the magnetic nanoszyme. After the antibody was coupled, the average particle size of the magnetic nanoszyme became 375 nm, and the magnetic separation could be completed within 30 seconds.

[0096] The catalytic effect of the prepared magnetic nanoszyme on different substrates was as shown in Figure 4 and 5 . Figure 4 , 5 The color development results of the nanoszyme catalyzing H2O2 and TMB substrates and the fluorescence results of H2O2 and HPA substrates at different concentrations of the nanoszyme were shown in Table 1 and Table 2, respectively. The nanoszyme had good catalytic performance when the concentration was above 2.5 mg / mL. The results showed that the magnetic nanoszyme could provide color signals and fluorescence signals by catalyzing different substrates.

[0097] Example 2 Preparation of manganese-iron oxygen magnetic nanoszyme

[0098] The manganese-iron oxide magnetic nanoscale enzyme was prepared according to the preparation method of the manganese-iron oxide magnetic nanoscale enzyme described in Embodiment 1, except that the volume ratio of the ethylene glycol and diethylene glycol in the mixed solution of ethylene glycol and diethylene glycol was 1:2, the molar ratio of FeCl3.6H2O to MnCl2.xH2O was 1:1, and the reaction was performed at 100°C for 12h. The average particle size of the prepared magnetic nanoscale enzyme was 200-400nm.

[0099] Embodiment 3 A multi-channel microfluidic detection method based on magnetic nanoscale enzyme for colorimetric detection of human IgG

[0100] A microfluidic immunoassay method based on the manganese-iron oxide magnetic nanoscale enzyme prepared in Embodiment 1 for catalyzing color development. As shown in Figure 3 , the specific detection method is as follows:

[0101] S1, the manganese-iron oxide magnetic nanoscale enzyme was coupled with the antibody, and the method was as follows: 1mL of 1mg / mL nanoscale enzyme solution was mixed with 100μL of 1mg / mL goat anti-human IgG solution, and incubated at room temperature for 2h. After completion, the nanoscale enzyme was adsorbed by a magnet, and the supernatant was removed. Then, 1mL of blocking solution containing 1% BSA was added, and incubated at room temperature for 2h. The magnet was used for separation and washing, and 1mL of washing solution was added for washing three times.

[0102] S2, 50μL of the antibody-coupled magnetic nanoscale enzyme solution was mixed with 50μL of the human IgG solution to be detected, and incubated for 15min. The magnetic immunocomplex was adsorbed by a magnet, and the supernatant was removed. Then, the washing solution was added for repeated washing three times. 50μL of PBS was added to disperse the immunocomplex.

[0103] S3, 50μL of the immunocomplex solution was loaded into a microfluidic chip coated with rabbit anti-human IgG antibody, and the immunocomplex was captured by reciprocating flow. After completion, it was washed three times.

[0104] S4, 50μL of H2O2 and TMB substrate solution mixed at a ratio of 1:1 was added, and the color development was catalyzed by reciprocating flow. After completion, the color picture was taken in a photo studio, and the detection was completed. The color picture can be analyzed by ImageJ to quantify the results.

[0105] The microfluidic chip structure in step S3 consists of a substrate, a channel, and a top layer. The channel length is 20mm, and the height is 0.3-2mm. The microfluidic chip material can be one or more of PMMA, PC, and PVC plastic. Preferably, the chip can have multiple channels at the same time, and multiple indicators can be detected at the same time.

[0106] The control device for reciprocating flow in steps S3 and S4 is a magnetic control.

[0107] From Figure 4The results showed that the colorimetric signals catalyzed by magnetic nanozymes could be visually distinguished from negative and positive signals of different concentrations. Further quantitative analysis of the grayscale values ​​could be performed using software, demonstrating that this magnetic nanozyme, conjugated with antibodies, could replace HRP enzyme for the colorimetric detection of human IgG immunomarkers. Furthermore, the nanozyme could be used with a multi-channel chip for catalytic detection, eliminating the need for incubation with enzyme-labeled secondary antibodies. The detection speed and throughput were superior to conventional enzyme-linked immunosorbent assay (ELISA), demonstrating the beneficial effect of accelerating immunoassay efficiency.

[0108] Example 4: A microfluidic detection method based on magnetic nanozymes for catalytic fluorescence immunoassay.

[0109] A microfluidic immunoassay method based on magnetic nanozyme-catalyzed fluorescence is presented. Following the detection method in Example 2, H₂O₂ and HPA solution are added to S4 as substrates. After cyclic flow catalysis, the chip is placed in a dark chamber, and fluorescence is excited using a 295nm ultraviolet flashlight. Fluorescence images are then captured by a mobile phone. The fluorescence images can be analyzed using ImageJ to determine grayscale quantification results.

[0110] Depend on Figure 5 The results showed that magnetic nanozymes can catalyze H2O2 and HPA to generate fluorescent signals for immunoassay. The intensity of the fluorescence could be quantitatively analyzed using software, proving that this magnetic nanozyme can be used for catalytic fluorescence immunoassay. Multi-channel fluorescence detection helps to further improve the throughput and sensitivity of the detection.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An immunoassay method based on manganese ferrooxy magnetic nanozyme catalysis and a multi-channel microfluidic chip, characterized in that, Includes the following steps: S1. The manganese ferromagnetic nanozyme was conjugated with the first antibody; S2. Mix the manganese iron oxide magnetic nanozyme coupled with the first antibody with the antigen solution to be tested, incubate, and obtain the magnetic immune complex by magnetic adsorption separation. S3. Load the magnetic immune complex solution into a microfluidic chip pre-coated with a second antibody and allow it to flow back and forth, so that the second antibody captures the magnetic immune complex. S4. Add the chromogenic / fluorescent substrate solution, and perform reciprocating flow catalysis to amplify the signal. Read the signal to complete the detection. The manganese iron oxide magnetic nanozyme is a solid microsphere of manganese iron oxide (MnFe2O4NPs-PDA) with dopamine groups modified on its surface, and its average particle size is 200-400 nm. The preparation method of the manganese iron oxide magnetic nanoenzyme is as follows: iron salt and manganese salt are dissolved using a dual solvent method to obtain a reaction solution; the dual solvent is ethylene glycol and diethylene glycol; the obtained reaction solution is subjected to a hydrothermal reaction to obtain a synthetic product MnFe2O4NPs with an average particle size of 100-200 nm; the synthetic product MnFe2O4NPs is mixed and reacted with dopamine hydrochloride, separated by magnetic adsorption, washed, and dried to obtain solid manganese iron oxide microspheres MnFe2O4NPs-PDA with dopamine groups modified on the surface.

2. The immunoassay method according to claim 1, characterized in that, The molar ratio of the iron salt to the manganese salt is 1 to 2:

1.

3. The immunoassay method according to claim 1, characterized in that, Step S1 involves mixing the manganese iron oxide nanozyme solution with the first antibody, incubating with shaking at room temperature, using magnetic adsorption to remove the supernatant, adding blocking solution, incubating with shaking at room temperature, and obtaining the manganese iron oxide magnetic nanozyme coupled with the first antibody by magnetic adsorption and separation.

4. The immunoassay method according to claim 1, characterized in that, The magnetic adsorption mentioned in step S2 is magnetic adsorption.

5. The immunoassay method according to claim 1, characterized in that, The control device for the reciprocating flow mentioned in steps S3 and S4 is any one of an air pump, a water bottle, or a magnetic control device.

6. The immunoassay method according to claim 1, characterized in that, The signal mentioned in step S4 is a colorimetric or fluorescent signal.

7. The application of the immunoassay method according to any one of claims 1 to 6 in the detection of macromolecular biomarkers.

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