CN109682964B - Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen - Google Patents
Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention relates to Au @ Fe3O4MNPs‑Ab2The invention discloses a preparation method of a nano enzyme detection probe and a method for detecting multi-component antigen3O4The nano particle probe is successfully applied to the construction of a chemiluminescence array immunosensor with amplified nano enzyme signals. Secondly, fixing different capture antibodies on different solid phase interfaces of the immune array sensor, and then respectively introducing an antigen sample and Au @ Fe3O4MNPs‑Ab2The nano signal amplification probe is incubated on line to form a stable sandwich immunoreaction compound, and an optical signal generated after a chemiluminescent substrate is introduced is collected by a charge coupled CCD (charge coupled device) camera, so that the multi-component antigen chemiluminescence immunoassay with nano enzyme catalysis signal amplification is realized, and the nano signal amplification probe is suitable for simultaneous detection of multiple analyte antigens. The analysis method has the advantages of low detection cost, less sample consumption, short consumed time, good stability, simple operation and the like, and provides a detection platform with a deep prospect for the clinical detection of poultry diseases.
Description
Technical Field
The invention relates to the technical field of biomolecule chemiluminescence immunoassayA domain, in particular Au @ Fe3O4MNPs-Ab2A method for preparing a nano enzyme detection probe and a method for detecting multi-component antigens by using a luminescent array thereof.
Background
Chemiluminescence Immunoassay (CLIA) is an analysis method combining Chemiluminescence with high-specificity immunoreaction with high sensitivity, and is a novel labeling Immunoassay technology for detecting trace antigens and antibodies, which is developed after the traditional Immunoassay methods such as radioimmunoassay, enzyme-linked Immunoassay, fluorescence Immunoassay, time-resolved fluorescence Immunoassay and the like. Chemiluminescence immune analysis is one of the most concerned methods in chemiluminescence immune analysis detection at present, combines chemiluminescence immune analysis and imaging technology, and has the advantages of chemiluminescence immune analysis, high imaging analysis flux and simultaneous detection of multiple components. Has been successfully used for detecting trace substances such as protein, hormone, cell factor and the like, and also plays an important role in the diagnosis of diseases, the detection of the immune function condition of the organism, the monitoring of the treatment effect and the monitoring of the disease condition. In order to improve the sensitivity of immunoassay, nano materials are generally used as carriers for signal amplification.
Compared with natural enzyme, the catalytic activity of the enzyme is easily lost due to the change of the structure in the preservation process, and the catalytic activity of the enzyme can influence other catalytic activities because the active sites of the macromolecular peptide are embedded. The mimic enzyme has the advantages of simple synthesis and preparation, better stability, difficult influence of environmental temperature and the like, simple modification method and the like. Therefore, it is necessary to research and develop a stable and efficient mimic enzyme.
In the practical application of immunoassay, a plurality of single-component analysis methods are commonly used for measuring the content of multiple components in a complex sample in parallel, and the method generally has the defects of complex operation, high analysis cost, time and labor consumption and the like. At present, the multi-component immunoassay technology arouses great research interest in the field of immunoassay, overcomes the defects of the traditional analysis mode, realizes simultaneous detection of multiple components in a single analysis process, and has the advantages of high analysis efficiency, less reagent consumption, short required time, low analysis cost and the like.
Disclosure of Invention
Aiming at the problem that the catalytic activity of a nano material amplification carrier used for improving the analysis sensitivity in the chemiluminescence immunoassay in the prior art is limited, the invention provides Au @ Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe to prepare Au and Fe3O4Nano enzyme detecting probe in conjuncted structure to make Fe3O4The catalytic activity of the (C) is not blocked, and the detection sensitivity is improved.
In order to achieve the above purpose of the invention, the invention Au @ Fe3O4MNPs-Ab2The preparation method of the nano enzyme detection probe comprises the following steps:
first, Au @ Fe was prepared3O4Nanoparticle: the volume ratio is (1-1.5): 20 oleic acid mixed with octadecene in N2Heating to 120 deg.C under flowing condition, and maintaining the temperature for 20 min under N2Injecting Fe (CO) into the mixed liquid below the layer5Of Fe (CO)5Stirring the mixture for 5 min, injecting oleylamine with a volume of half of that of oleic acid into the reacted mixture, and then adding Fe (CO) into the mixture5Uniformly mixing the Au nanoparticle dispersion 10-15 times, heating the mixed solution to 300-310 ℃, refluxing for 45 min, cooling to room temperature, adding isopropanol to separate the particles, separating discrete Au nanoparticles, and separating the Au nanoparticles to obtain the diad Au @ Fe3O4Dispersing the nanoparticles in hexane to make Au @ Fe3O4The dispersion concentration of (A) is 5 mg/mL; au @ Fe of diad structure is prepared by the step3O4Nanoparticles of Au and Fe3O4Are not wrapped mutually, and mutual catalytic activity and capturing capability are not influenced mutually.
To facilitate the preparation of Au @ Fe3O4MNPs-Ab2The nano enzyme detection probe continues to be prepared into Au @ Fe3O4The volume ratio of the solution of the nano particles is 30: (1-1.5) 100E200 μ g/mL Secondary antibody Ab2Slowly stirring for 2h, and centrifuging at 10000 rpm at 4 ℃ for 30min to remove excess Ab2Capturing the antibody; dispersing the centrifugate after precipitation in 0.01M phosphate buffer; the centrifugation and dispersion processes are repeated at least twice, and Au @ Fe with the dispersion concentration of 5mg/mL is finally obtained3O4MNPs-Ab2And (3) nano enzyme detection probe suspension dispersion liquid. The dispersion can be stored at 4 ℃.
Ab with capture antibody prepared by the above method2Of Au @ Fe3O4MNPs-Ab2Nano enzyme detection probe, Au @ Fe of diad structure3O4The MNPs magnetic nanoparticles have excellent catalytic activity and high stability, and overcome the defects of poor stability, low sensitivity, difficult acquisition and the like of natural enzymes.
To further achieve the object of the present invention, the preparation method of the Au nanoparticle dispersion comprises: adding HAuCl4·3H2Adding O into tetralin to prepare a solution with the concentration of 2.5-3 mmol/100 mL, adding oleylamine with the volume of 1/10 of tetralin to prepare a red solution, heating the solution at 0-70 ℃ for 5-6h, cooling to room temperature, adding ethanol into the solution, centrifugally separating gold particles, washing with ethanol, and then re-dispersing in hexane to prepare the Au colloidal dispersion with the dispersion concentration of 10 mg/mL.
To facilitate the utilization of the above Au @ Fe3O4MNPs-Ab2The invention also provides a nano enzyme detection probe for simultaneously detecting multi-component factors, and the invention further provides a nano enzyme detection probe based on Au @ Fe3O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano enzyme detection probe comprises the following steps:
b1: printing a reaction array for multi-point position detection on the silanized glass slide by using a screen printing technology;
b2: respectively dripping the chitosan solution with the same volume and mixed with the capture antibodies of different analytes on each point of the reaction array which is subjected to silk-screen printing in the step B1; blocking the active sites by using a blocking buffer solution to prepare an array immunosensor;
b3: sequentially dripping 5 mu L of antigen to be detected and Au @ Fe which correspond to each point in the step B2 into each point of the immune array3O4MNPs-Ab2A nano enzyme detection probe is incubated to form a stable sandwich immune complex, and a chemiluminescent substrate is added after the buffer solution is cleaned and dried;
b4: the luminescence signals are collected by integration with a CCD camera, the light intensity of each luminescence signal is detected, and the concentration of the antigen sample is calculated according to the linear relation between the light intensity of each luminescence signal and the concentration of the antigen.
The invention successfully constructs a catalyst based on Au @ Fe3O4The nano particle chemiluminescence immune array sensor is characterized in that a high-flux immune sensing array is manufactured on a silanized glass slide by means of a screen printing technology, and a capture antibody is fixed on a corresponding binding site by means of covalent binding of an epoxy group on the glass slide and an amino group carried by the capture antibody. A chemiluminescence imaging immunosensor capable of simultaneously detecting multiple cytokines is constructed. After the antigen and the enzyme-labeled antibody are introduced, based on a sandwich immunoreaction, a chemiluminescent substrate is added to initiate a chemiluminescent reaction catalyzed by a double-nano enzyme, an inductively coupled CCD is used for simultaneously detecting chemiluminescent signals of different cytokines, and the concentration of a detection factor is determined according to the linear relation between the intensity of the luminescent signal and the concentration of the detection factor, so that the method can be used for simultaneously detecting various cytokines, and compared with parallel multi-time single-component analysis, the multi-component immunoassay method can improve the detection flux, shorten the detection time, and reduce the sample consumption and the detection cost; meanwhile, the invention synthesizes Au @ Fe3O4The nano-particle is used for constructing a chemiluminescence array immunosensor simulating enzyme signal amplification, a chemiluminescence imaging immunoassay technology is utilized, a double-antibody sandwich composite structure is formed by means of a capture antibody, an antigen and an enzyme-labeled antibody, chemiluminescence signals of different cytokines are detected by combining an inductively coupled CCD, a good chemiluminescence imaging immunoassay system is constructed, the traditional biological detection method is broken through, the detection of the bottom concentration of 0.01ng/mL can be realized, and the chemiluminescence array immunosensor has high flux, low cost, less consumption, easy operation and high sensitivity on the combined detection of specific cytokines.
Further, in the detection method of the invention, in the step B1, the diameter of the micropore of each detection point of the reaction array subjected to screen printing is 2mm, the distance between the point of the edge and the edge of the glass slide is 4-6 mm, and the outer edge of each micropore is green insulating paint with hydrophobic effect. Therefore, the micropores are hydrophilic, the outer parts of the micropores are hydrophobic, the micropores can bear various reagents, and the problems of signal interference and the like are effectively avoided.
In step B1, the preparation method of the silanized slide glass comprises: and (2) soaking the disposable glass slide in a solution of a tiger fish acid to activate for 10-12 hours, so that the surface of the disposable glass slide is provided with amino, washing with deionized water and drying in the air, then soaking in a toluene solution containing 1% of gamma-glycidoxypropyltrimethoxysilane to silanize the disposable glass slide, then sequentially washing with toluene and ethanol respectively, and drying with nitrogen to prepare the silanized glass slide.
In the step B2), mixing different capture antibodies with the concentration of 100-200 mug/mL and 0.5 wt% of chitosan solution in equal volume, uniformly dripping the mixture into micropores of each array point prepared in the step B1, incubating the mixture for more than 12 hours at room temperature, flushing the glass slide by using phosphate buffer, drying the glass slide by using nitrogen, dripping blocking liquid to each detection point, reacting for 12 hours to block active points, and flushing the glass slide by using PBST solution for three times to prepare the array immunosensor; the PBST solution is 0.01 mol/L pH 7.4 phosphate buffer solution with the mass content of 0.05 percent Tween-20.
Further, the confining liquid is bovine serum albumin solution with the mass concentration of 1.0-3.0%.
Further, respectively dripping the antigen sample to be detected and Au @ Fe in the step B33O4MNPs-Ab2After the nano-enzyme is used for detecting the probes, the incubation time is 25-30 min.
In order to rapidly and accurately perform the simultaneous detection of multiple samples, in step B4, the method for determining the linear relationship between the intensity of the luminescence signal and the antigen concentration is as follows: in step B3, the test sample and Au @ Fe are added to the immunosensor array3O4MNPs-Ab2Before the nano enzyme detection probe, the immunosensor array is divided into a standard sample detection area and an unknown sample detection areaEach detection point position of the standard sample detection area is used for dripping antigen sample with known concentration and Au @ Fe3O4MNPs-Ab2A nano enzyme signal amplification probe; then in step B4, a standard curve of the anti-source concentration and the light intensity is fitted to the obtained light intensity at the known concentration point and the known concentration, and the standard curve is used to determine the concentration value from the light intensity value at the unknown concentration.
In order to quickly and accurately measure the luminous intensity of each detection point, in step B4, the luminous intensity of each image in the CCD camera is detected through software Alpha View SA identification and analysis.
Drawings
FIG. 1 is a light spot image array of luminescence signals when ChIL-4 and ChIFN-gamma standard samples with different concentrations are detected.
FIG. 2 is a plot of concentration versus luminescence intensity for ChIL-4 standards.
FIG. 3 is a graph of ChIFN- γ standard concentration versus luminescence intensity.
Detailed Description
Example 1
Au @ Fe was prepared by this example3O4MNPs-Ab2The nano enzyme detection probe comprises the following specific processes:
firstly, preparing Au nanoparticles: 1.0g of HAuCl4 & 3H2O (2.5 mmol) and 10 mL of oleylamine (30 mmol) were sequentially added to 100mL of tetralin, heated at 65 ℃ for 5H, cooled to room temperature, collected by centrifugation and washed with ethanol to give Au nanoparticles, which were dispersed in hexane to give a dispersion of Au nanoparticles having a dispersion concentration of 10 mg/mL.
Preparing Au @ Fe3O4Nanoparticle: 1 mL of oleic acid (3 mmol) was mixed with 20mL of octadecene in N2Heating to 120 deg.C under flowing condition, and maintaining the temperature for 20 min under N2Under the layer, 0.15 mL of 5mg/mL Fe (CO) solution was injected5. Stirring for 5 min, injecting 0.5 mL oleylamine into the reaction mixture, then injecting 2 mL of the dispersion of Au nanoparticles prepared as described above, heating the solution at about 310 deg.C under reflux for 45 min, cooling to room temperature, adding isopropanol to separate the particles, and separatingThe particles were dispersed in hexane to give a dispersed concentration of 5mg/mL Au @ Fe3O4Nanoparticles.
Finally, Au @ Fe was prepared3O4MNPs-Ab2Nano enzyme detection probe: to the aforementioned Au @ Fe3O4 Add 10. mu.L of 100. mu.g/mL capture antibody Ab to nanoparticle dispersion2Slowly stir for 2h, centrifuge the solution at 10000 rpm for 30min at 4 ℃ to remove excess Ab2(ii) a The pellet was redispersed in 0.01M PBS phosphate buffer. Repeating the centrifugation and dispersion processes twice to finally obtain Au @ Fe3O4MNPs-Ab2The biological complexes were suspended and dispersed in 1.0 mL of 0.01M PBS to make Au @ Fe3O4The dispersion concentration of MNPs-Ab is 5 mg/mL.
Ab with capture antibody prepared by the above procedure of this example2Of Au @ Fe3O4MNPs-Ab2Nano enzyme detection probe, Au @ Fe of diad structure3O4The MNPs magnetic nanoparticles have excellent catalytic activity and high stability, and overcome the defects of poor stability, low sensitivity, difficult acquisition and the like of natural enzymes.
Example 2
In this example, Au @ Fe obtained in example 1 was used3O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor prepared by the nano enzyme detection probe comprises the following steps:
b1: soaking the disposable glass slide in H with the volume ratio of 7:32SO4And 30% H2O2Activating the tiger fish in an acid solution for 10-12 hours to enable the surface of the tiger fish to have amino groups, washing the tiger fish with deionized water, airing the tiger fish, soaking the tiger fish in a toluene solution containing 1% of GPTMS (gamma-glycidyl ether oxypropyltrimethoxysilane) to silanize the tiger fish, sequentially washing the tiger fish with toluene and ethanol respectively, and drying the tiger fish with nitrogen to prepare a silanized glass slide; the silanized glass slide was then printed by screen printing technique and by means of a stencil into a reaction array of 48 detection sites in a 4-line x 12-column format in which the holes formed in the hydrophobic, non-photoactive membrane had a diameter of 2mm and the holes at the edgesThe distance between the edge of the glass slide and the edge of the glass slide is 4 mm;
b2: respectively dripping 5 mu L of chitosan solution mixed with capture antibodies of different chicken cytokines in equal volume on each point of a reaction array which is subjected to silk-screen printing in the step B1; wherein, two rows of 24 point sites are dripped with ChIL-4 capture antibody, the other two rows are dripped with ChIFN-gamma, the concentration of the capture antibody is 100 mug/mL, the incubation is carried out for 12 hours at room temperature, then after a glass slide is washed by phosphate buffer, nitrogen is dried, bovine serum albumin solution with the mass concentration of 2.0 percent is dripped to each detection site as a blocking solution to react for 12 hours so as to block active sites, and PBST (0.01 mol/L of 0.05 percent Tween-20 and pH 7.4 PBS) is used for washing three times to prepare the array immunosensor;
b3: an immunosensor array is divided into a standard sample detection area and an unknown sample detection area, wherein the standard sample detection area is used for detecting the immune luminous intensity of a subsequent sample with a known concentration, 5 mu L of ChIL-4 and ChIFN-gamma antigen samples with different known concentrations are respectively dripped into each detection point position of the standard sample detection area, 5 mu L of ChIL-4 and ChIFN-gamma with different unknown concentrations are respectively dripped into the unknown sample detection area and each point position, then the incubation is carried out for 25 min at room temperature, and 5 mu L of Au @ Fe prepared in the example 1 is dripped into each point position of the standard sample detection area and each point position of the unknown sample detection area respectively3O4MNPs-Ab2Incubating the nano enzyme detection probe for 30min at room temperature; in the step, antigen samples with different known concentrations are prepared by diluting 0.01 mol/L PBS solution with pH 7.4; after the incubation treatment, stable ChIL-4 and ChIFN-gamma core immune complexes are formed, and after being cleaned by 0.01 mol/L PBS phosphate buffer solution and dried by blowing nitrogen, chemiluminescent substrates are added to each detection point; the chemiluminescent substrates used were luminol, p-iodophenol and H2O2Tris-HCl buffer solution of (1);
b4: collecting the generated luminescence signal by using a CCD camera for dynamic integration for 10 min, displaying the luminescence signal into light spot diagrams with different intensities, inputting each light spot diagram into a matched software Alpha View SA to identify and analyze the luminescence point of the image, taking a circle with a fixed diameter at the center of each micropore, and calculating the chemiluminescence intensity of each point by using the average pixel intensity, wherein the image of each antigen sample point dropped in a standard sample detection area is shown in figure 1, figures 2 and 3 are ChIL-4 and ChIFN-gamma standard curves fitted according to the light intensity of each light spot image, and the concentration of a sample with unknown concentration can be determined according to the luminescence intensity of each light spot image in an unknown sample detection area through the standard curves.
Meanwhile, in order to investigate the accuracy and the practical application value of the immune array sensor constructed by the invention, five ChIL-4 and ChIFN-gamma samples with standard concentrations are respectively selected in a standard sample detection zone, a standard addition recovery experiment is carried out, and the obtained result is compared with the standard addition amount. As shown in Table 1, the concentration of the compound was measured at 0.01ng/mL, 0.02 ng/mL, 0.04 ng/mL, 0.08 ng/mL, 0.10 ng/mL (ChIL-4); recovery experiments carried out on 0.01ng/mL, 0.04 ng/mL, 0.08 ng/mL, 0.12 ng/mL, 0.16 ng/mL (ChIFN-. gamma.) standard samples resulted in recoveries of 96% -104% and 94% -104.5%, respectively, showing good agreement with standard additions. The multicomponent chemiluminescence imaging immune system constructed based on the Au @ Fe3O4MNPs mimic enzyme has acceptable actual sample detection capability.
TABLE 1 spiking recovery assay determination of ChIL-4, ChIFN- γ
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. Au @ Fe3O4MNPs-Ab2The preparation method of the nano enzyme detection probe comprises the following steps:
a1: preparation of Au @ Fe3O4Nanoparticle: the volume ratio is (1-1.5): 20 oleic acid mixed with octadecene in N2Heating under flowing downKeeping the temperature at 120 ℃ for 20 min at N2Injecting Fe (CO) into the mixed liquid below the layer5Of Fe (CO)5The concentration of the mixed solution is 35-40 mg/mL, after stirring for 5 min, oleylamine with the volume being half of that of oleic acid is injected into the reacted mixture, and then Fe (CO) is added into the mixture5Uniformly mixing the Au nanoparticle dispersion 10-15 times, heating the mixed solution to 300-310 ℃, refluxing for 45 min, cooling to room temperature, adding isopropanol to separate the particles, separating discrete Au nanoparticles, and separating the Au nanoparticles to obtain the diad Au @ Fe3O4Dispersing the nanoparticles in hexane to make Au @ Fe3O4The dispersion concentration of (A) is 5 mg/mL;
a2: preparation of Au @ Fe3O4MNPs-Ab2Nano enzyme detection probe: to Au @ Fe prepared in step A13O4The volume ratio of the solution of the nano particles is 30: (1-1.5) adding a secondary antibody Ab with a concentration of 100-200 [ mu ] g/mL2Slowly stirring for 2h, and centrifuging at 10000 rpm at 4 ℃ for 30min to remove excess Ab2Capturing the antibody; dispersing the centrifugate after precipitation in 0.01M phosphate buffer; the centrifugation and dispersion processes are repeated at least twice, and Au @ Fe with the dispersion concentration of 5mg/mL is finally obtained3O4MNPs-Ab2And (3) nano enzyme detection probe suspension dispersion liquid.
2. Au @ Fe as set forth in claim 13O4MNPs-Ab2The preparation method of the nano enzyme detection probe comprises the following steps: adding HAuCl4·3H2Adding O into tetralin to prepare a solution with the concentration of 2.5-3 mmol/100 mL, adding oleylamine with the volume of 1/10 of tetralin to prepare a red solution, heating the solution at 0-70 ℃ for 5-6h, cooling to room temperature, adding ethanol into the solution, centrifugally separating gold particles, washing with ethanol, and then re-dispersing in hexane to prepare the Au nanoparticle dispersion with the dispersion concentration of 10 mg/mL.
3. Au @ Fe as claimed in claim 1 or 23O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano enzyme detection probe is characterized by comprising the following steps:
b1: printing a reaction array for multi-point position detection on a silanization glass slide by using a screen printing technology;
b2: dripping the capture antibody of the chitosan solution mixed with different analytes in equal volume on each spot of the reaction array which is silk-screen printed in the step B1; blocking the active sites by using a blocking buffer solution to prepare an array immunosensor;
b3: sequentially dripping 5 mu L of antigen to be detected and Au @ Fe which correspond to each point in the step B2 into each point of the immune array3O4MNPs-Ab2A nano enzyme detection probe is incubated to form a stable sandwich immune complex, and a chemiluminescent substrate is added after the buffer solution is cleaned and dried;
b4: collecting luminescence signals by using a CCD camera, detecting the light intensity of each luminescence signal, and calculating the concentration of the original antigen sample according to the linear relation between the light intensity of each luminescence signal and the antigen concentration.
4. Au @ Fe-based according to claim 33O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nanoenzyme detection probe is characterized in that in the step B1, the diameter of a micropore of each detection point of a silk-screen printing reaction array is 2mm, the distance between the point of the edge and the edge of a glass slide is 4-6 mm, and the outer edge of the micropore is green insulating paint with a hydrophobic effect.
5. Au @ Fe-based according to claim 33O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano-enzyme detection probe is characterized in that in the step B1, the preparation method of the silanization glass slide comprises the following steps: soaking the disposable glass slide in a goby acid solution for activation for 10-12 hours to enable the surface of the disposable glass slide to have amino groups, washing the disposable glass slide with deionized water, drying the disposable glass slide in the air, and then soaking the disposable glass slide in the deionized waterSoaking in toluene solution containing 1% gamma-glycidoxypropyltrimethoxysilane, silanizing, washing with toluene and ethanol, and blowing with nitrogen to dry to obtain silanized glass slide.
6. Au @ Fe-based according to claim 33O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano enzyme detection probe is characterized in that in the step B2), different capture antibodies with the concentration of 100-200 mug/mL and 0.5 wt% chitosan solution are mixed in the same volume and then are uniformly dripped into micropores of each array point position prepared in the step B1, the mixture is incubated for 12 hours at room temperature, then a glass slide is washed by phosphate buffer solution, nitrogen is used for blow-drying, then a sealing liquid is dripped to each detection point position for reaction for 12 hours to seal active points, and then PBST solution is used for washing three times to prepare the array immunosensor; the PBST solution is 0.01 mol/L pH 7.4 phosphate buffer solution with the mass content of 0.05 percent Tween-20.
7. Au @ Fe-based according to claim 63O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano-enzyme detection probe is characterized in that the confining liquid is bovine serum albumin solution with the mass concentration of 1.0-3.0%.
8. Au @ Fe-based according to claim 33O4MNPs-Ab2A method for detecting multi-component antigen by a chemiluminescence array immunosensor of a nano enzyme detection probe comprises the step B3 of respectively dripping an antigen sample to be detected and Au @ Fe3O4MNPs-Ab2After the nano-enzyme is used for detecting the probes, the incubation time is 25-30 min.
9. Au @ Fe-based according to claim 63O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano enzyme detection probe is characterized in that in the step B4, the method for determining the linear relation between the intensity of the luminescence signal and the antigen concentrationThe method comprises the following steps: in step B3, the test sample and Au @ Fe are added to the immunosensor array3O4MNPs-Ab2Before the nano enzyme detection probe, the immunosensor array is divided into a standard sample detection area and an unknown sample detection area, and each detection point of the standard sample detection area is used for dropwise adding an antigen sample with known concentration and Au @ Fe3O4MNPs-Ab2A nano enzyme signal amplification probe; then in step B4, a standard curve of the anti-source concentration and the light intensity is fitted to the obtained light intensity at the known concentration point and the known concentration, and the standard curve is used to determine the concentration value from the light intensity value at the unknown concentration.
10. Au @ Fe-based according to claim 63O4MNPs-Ab2The method for detecting the multi-component antigen by the chemiluminescence array immunosensor of the nano enzyme detection probe is characterized in that in the step B4, the luminous intensity of each image in the CCD camera is detected and obtained through the identification and analysis of software Alpha View SA.
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