CN110687182A - Preparation method of electrochemical immunosensor for detecting prostate specific antigen - Google Patents
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Abstract
The invention provides a preparation method of an electrochemical immunosensor for detecting prostate specific antigen, which uses Al for a glassy carbon electrode2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning; sequentially dripping gold nanoparticle-loaded black phosphorus nanosheet nanocomposite dispersion liquid, a prostate specific antibody, an ovalbumin solution, a prostate specific antigen solution and an Ab2 bioprobe conjugate onto the surface of a glassy carbon electrode, and washing the surface of the electrode by using a phosphate buffer solution with the pH value of 7.2 to prepare the sandwich type electrochemical immunosensor for detecting the prostate specific antigen; the electrochemical immunosensor prepared by the invention has the advantages of higher sensitivity, wider detection range, higher detection speed, lower detection limit, convenient operation and the like.
Description
Technical Field
The invention relates to the technical field of functional nano composite materials, immunoassay and biosensing, in particular to a preparation method of an electrochemical immunosensor for detecting prostate specific antigen.
Background
Prostate cancer is one of common malignant tumors of the male urinary system, seriously affects the life quality of patients and even threatens the lives of the patients. Early diagnosis is carried out through specific and sensitive biomarkers and analysis tools, the incidence rate of the prostate cancer can be reduced, and the survival rate can be effectively improved. Therefore, the nature and status of prostate cancer can be judged by detecting the content and amount changes of prostate specific antigen, and the early diagnosis of prostate cancer becomes the focus of clinical attention at present by quantitatively detecting prostate specific antigen in serum in medicine. The method for detecting the prostate antigen by combining the electrochemical method and the immunological method to prepare the electrochemical immunosensor has the advantages of higher sensitivity, lower price, higher detection speed, special selectivity, convenient operation and the like, and becomes an important method for detecting disease markers.
The technologies such as radioimmunoassay and chromatographic analysis are reported to be used for detecting prostate antigen processed by a sample in a serum sample. The radioactive immunoassay method has the advantages of rapidness, sensitivity, strong specificity, simplicity, practicality, low cost and the like. The main disadvantages are the problems of protection and pollution prevention, the reagent kit has short service time and relatively narrow measurable range, and the automation of operation and measurement is difficult to realize. The chromatographic analysis is used for analyzing and determining the prostate specific antigen and has the advantages of high analysis speed, small sample consumption and the like, but the detection process is time-consuming and the processing time is long. The application of the technologies such as surface plasma fluorescence method, surface enhanced Raman spectroscopy and the like enables the prostate antigen detection to be further developed. However, these methods not only require expensive automated kit instruments, but also are complicated and time-consuming to operate.
The electrochemical immunosensor is an analysis method based on the specific combination of antigen and antibody, and has the advantages of rapid detection, low detection limit, high sensitivity, simple operation and low preparation cost. In recent years, electrochemical immunosensors have attracted attention and are widely used for detecting tumor markers.
Disclosure of Invention
Aiming at the problems of high cost, low sensitivity, poor stability and the like of the current prostate specific antigen detection, the invention provides a preparation method of an electrochemical immunosensor for detecting prostate specific antigens, which comprises the following specific steps:
(1) al for glassy carbon electrode with diameter of 3-5mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in a nitric acid solution with the mass fraction of 50%, an ethanol water solution and pure water in sequence;
(2) dripping 10 mu L of gold nanoparticle-loaded black phosphorus nanosheet nanocomposite (Au @ BP) dispersion liquid with the concentration of 0.5-2mg/mL onto the surface of the glassy carbon electrode treated in the step (1), airing at room temperature, washing the surface of the electrode by using phosphate buffer solution with the pH value of 7.2, and airing;
(3) dripping 10 mu L of prostate specific antibody (Ab1) with the concentration of 8-12 mu g/mL on the surface of the electrode treated in the step (2), and incubating for 12h at 4 ℃; washing the surface of the electrode with phosphate buffer solution with pH 7.2, and drying in the air;
(4) dripping 10 mu L of Ovalbumin (OVA) solution with the mass fraction of 0.1-0.3 wt% onto the surface of the electrode treated in the step (3), incubating at room temperature for 40min to block nonspecific active sites, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying in the air;
(5) dripping 10 μ L of Prostate Specific Antigen (PSA) solution with a concentration of 0.0001-10ng/mL onto the electrode surface treated in step (4), incubating at room temperature for 1h, washing the electrode surface with phosphate buffer solution with pH of 7.2, and air-drying;
(6) and (3) dropwise adding 10 mu L of Ab2 biological probe conjugate to the electrode surface treated in the step (5), incubating at room temperature for 1h, washing the electrode surface with phosphate buffer solution with the pH value of 7.2, and airing to obtain the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
The ethanol aqueous solution in the step (1) is a mixed solution of absolute ethanol and ultrapure water in a volume ratio of 1: 1.
The preparation method of the Au @ BP dispersion liquid comprises the following specific steps:
(1) preparation of AuNPs: under stirring, 1.0-1.5mL HAuCl with the concentration of 23-25mmol/L4Adding the solution into 80-120mL of boiling deionized water, stirring at 80-150 ℃ for 5-15min, rapidly adding 8-12mL of 14-16mmol/L sodium citrate solution, refluxing for 20-40min until the solution turns to wine red, naturally cooling the solution to room temperature to obtain AuNPs dispersion, and storing at 4 ℃;
(2) preparation of black phospholene (BPene): weighing 10-20mg of black phosphorus crystals, grinding the black phosphorus crystals into powder, dispersing the powder in 20-40mL of deionized water, carrying out ice bath and dark ultrasound for 5-8h, wherein the ultrasound power is 300-500W, centrifuging at 3000rpm for 5-20min, removing residual non-falling blocky black phosphorus particles, centrifuging the collected supernatant at 9000rpm for 5-20min, and carrying out freeze drying to obtain black phosphorus alkene for later use;
(3) preparation of Au @ BP nanocomposite: and (2) preparing the black phosphorus alkene prepared in the step (2) into BP dispersion liquid with the concentration of 1mg/mL, dropwise adding 100-300 mu L of BP dispersion liquid into 1-3mL of AuNPs dispersion liquid prepared in the step (1), stirring for 12h at 4 ℃, centrifugally cleaning for 3 times to obtain an Au @ BP nano composite material, and dispersing the Au @ BP nano composite material in water to obtain the Au @ BP dispersion liquid of the gold nanoparticle-loaded black phosphorus nanosheet nano composite material with the concentration of 0.5-2 mg/mL.
Step (6) Ab2 biological probe conjugate preparation method, the concrete steps are as follows;
taking 4-6mg of Au @ Fe3O4Dispersing the @ COF nano composite material in 4-6mL of deionized water, performing ultrasonic treatment for 20-50min to obtain a mixed solution, adding 1-3mL of Methylene Blue (MB) aqueous solution with the concentration of 1mg/mL into the mixed solution, stirring for 8-16h at 4 ℃, performing centrifugal washing for 3 times, the product was dissolved in 0.5mL of 0.1mol/L phosphate buffer solution at pH 7.2, then adding 100 mul total amount of prostate specific antigen antibody with concentration of 0.8-1.2mg/mL into the solution for labeling by 5 times, after 12h of shake reaction at 4 ℃, 100 μ L of 8-12 wt% bovine serum albumin BSA solution was added, shake reaction was performed for 12h, the resulting solution was centrifuged and the precipitate was dispersed in 0.5mL of 0.1mol/L phosphate buffer solution at pH 7.2 to give Ab2 bioprobe conjugate and stored at 4 ℃ for further use.
The Au @ Fe3O4The preparation method of the @ COF nano composite material comprises the following specific steps:
(1)Fe3O4preparing nano clusters: weighing 1-3g of ferric trichloride hexahydrate, 2-5g of ammonium acetate and 0.1-0.5g of sodium citrate, dissolving the raw materials in 70mL of absolute ethyl alcohol, stirring the mixture at 160 ℃ for 1h, carrying out solvothermal reaction at 200 ℃ for 10-20h, naturally cooling the mixture to room temperature, centrifuging the mixture for 3 times by using ethanol and water respectively, and drying the mixture in vacuum to obtain Fe3O4Nano-cluster for later use;
(2)Fe3O4preparation of @ COF nanocomposites: weighing 12-20mg of Fe obtained in step (1)3O4Dissolving nanoclusters and 10-18mg of Benzidine (BD) in 15mL of Tetrahydrofuran (THF), ultrasonically mixing for 5-15min to form a brown uniform solution, stirring at 50 ℃ for 30min to obtain a dispersion, adding a 1,3, 5-trialdehyde phloroglucinol (Tp) -Tetrahydrofuran (THF) solution into the dispersion at a feeding rate of 0.4mL/min, reacting for 12-24h, and performing suction filtration to obtain a yellow product Fe3O4@ polyimide microspheres, which are dispersed in an o-dichlorobenzene-n-butanol mixed solvent, followed by addition of 0.15mL of pyrrolidine as a catalyst; then, degassing in a 77K liquid nitrogen bath for three freeze-pump-thaw cycles, sealing, heating at 120 deg.C for 24-48h, filtering, washing with acetone, and vacuum drying at 40 deg.C for 48h to obtain Fe3O4@ COF nanocomposites;
(3)Au@Fe3O4preparation of @ COF nanocomposites: fe obtained in the step (2)3O4The @ COF nano composite material is prepared into Fe with the concentration of 1mg/mL by adding water3O4@ COF solution, 0.1-0.7mL of Fe3O4Dropping the @ COF solution into 1.5-4.5mL AuNPs dispersion, stirring at 4 ℃ for 12h, and centrifuging and washing to obtain Au @ Fe3O4@ COF nanocomposites.
The 1,3, 5-trialdehyde phloroglucinol-tetrahydrofuran solution in the step (2) is 4mL of tetrahydrofuran solution in which 6-18mg of 1,3, 5-trialdehyde phloroglucinol is dissolved.
The mixed solvent of the o-dichlorobenzene and the n-butyl alcohol in the step (2) is prepared by mixing 1.35mL of o-dichlorobenzene and 0.15mL of n-butyl alcohol.
The application method of the electrochemical immunosensor for the prostate specific antigen comprises the following specific steps:
(1) testing in 10mL phosphate buffer solution with concentration of 0.1mol/L and pH value of 7.2 by using an electrochemical workstation and taking the prepared electrochemical immunosensor as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode in a three-electrode system;
(2) detecting a target analyte by using a Differential Pulse Voltammetry (DPV) method, scanning a voltage range of-0.1-0.5V, a pulse amplitude of 0.05V and a pulse width of 0.05s, and recording a current peak value;
(3) recording current peak values corresponding to prostate specific antigens under different concentrations;
(4) the concentration of the prostate specific antigen in the sample to be detected is obtained by using a working curve method, and the result shows that the detection range is 0.0001ng/mL-10ng/mL, and the lower limit of detection (LOD) reaches 30fg/mL (S/N is 3).
The invention has the beneficial effects that:
(1) the invention sequentially modifies the surface of a working electrode with gold nanoparticles to load black phosphorus nanosheets (Au @ BP) with better conductivity and gold nanoparticles modified magnetic covalent organic frameworks (Au @ Fe) with excellent enrichment effect on signal molecule methylene blue3O4@ COF); the two materials are used for realizing the amplification effect of the electrochemical signal probe, the constructed sandwich type electrochemical immunosensor realizes the purpose of accurately and quantitatively detecting the prostate specific antigen, and the invention has excellent stability and good reproducibility3O4The @ COF nano composite material is used for marking a secondary antibody as a signal amplification strategy of the sensor, has a good enrichment effect on guest signal molecules, has a large number of catalytic active sites, and shows excellent catalytic performance; au @ BP shows excellent conductivity as a substrate material, and can effectively improve the sensitivity of the immunosensor; the linear detection range of the prepared sensor is 0.0001ng/mL-10ng/mL, and the lowest detection lower limit is 30fg/mL (3 multiplied by 10)-14Mole/liter), successfully detects the content of PSA in an actual serum sample, is lower than the detection limit of the existing electrochemical immunosensor, shows higher detection sensitivity, and provides a cheap, rapid and sensitive method for detecting the PSA.
(2) The electrochemical immunosensor constructed by the invention has the advantages of simple operation, rapid detection, low cost and the like, and can be used for rapid detection of actual samples.
(3) The invention adopts the magnetic covalent organic framework material with excellent enrichment effect on signal molecules and stable performance to construct the sensor for amplifying electrochemical signals, and can effectively catalyze the electrochemical reaction so as to improve the electrical signals.
(4) The invention uses the gold nano particle/black phosphorus nano sheet composite material with excellent conductivity as the substrate material of the constructed electrochemical immunosensor, and has promotion effect on the amplification of the electric signal.
(5) The method for detecting the prostate antigen by combining the electrochemical method and the immunological method to prepare the electrochemical immunosensor has the advantages of higher sensitivity, wider detection range, higher detection speed, lower detection limit, special selectivity, convenient operation and the like, and is expected to become an important method for detecting the marker.
Drawings
FIG. 1 is a schematic diagram of the construction of a PSA electrochemical immunosensor;
FIG. 2 is a Raman spectrum of black phosphorus crystals and black phosphorus nanoplates of example 3;
FIG. 3 shows Fe in example 33O4Nanoclusters and Fe3O4@ COF IR spectrum;
FIG. 4 shows Fe in example 33O4@COF、Au@Fe3O4Transmission Electron microscopy of @ COF, BP and Au @ BP (in the figure, A is Fe)3O4@ COF; b is Au @ Fe3O4@ COF; c is BP; d is Au @ BP);
FIG. 5 is an impedance spectrum of different modified electrodes;
FIG. 6 is a CV diagram (A) and a DPV diagram (B) for different modified electrodes;
FIG. 7 shows a DPV graph (A) and a calibration curve (B) for PSA-modified electrodes at different concentrations.
Detailed Description
The chemical reagents and solvents used in the examples were all analytical grade; the raw materials can be purchased from chemical agents companies or biopharmaceutical companies; the stirring mode adopts a magnetic stirrer; phosphate buffer at pH 7.2 was formulated according to the prior art.
FIG. 1 is a schematic diagram showing the construction of a sandwich-type electrochemical immunosensor for detecting prostate-specific antigens, from which a platform-immobilized antibody A using Au @ BP as an immunoreaction is shownb1, fixing the gold-modified black phosphorus nanosheet on the surface of the electrode, accelerating electron transfer, providing a biocompatible microenvironment for fixing Ab1 and PSA as an analyte, and performing specific reaction of antibody antigen to Au @ Fe3O4@ COF @ MB @ Ab2 as an electrochemical label and finally, the target analyte PSA was detected by electrochemical differential pulse voltammetry.
Example 1
A preparation method of an electrochemical immunosensor for detecting prostate specific antigen comprises the following specific steps:
(1) preparation of AuNPs: 1.0mL of HAuCl at a concentration of 23.46mmol/L was added under stirring4Adding the solution into 80mL of boiling deionized water, stirring for 15min at 80 ℃, rapidly adding 8mL of sodium citrate solution with the concentration of 14.55mmol/L, refluxing for 20min until the solution turns to wine red, naturally cooling the solution to room temperature to obtain AuNPs dispersion, and storing at 4 ℃;
(2) preparation of black phospholene (BPene): weighing 10mg of black phosphorus crystal, grinding the black phosphorus crystal into powder, dispersing the powder in 20mL of deionized water, carrying out ice-bath dark ultrasound for 5h, carrying out centrifugation at 3000rpm for 5min under the ultrasonic power of 300W, removing residual non-shedding blocky black phosphorus particles, centrifuging the collected supernatant at 9000rpm for 5min, and carrying out freeze drying to obtain black phosphorus alkene for later use;
(3) preparation of Au @ BP nanocomposite: preparing the black phosphorus alkene prepared in the step (2) into BP dispersion liquid with the concentration of 1mg/mL, dropwise adding 100 mu L of prepared BP dispersion liquid into 1mL of AuNPs dispersion liquid prepared in the step (1), stirring for 12h at 4 ℃, centrifugally cleaning for 3 times to obtain an Au @ BP nano composite material, and dispersing the Au @ BP nano composite material in water to prepare the Au @ BP dispersion liquid with the concentration of 0.5mg/mL for later use;
(4)Fe3O4preparing nano clusters: weighing 1g of ferric trichloride hexahydrate, 2g of ammonium acetate and 0.1g of sodium citrate, dissolving the raw materials in 70mL of absolute ethyl alcohol, stirring the mixture at 160 ℃ for 1h, carrying out solvothermal reaction at 200 ℃ for 10h, naturally cooling the mixture to room temperature, centrifuging the mixture for 3 times by using ethanol and water respectively, and carrying out vacuum drying to obtain Fe3O4Nano-cluster for later use;
(5)Fe3O4@ COF nmPreparing a composite material: weighing 12mg of Fe prepared in step (1)3O4Dissolving nanoclusters and 10mg of Benzidine (BD) in 15mL of Tetrahydrofuran (THF), ultrasonically mixing for 5min to form a brown uniform solution, stirring at 50 ℃ for 30min to obtain a dispersion, adding 6mg of a 4mL Tetrahydrofuran (THF) solution of 1,3, 5-trialdehyde phloroglucinol (Tp) into the dispersion at a feeding rate of 0.4mL/min, reacting for 12h, and performing suction filtration to obtain a yellow product Fe3O4@ polyimide microspheres, which are dispersed in a mixed solvent of o-dichlorobenzene-n-butanol (1.35mL-0.15mL), wherein the mixed solvent is formed by mixing 1.35mL of o-dichlorobenzene and 0.15mL of n-butanol, and then 0.15mL of pyrrolidine serving as a catalyst is added; then, degassing in a 77K liquid nitrogen bath for three freeze-pump-thaw cycles, sealing, heating at 120 deg.C for 24h, filtering, washing with acetone, and vacuum drying at 40 deg.C for 48h to obtain Fe3O4@ COF nanocomposites;
(6)Au@Fe3O4preparation of @ COF nanocomposites: obtaining Fe in the step (5)3O4The @ COF nano composite material is prepared into Fe with the concentration of 1mg/mL by adding water3O4@ COF solution, 0.1mL Fe3O4Dripping the @ COF solution into 1.5mL of gold nanoparticle dispersion liquid, preparing the gold nanoparticle dispersion liquid from the step (1), stirring for 12h at 4 ℃, and centrifugally washing to obtain Au @ Fe3O4@ COF nanocomposites;
(7) taking 4mg of Au @ Fe prepared in the step (6)3O4@ COF nano composite material is dispersed in 4mL deionized water, ultrasonic treatment is carried out for 20min to obtain a mixed solution, 1mL Methylene Blue (MB) aqueous solution with the concentration of 1mg/mL is added into the mixed solution, stirring is carried out for 8h at 4 ℃, after centrifugal washing is carried out for 3 times, the product is dissolved in 0.5mL phosphate buffer solution with the concentration of 0.1mol/L and the pH value of 7.2, then 100 muL total prostate specific antigen PSA antibody with the concentration of 0.8mg/mL is added into the mixed solution in 5 times of average amount for marking, after shaking table reaction is carried out for 12h at 4 ℃, 100 muL bovine serum BSA solution with the mass fraction of 8 wt% is added, shaking table reaction is carried out for 12h, the obtained solution is centrifugally separated, and sediment is dispersed in 0.5mL bovine serum albumin BSA solution with the concentration of 0.1mol/L and the pH value of 7.2Obtaining Ab2 biological probe conjugate in phosphate buffer solution and storing at 4 ℃ for later use;
(8) al for glassy carbon electrode with diameter of 3mm2O3Polishing the polishing powder into a mirror surface, and carrying out ultrasonic cleaning in a mixture of a nitric acid solution with the mass fraction of 50%, absolute ethyl alcohol and ultrapure water in a volume ratio of 1:1 and pure water in sequence to prepare a bare glassy carbon electrode GCE;
(9) dripping 10 mu L of Au @ BP dispersion liquid with the concentration of 0.5mg/mL prepared in the step (3) onto the surface of the glassy carbon electrode treated in the step (8), airing at room temperature, washing the surface of the electrode by using phosphate buffer solution with the pH value of 7.2, and airing to prepare GCE/Au @ BP;
(10) dripping 10 mu L of prostate specific antibody Ab1 with the concentration of 8 mu g/mL on the surface of the glassy carbon electrode treated in the step (9), incubating at 4 ℃ for 12h, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying in the air to prepare GCE/Au @ BP/Ab 1;
(11) dripping 10 mu L of ovalbumin OVA solution with the mass fraction of 0.1 wt% onto the surface of the glassy carbon electrode treated in the step (10), incubating at room temperature for 40min to block nonspecific active sites, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying to prepare GCE/Au @ BP/Ab 1/OVA;
(12) dripping 10 mu L of prostate specific antigen PSA solution with the concentration of 10ng/mL on the surface of the glassy carbon electrode treated in the step (10), incubating for 1h at room temperature, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and airing to prepare GCE/Au @ BP/Ab 1/OVA/PSA;
(13) and (3) dropwise adding 10 mu L of Ab2 biological probe conjugate prepared in the step (7) to the surface of the glassy carbon electrode treated in the step (5), incubating at room temperature for 1h, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and airing to obtain the sandwich type electrochemical immunosensor GCE/Au @ BP/Ab1/OVA/PSA/Ab2 for detecting prostate specific antigen.
The application of the electrochemical immunosensor for prostate specific antigen in the embodiment comprises the following specific steps:
(1) testing in 10mL phosphate buffer solution with concentration of 0.1mol/L and pH value of 7.2 by using an electrochemical workstation and taking the prepared electrochemical immunosensor as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode in a three-electrode system;
(2) detecting a target analyte by using a Differential Pulse Voltammetry (DPV) method, scanning a voltage range of-0.1-0.5V, a pulse amplitude of 0.05V and a pulse width of 0.05s, and recording a current peak value;
(3) recording current peak values corresponding to prostate specific antigens under different concentrations;
(4) the concentration of the prostate specific antigen in the sample to be detected is obtained by using a working curve method, and the result shows that the detection range is 0.0001ng/mL-10ng/mL, and the lower limit of detection (LOD) reaches 30fg/mL (S/N is 3).
Example 2
A preparation method of an electrochemical immunosensor for detecting prostate specific antigen comprises the following specific steps:
(1) preparation of Au NPs: 1.2mL of 23mmol/L HAuCl were added under stirring4Adding the solution into 100mL of boiling deionized water, stirring at 100 ℃ for 10min, quickly adding 10mL of sodium citrate solution with the concentration of 16mmol/L, refluxing for 30min until the solution turns into wine red, naturally cooling the solution to room temperature to obtain AuNPs dispersion, and storing at 4 ℃;
(2) preparation of black phospholene (BPene): weighing 15mg of black phosphorus crystal, grinding the black phosphorus crystal into powder, dispersing the powder in 30mL of deionized water, carrying out ice-bath dark ultrasound for 6h, wherein the ultrasound power is 400W, centrifuging the powder at 3000rpm for 10min, removing residual and non-shedding blocky black phosphorus particles, centrifuging the collected supernatant at 9000rpm for 10min, and carrying out freeze drying to obtain black phosphorus alkene for later use;
(3) preparation of Au @ BP nanocomposite: preparing the black phosphorus alkene prepared in the step (2) into BP dispersion liquid with the concentration of 1mg/mL, dropwise adding 200 mu L of prepared BP dispersion liquid into 2mL of AuNPs dispersion liquid prepared in the step (1), stirring for 12h at 4 ℃, centrifugally cleaning for 3 times to obtain an Au @ BP nano composite material, and dispersing the Au @ BP nano composite material in water to prepare the Au @ BP dispersion liquid with the concentration of 1mg/mL for later use;
(4)Fe3O4preparing nano clusters: weighing 2g ferric trichloride hexahydrate, 3g ammonium acetate and 0.3g sodium citrate, and dissolving in the mixture70mL of absolute ethyl alcohol, stirring at 160 ℃ for 1h, carrying out solvothermal reaction at 200 ℃ for 15h, naturally cooling to room temperature, centrifuging for 3 times by using ethanol and water respectively, and carrying out vacuum drying to obtain Fe3O4Nano-cluster for later use;
(5)Fe3O4preparation of @ COF nanocomposites: weighing 15mg of Fe prepared in step (1)3O4Dissolving nanoclusters and 15mg of Benzidine (BD) in 15mL of Tetrahydrofuran (THF), ultrasonically mixing for 10min to form a brown uniform solution, stirring at 50 ℃ for 30min to obtain a dispersion, slowly adding 10mg of 1,3, 5-trialdehyde phloroglucinol (Tp) -dissolved 4mL of Tetrahydrofuran (THF) solution into the dispersion at a feeding rate of 0.4mL/min, reacting for 15h, and performing suction filtration to obtain a yellow product Fe3O4@ polyimide microspheres, which are dispersed in a mixed solvent of o-dichlorobenzene-n-butanol (1.35mL-0.15mL), wherein the mixed solvent is formed by mixing 1.35mL of o-dichlorobenzene and 0.15mL of n-butanol, and then 0.15mL of pyrrolidine serving as a catalyst is added; then, degassing in a 77K liquid nitrogen bath for three freeze-pump-thaw cycles, sealing, heating at 120 deg.C for 30h, filtering, washing with acetone, and vacuum drying at 40 deg.C for 48h to obtain Fe3O4@ COF nanocomposites;
(6)Au@Fe3O4preparation of @ COF nanocomposites: obtaining Fe in the step (5)3O4The @ COF nano composite material is prepared into Fe with the concentration of 1mg/mL by adding water3O4@ COF solution, 0.57mL Fe3O4Dripping the @ COF solution into 3mL of gold nanoparticle dispersion liquid, preparing the gold nanoparticle dispersion liquid from the step (1), stirring for 12h at 4 ℃, and centrifugally washing to obtain Au @ Fe3O4@ COF nanocomposites;
(7) dispersing 4.5mg of the Au @ Fe3O4@ COF nano composite material prepared in the step (6) in 5mL of deionized water, performing ultrasonic treatment for 30min to obtain a mixed solution, adding 2mL of Methylene Blue (MB) aqueous solution with the concentration of 1mg/mL into the mixed solution, after stirring for 12h at 4 ℃ and 3 times of centrifugal washing, the product was dissolved in 0.5mL of 0.1mol/L phosphate buffer solution with pH 7.2, then adding 100 mu L of prostate specific antigen PSA antibody with the concentration of 1mg/mL into the solution for labeling by 5 times, placing the mixture in a shaking table at 4 ℃ for reaction for 12h, adding 100 mu L of 10 wt% bovine serum albumin BSA solution, carrying out shaking table reaction for 12h, carrying out centrifugal separation on the obtained solution, dispersing the precipitate in 0.5mL of 0.1mol/L phosphate buffer solution with pH of 7.2 to obtain Ab2 biological probe conjugate, and storing the Ab2 biological probe conjugate at 4 ℃ for later use;
(8) al for glassy carbon electrode with diameter of 4mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in a mixed solution and pure water which are mixed by 50 percent of nitric acid solution, absolute ethyl alcohol and ultrapure water in a volume ratio of 1:1 in sequence to prepare a bare glassy carbon electrode GCE;
(9) dripping 10 mu L of the Au @ BP dispersion liquid with the concentration of 1mg/mL prepared in the step (3) onto the surface of the glassy carbon electrode treated in the step (8), airing at room temperature, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and airing to prepare GCE/Au @ BP;
(10) dripping 10 mu L of prostate specific antibody Ab1 with the concentration of 10 mu g/mL on the surface of the glassy carbon electrode treated in the step (9), incubating at 4 ℃ for 12h, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying in the air to prepare GCE/Au @ BP/Ab 1;
(11) dripping 10 mu L of ovalbumin OVA solution with the mass fraction of 0.2 wt% onto the surface of the glassy carbon electrode treated in the step (10), incubating at room temperature for 40min to block nonspecific active sites, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying to prepare GCE/Au @ BP/Ab 1/OVA;
(12) dripping 10 mu L of prostate specific antigen PSA solution with the concentration of 10ng/mL on the surface of the glassy carbon electrode treated in the step (10), incubating for 1h at room temperature, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and airing to prepare GCE/Au @ BP/Ab 1/OVA/PSA;
(13) and (3) dropwise adding 10 mu L of Ab2 biological probe conjugate prepared in the step (7) to the surface of the glassy carbon electrode treated in the step (5), incubating at room temperature for 1h, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and airing to obtain the sandwich type electrochemical immunosensor GCE/Au @ BP/Ab1/OVA/PSA/Ab2 for detecting prostate specific antigen.
The application of the electrochemical immunosensor for prostate specific antigen in the embodiment comprises the following specific steps:
(1) testing in 10mL phosphate buffer solution with concentration of 0.1mol/L and pH value of 7.2 by using an electrochemical workstation and taking the prepared electrochemical immunosensor as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode in a three-electrode system;
(2) detecting a target analyte by using a Differential Pulse Voltammetry (DPV) method, scanning a voltage range of-0.1-0.5V, a pulse amplitude of 0.05V and a pulse width of 0.05s, and recording a current peak value;
(3) recording current peak values corresponding to prostate specific antigens under different concentrations;
(4) the concentration of the prostate specific antigen in the sample to be detected is obtained by using a working curve method, and the result shows that the detection range is 0.0001ng/mL-10ng/mL, and the lower limit of detection (LOD) reaches 30fg/mL (S/N is 3).
Example 3
A preparation method of an electrochemical immunosensor for detecting prostate specific antigen comprises the following specific steps:
(1) preparation of Au NPs: 1.5mL of 25mmol/L HAuCl were added under stirring4Adding the solution into 120mL of boiling deionized water, stirring for 5min at 150 ℃, rapidly adding 12mL of sodium citrate solution with the concentration of 14mmol/L, refluxing for 40min until the solution turns to wine red, naturally cooling the solution to room temperature to obtain AuNPs dispersion, and storing at 4 ℃;
(2) preparation of black phospholene (BPene): weighing 20mg of black phosphorus crystal, grinding the black phosphorus crystal into powder, dispersing the powder in 40mL of deionized water, carrying out ice bath and dark ultrasound for 8h, carrying out centrifugation at 3000rpm for 20min under the condition that the ultrasonic power is 500W, removing residual and non-shedding blocky black phosphorus particles, carrying out centrifugation on the collected supernatant at 9000rpm for 20min, and carrying out freeze drying to obtain black phosphorus alkene for later use;
(3) preparation of Au @ BP nanocomposite: preparing the black phosphorus alkene prepared in the step (2) into BP dispersion liquid with the concentration of 1mg/mL, dropwise adding 300 mu L of prepared BP dispersion liquid into 3mL of prepared AuNPs dispersion liquid, stirring for 12h at 4 ℃, centrifugally cleaning for 3 times to obtain an Au @ BP nano composite material, and dispersing the Au @ BP nano composite material into water to prepare the Au @ BP dispersion liquid with the concentration of 2mg/mL for later use;
(4)Fe3O4preparing nano clusters: weighing 3g of ferric trichloride hexahydrate, 5g of ammonium acetate and 0.5g of sodium citrate, dissolving the materials in 70mL of absolute ethyl alcohol, stirring the materials at 160 ℃ for 1h, carrying out solvothermal reaction at 200 ℃ for 20h, naturally cooling the reaction product to room temperature, centrifuging the reaction product for 3 times by using ethanol and water respectively, and carrying out vacuum drying to obtain Fe3O4Nano-cluster for later use;
(5)Fe3O4preparation of @ COF nanocomposites: weighing 20mg of Fe prepared in step (1)3O4Dissolving nanoclusters and 18mg of Benzidine (BD) in 15mL of Tetrahydrofuran (THF), ultrasonically mixing for 15min to form a brown uniform solution, stirring at 50 ℃ for 30min to obtain a dispersion, slowly adding a 4mL Tetrahydrofuran (THF) solution of 18mg of 1,3, 5-trialdehyde phloroglucinol (Tp) into the dispersion at a feeding rate of 0.4mL/min, reacting for 24h, and performing suction filtration to obtain a yellow product Fe3O4@ polyimide microspheres, which are dispersed in a mixed solvent of o-dichlorobenzene-n-butanol (1.35mL-0.15mL), wherein the mixed solvent is formed by mixing 1.35mL of o-dichlorobenzene and 0.15mL of n-butanol, and then 0.15mL of pyrrolidine serving as a catalyst is added; then, degassing in a 77K liquid nitrogen bath for three freeze-pump-thaw cycles, sealing, heating at 120 deg.C for 48h, filtering, washing with acetone, and vacuum drying at 40 deg.C for 48h to obtain Fe3O4@ COF nanocomposites;
(6)Au@Fe3O4preparation of @ COF nanocomposites: obtaining Fe in the step (5)3O4The @ COF nano composite material is prepared into Fe with the concentration of 1mg/mL by adding water3O4@ COF solution, 0.7mL Fe3O4Dripping the @ COF solution into 4.5mL of gold nanoparticle dispersion liquid, preparing the gold nanoparticle dispersion liquid from the step (1), stirring for 12h at 4 ℃, and centrifugally washing to obtain Au @ Fe3O4@ COF nanocomposites;
(7) taking 6mg of Au @ Fe prepared in the step (6)3O4Dispersing the @ COF nano composite material in 6mL of deionized water, and performing ultrasonic treatment for 50min to obtain a mixtureAdding 3mL of Methylene Blue (MB) aqueous solution with the concentration of 1mg/mL into the mixed solution, stirring for 16h at 4 ℃, centrifugally washing for 3 times, dissolving the product in 0.5mL of phosphate buffer solution with the concentration of 0.1mol/L and the pH value of 7.2, then adding 100 mu L of prostate specific antigen PSA antibody with the concentration of 1.2mg/mL into the solution in 5 times of average amount for labeling, placing the solution in a shaking table at 4 ℃ for reaction for 12h, adding 100 mu L of bovine serum albumin BSA solution with the mass fraction of 12 wt%, carrying out shaking table reaction for 12h, centrifugally separating the obtained solution, dispersing the precipitate in 0.5mL of phosphate buffer solution with the concentration of 0.1mol/L and the pH value of 7.2 to obtain an Ab2 biological probe conjugate, and storing the Ab2 biological probe conjugate at 4 ℃ for later use;
(8) al for glassy carbon electrode with diameter of 5mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in a mixed solution and pure water which are mixed by 50 percent of nitric acid solution, absolute ethyl alcohol and ultrapure water in a volume ratio of 1:1 in sequence to prepare a bare glassy carbon electrode GCE;
(9) dripping 10 mu L of Au @ BP dispersion liquid with the concentration of 2mg/mL prepared in the step (3) onto the surface of the glassy carbon electrode treated in the step (8), airing at room temperature, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and airing to prepare GCE/Au @ BP;
(10) dripping 10 mu L of prostate specific antibody Ab1 with the concentration of 12 mu g/mL on the surface of the glassy carbon electrode treated in the step (9), incubating at 4 ℃ for 12h, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying in the air to prepare GCE/Au @ BP/Ab 1;
(11) dripping 10 mu L of ovalbumin OVA solution with the mass fraction of 0.3 wt% onto the surface of the glassy carbon electrode treated in the step (10), incubating at room temperature for 40min to block nonspecific active sites, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and drying to prepare GCE/Au @ BP/Ab 1/OVA;
(12) dripping 10 mu L of prostate specific antigen PSA solution with the concentration of 10ng/mL on the surface of the glassy carbon electrode treated in the step (10), incubating for 1h at room temperature, washing the surface of the electrode with phosphate buffer solution with the pH of 7.2, and airing to prepare GCE/Au @ BP/Ab 1/OVA/PSA;
(13) and (3) dropwise adding 10 mu L of Ab2 biological probe conjugate prepared in the step (7) to the surface of the glassy carbon electrode treated in the step (5), incubating at room temperature for 1h, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and airing to obtain the sandwich type electrochemical immunosensor GCE/Au @ BP/Ab1/OVA/PSA/Ab2 for detecting prostate specific antigen.
The application of the electrochemical immunosensor for prostate specific antigen in the embodiment comprises the following specific steps:
(1) testing in 10mL phosphate buffer solution with concentration of 0.1mol/L and pH value of 7.2 by using an electrochemical workstation and taking the prepared electrochemical immunosensor as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode in a three-electrode system;
(2) detecting a target analyte by using a Differential Pulse Voltammetry (DPV) method, scanning a voltage range of-0.1-0.5V, a pulse amplitude of 0.05V and a pulse width of 0.05s, and recording a current peak value;
(3) recording current peak values corresponding to prostate specific antigens under different concentrations;
(4) the concentration of the prostate specific antigen in the sample to be detected is obtained by using a working curve method, and the result shows that the detection range is 0.0001ng/mL-10ng/mL, and the lower limit of detection (LOD) reaches 30fg/mL (S/N is 3).
FIG. 2 is the Raman spectrum of the black phosphorus crystal and the stripped black phosphorus alkene in step (2) of example 3, at 320--1An out-of-plane vibration peak (361 cm) appears in the range-1) And two in-plane vibration peaks (438 cm)-1)、(465.4cm-1) As shown in the figure, three vibration peaks of the black phosphorus alkene are shifted to a certain extent compared with the black phosphorus crystal, and are respectively 1.3 cm, 1.4 cm and 1.9cm-1It is demonstrated that the black phosphorus crystals are exfoliated into black phosphorus alkene, and van der waals force between the black phosphorus crystals is reduced, thereby proving successful preparation of the black phosphorus alkene.
FIG. 3 shows Fe prepared in step (4) of example 33O4Nanoclusters and Fe prepared in step (5)3O4Infrared spectrum of @ COF; due to Fe3O4Nanoclusters and Fe3O4The @ COF has characteristic absorption peaks in the infrared visible region, so that the infrared visible absorption spectrum can be used for monitoring the recombinationThe synthesis of the compound is shown in the figure as Fe3O4At 3429cm-1(O-H) and 586cm-1(Fe-O) has an absorption peak, Fe3O4@ COF spectrum showing imine 1598cm-1Characteristic peak of the (C ═ N) group, 1448cm-1And 1285cm-1The two peaks at (a) correspond to the aromatic C ═ C and the newly formed C — N bond of the keto form, indicating Fe3O4@ COF complexation was successful.
FIG. 4 shows Fe in example 33O4@COF、Au@Fe3O4Transmission electron micrographs of @ COF, BP and Au @ BP; in the figure, A is Fe3O4@ COF; b is Au @ Fe3O4@ COF; c is BP; d is Au @ BP; as shown in FIG. 4A, the polyimide microspheres were deposited on Fe3O4An organic shell layer with the thickness of 100nm is formed on the particles, the appearance is continuous and smooth, and the particle size distribution is uniform; FIG. 4B shows that in Fe3O4The AuNPs with the size of about 5nm is attached to the surface of @ COF, which shows that the nano material Au @ Fe3O4@ COF recombination was successful; FIG. 4C shows that BP prepared is in a lamellar structure; FIG. 4D also shows that AuNPs with a size of about 5nm are attached to the surface of BP, indicating that the nanomaterial Au @ BP is successfully complexed.
The impedance spectra of the GCE, GCE/Au @ BP/Ab1, GCE/Au @ BP/Ab1/OVA, GCE/Au @ BP/Ab1/OVA/PSA and GCE/Au @ BP/Ab1/OVA/PSA/Ab2 prepared in step (8) to step (11) of example 3 were tested with a frequency range of 10, respectively-1-105Hz, as shown in FIG. 5, wherein a-f represent impedance curves of GCE, GCE/Au @ BP/Ab1, GCE/Au @ BP/Ab1/OVA, GCE/Au @ BP/Ab1/OVA/PSA, and GCE/Au @ BP/Ab1/OVA/PSA/Ab2, respectively, the results show that the Rct of Au @ BP/GCE is significantly reduced compared with that of naked GCE because AuNPs can promote electron transfer; when the modified electrode is respectively incubated in Ab1, OVA and PSA solutions, Rct is increased sequentially because protein biomacromolecules obstruct charge transfer, and GCE/Au @ BP/Ab1/OVA/PSA modified electrode is incubated with Ab2 biological probe conjugateThen, due to the high specific surface area of Fe3O4The @ COF has strong adsorption capacity to ferricyanide ions, AuNPs have good electron transfer capacity in the Ab2 bioconjugate, and the Rct value is obviously reduced.
The GCE, GCE/Au @ BP/Ab1, GCE/Au @ BP/Ab1/OVA, GCE/Au @ BP/Ab1/OVA/PSA and GCE/Au @ BP/Ab1/OVA/PSA/Ab2 prepared in step (8) to step (11) of example 3 were subjected to CV (potential range of 0.5 to 0.1V) and DPV (potential range of-0.2 to 0.5V), respectively, since the electrochemical behavior of the sensor could be revealed by measuring the change in the current intensity of the modified electrode, as shown in FIG. 6A as a CV curve, as shown in FIG. 6B as a DPV curve, and as shown in FIGS. 6B as a to f as GCE, GCE/Au @ BP, GCE/Ab @ BP/1, GCE/Au @ BP/5634/Ab, GCE/Au @ BP/3526/Ab, it can be seen from the figure that after a layer of Au @ BP is fixed on a bare GCE, the electric signal response is obviously improved because Au @ BP can promote electron transfer, and after the modified electrode is respectively incubated in Ab1, OVA and PSA solutions, the peak current response is reduced in sequence because protein biomacromolecules can inhibit charge transfer, and when an Ab2 bioprobe conjugate is added, the peak current response is obviously increased probably because Fe is in the Ab2 bioprobe conjugate3O4The high specific surface area of @ COF can absorb a large amount of sodium ferricyanide ions, thereby improving the electron transfer efficiency.
Examples 4 to 11
The electrochemical immunosensor prepared in example 3 was used in the same manner as in example 3 except that the concentration of the prostate-specific antigen (PSA) solution in step (10) of example 3 was 0, 0.0001, 0.0002, 0.0005, 0.001, 0.01, 0.1, and 1ng/mL, respectively, and the lower limit of detection (LOD) was 30fg/mL (S/N ═ 3).
The sensors prepared in examples 4 to 11 plus the sensor prepared in example 3 yielded 9 electrochemical sensors, which were subjected to a DPV (potential range-0.4- -0.1V) test, as shown in FIG. 7A, the value of the current response increased with increasing PSA concentration in the concentration range of 0.0001 to 10ng/mL, due to the fact that the signal molecule immobilized MB on the electrode had a large amount of Ab2 signal probe complexes at higher concentrations of PSA solution; as shown in the standard curve of FIG. 7B, the current is strongDegree (I) and log CPSAThere is a good linear relationship between them, according to the 3 σ criterion, the lower limit of detection (LOD) is 30fg/mL (S/N-3); coefficient of correlation (R)2) A linear relationship of 0.996 is-0.899 log CPSA-6.319, based on Au @ Fe3O4The @ COF nano material has larger pore diameter and good biocompatibility, and the prepared immunosensor has stronger analysis performance, lower detection limit, higher sensitivity and wider linear range, so that the immunosensor is favorable for capturing more signal molecules, and further the dissolution signal is increased and the detection sensitivity is improved.
Claims (7)
1. A preparation method of an electrochemical immunosensor for detecting prostate specific antigens is characterized by comprising the following specific steps:
(1) al for glassy carbon electrode with diameter of 3-5mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in a nitric acid solution with the mass fraction of 50%, an ethanol water solution and pure water in sequence;
(2) dripping 10 mu L of gold nanoparticle-loaded black phosphorus nanosheet nanocomposite dispersion liquid with the concentration of 0.5-2mg/mL onto the surface of the glassy carbon electrode treated in the step (1), airing at room temperature, washing the surface of the electrode with a phosphate buffer solution with the pH value of 7.2, and airing;
(3) dripping 10 μ L of prostate specific antibody with a concentration of 8-12 μ g/mL onto the electrode surface treated in step (2), incubating at 4 ℃ for 12h, washing the electrode surface with phosphate buffer solution with pH 7.2, and air-drying;
(4) dripping 10 mu L of ovalbumin solution with the mass fraction of 0.1-0.3% onto the surface of the electrode treated in the step (3), incubating at room temperature for 40min, washing the surface of the electrode with phosphate buffer solution with the pH value of 7.2, and drying in the air;
(5) dripping 10 μ L of prostate specific antigen solution with concentration of 0.0001-10ng/mL onto the electrode surface treated in step (4), incubating at room temperature for 1h, washing the electrode surface with phosphate buffer solution with pH 7.2, and air drying;
(6) and (3) dropwise adding 10 mu L of Ab2 biological probe conjugate to the electrode surface treated in the step (5), incubating at room temperature for 1h, washing the electrode surface with phosphate buffer solution with the pH value of 7.2, and airing to obtain the sandwich type electrochemical immunosensor for detecting the prostate specific antigen.
2. The method for preparing an electrochemical immunosensor for detecting prostate specific antigens according to claim 1, wherein the ethanol aqueous solution of step (1) is a mixture of absolute ethanol and ultrapure water in a volume ratio of 1: 1.
3. The method for preparing an electrochemical immunosensor for detecting prostate specific antigen according to claim 1, wherein the method for preparing the gold nanoparticle-loaded black phosphorus nanosheet nanocomposite dispersion liquid in step (2) comprises the following specific steps:
(1) under stirring, 1.0-1.5mL HAuCl with the concentration of 23-25mmol/L4Adding the solution into 80-120mL of boiling deionized water, stirring at 80-150 ℃ for 5-15min, adding 8-12mL of 14-16mmol/L sodium citrate solution, refluxing for 20-40min, naturally cooling to room temperature to obtain AuNPs dispersion, and storing at 4 ℃;
(2) weighing 10-20mg of black phosphorus crystal, grinding the black phosphorus crystal into powder, dispersing the powder in 20-40mL of deionized water, carrying out ice bath and dark ultrasound for 5-8h, wherein the ultrasound power is 300 and 500W, centrifuging for 5-20min at 3000rpm, centrifuging the collected supernatant for 5-20min at 9000rpm, and carrying out freeze drying to obtain black phosphorus alkene for later use;
(3) and (2) preparing the black phosphorus alkene prepared in the step (2) into BP dispersion liquid with the concentration of 1mg/mL, dropwise adding 100-300 mu L of BP dispersion liquid into 1-3mL of AuNPs dispersion liquid prepared in the step (1), stirring for 12h at 4 ℃, centrifugally cleaning for 3 times to obtain an Au @ BP nano composite material, and dispersing the Au @ BP nano composite material in water to obtain gold nanoparticle-loaded black phosphorus nanosheet nano composite material dispersion liquid with the concentration of 0.5-2 mg/mL.
4. The method for preparing an electrochemical immunosensor for detecting prostate-specific antigen (PSA) according to claim 3, wherein the Ab2 bioprobe conjugate is prepared by the following steps;
taking 4-6mg of Au @ Fe3O4Dispersing the @ COF nano composite material in 4-6mL of deionized water, performing ultrasonic treatment for 20-50min to obtain a mixed solution, adding 1-3mL of methylene blue solution with the concentration of 1mg/mL into the mixed solution, stirring for 8-16h at 4 ℃, performing centrifugal washing for 3 times, the product was dissolved in 0.5mL of 0.1mol/L phosphate buffer solution at pH 7.2, then adding 100 μ L total prostate specific antigen antibody with concentration of 0.8-1.2mg/mL into the above solution by 5 times, after 12h of shake reaction at 4 ℃, 100 μ L of 8-12% by mass bovine serum albumin solution was added, shake reaction was performed for 12h, the resulting solution was centrifuged and the precipitate was dispersed in 0.5mL of 0.1mol/L phosphate buffer solution at pH 7.2 to give Ab2 bioprobe conjugate and stored at 4 ℃ for further use.
5. The method of claim 4, wherein the electrochemical immunosensor comprises Au @ Fe3O4The preparation method of the @ COF nano composite material comprises the following specific steps:
(1) weighing 1-3g of ferric trichloride hexahydrate, 2-5g of ammonium acetate and 0.1-0.5g of sodium citrate, dissolving the raw materials in 70mL of absolute ethyl alcohol, stirring the mixture at 160 ℃ for 1h, carrying out solvothermal reaction at 200 ℃ for 10-20h, naturally cooling the mixture to room temperature, centrifuging the mixture for 3 times by using ethanol and water respectively, and drying the mixture in vacuum to obtain Fe3O4Nano-cluster for later use;
(2) weighing 12-20mg of Fe prepared in the step (1)3O4Dissolving nanoclusters and 10-18mg of benzidine in 15mL of tetrahydrofuran together, ultrasonically mixing for 5-15min, stirring for 30min at 50 ℃ to obtain a dispersion liquid, adding a 1,3, 5-trialdehyde phloroglucinol-tetrahydrofuran solution into the dispersion liquid at a feeding rate of 0.4mL/min, reacting for 12-24h, and performing suction filtration to obtain a yellow product Fe3O4@ polyimide microspheres, which are dispersed in an o-dichlorobenzene-n-butanol mixed solvent, and then 0.15mL of pyrrolidine is added; then, degassing in a 77K liquid nitrogen bath for three freeze-pump-thaw cycles, sealing, heating at 120 deg.C for 24-48h, filtering, washing with acetone, and vacuum drying at 40 deg.C for 48h to obtain Fe3O4@ COF nanocomposite；
(3) Fe obtained in the step (2)3O4The @ COF nano composite material is prepared into Fe with the concentration of 1mg/mL by adding water3O4@ COF solution, 0.1-0.7mL of Fe3O4Dropping the @ COF solution into 1.5-4.5mL AuNPs dispersion, stirring at 4 ℃ for 12h, and centrifuging and washing to obtain Au @ Fe3O4@ COF nanocomposites.
6. The method of claim 5, wherein the 1,3, 5-trialdehyde phloroglucinol-tetrahydrofuran solution of step (2) is 4mL of tetrahydrofuran solution containing 6-18mg of 1,3, 5-trialdehyde phloroglucinol.
7. The method of claim 5, wherein the mixed solvent of o-dichlorobenzene and n-butanol of step (2) is a mixture of 1.35mL of o-dichlorobenzene and 0.15mL of n-butanol.
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