阅读说明：本技术 一种检测***特异性抗原的电化学免疫传感器的制备方法 (Preparation method of electrochemical immunosensor for detecting prostate specific antigen ) 是由 李灿鹏 梁还 于 2019-10-30 设计创作，主要内容包括：本发明提供一种检测前列腺特异性抗原的电化学免疫传感器的制备方法,将玻碳电极用Al<Sub>2</Sub>O<Sub>3</Sub>抛光粉抛光成镜面,超声清洗；依次将金纳米粒子负载黑磷纳米片纳米复合材料分散液、前列腺特异性抗体、卵白蛋白溶液、前列腺特异性抗原溶液、Ab2生物探针共轭物滴加到玻碳电极表面上,并用pH＝7.2的磷酸盐缓冲液冲洗电极表面,制备得到夹心型检测前列腺特异性抗原的电化学免疫传感器；本发明制成电化学免疫传感器检测前列腺抗原具有较高的灵敏度、较宽的检测范围、较快的检测速度、较低的检出限以及操作方便等优点。(The invention provides a preparation method of an electrochemical immunosensor for detecting prostate specific antigen, which uses Al for a glassy carbon electrode 2 O 3 Polishing 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.)

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.

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)[email protected]₃O₄preparation 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、[email protected]₃O₄Transmission 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