Preparation method and application of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

Abstract

The invention belongs to the technical field of electrochemical biosensors, and particularly relates to a preparation method and application of a self-supporting electrochemical immunosensor based on an Au-modified Cu2O / CuO heterostructure. The preparation method comprises the following steps: dissolving soluble copper salt, a surfactant, alkali and a reducing agent in water to prepare a mixed solution, standing for reaction, centrifugally washing and drying to obtain a cuprous oxide polyhedron; the preparation method comprises the following steps: preparing cuprous oxide into a dispersion liquid, then adding chloroauric acid, carrying out a stirring reaction in an ice-water bath, and carrying out centrifugal washing and drying to obtain an Au-modified Cu2O / CuO heterostructure; a heterostructure, an OTA antibody and bovine serum albumin are gradually modified on a carbon paper electrode, and the self-supporting electrochemical immunosensor is obtained. According to the invention, the reduction potential of gold is higher than that of copper, the use of an extra reducing agent is avoided by using an electric replacement method, the reaction time is shortened, and the prepared heterostructure self-supporting electrode has good performance and can be used for specific sensitive detection of ochratoxin A in food.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical biosensors, and in particular relates to a self-supporting electrochemical immunosensor based on a Cu2O / CuO heterostructure modified by Au, and a preparation method and application thereof. Background Art

[0002] Ochratoxin A (OTA) has been classified as a Class 2B carcinogen by the International Agency for Research on Cancer due to its toxicity and carcinogenicity to human kidneys and livers, and has been widely concerned by people. OTA is usually found in agricultural products, which has a huge impact on the quality of agricultural products. It can also enter the body of animals through feed, thereby threatening human life and health.

[0003] Currently, many methods have been applied to the determination of OTA, such as enzyme-linked immunosorbent assay, thin-layer chromatography, photoelectrochemical immunoassay, high-performance liquid chromatography, electrochemical immunoassay, etc. Despite the high sensitivity of these methods, the food industry is still focusing on developing simpler, faster and more cost-effective methods for the determination of OTA. Electrochemical biosensors have become a promising analytical technology for the specific detection and analysis of ochratoxin A in the food industry due to their special advantages such as simple operation, good selectivity, rapid response and wide dynamic concentration response range.

[0004] The core of assembling electrochemical biosensors for electrochemical analysis and testing is sensitive signal generators, including enzymes, dyes and metal ions, which have been used in these sensors. Metal ions have excellent conductivity and electroactivity and do not require complex modification, so they can be directly used for electrochemical testing. However, the unsustainable nature of metal materials limits widespread practical implementation. For example, patent CN106855534B discloses the construction of an ochratoxin A immunosensor based on Au octahedral plasma colloids, using Au octahedral dispersion as a base material, Au octahedral plasma colloid-loaded toluidine blue as a secondary antibody marker, and constructing a sandwich electrochemical immunosensor through layer-by-layer self-assembly. The construction method of this immunosensor is complex and costly, which is not conducive to promotion and application. Therefore, it is necessary to provide a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure and its preparation method and application. The reduction potential of gold is higher than that of copper. The use of an electrical replacement method avoids the use of an additional reducing agent and shortens the reaction time. The prepared heterostructure self-supporting electrode has good biosensing performance.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a Cu2O / CuO heterostructure based on Au modification, comprising the following steps:

[0007] S1, dissolving a soluble copper salt, a surfactant, an alkali and a reducing agent in water to prepare a mixed solution, and then standing to react, centrifuging, washing and drying after the reaction is completed to obtain cuprous oxide polyhedrons;

[0008] S2, forming a dispersion of the cuprous oxide polyhedron in a solvent, then adding chloroauric acid, stirring and reacting in an ice-water bath, and after the reaction is completed, centrifuging, washing, and drying to obtain an Au-modified Cu2O / CuO heterostructure.

[0009] Preferably, in step S1, a soluble copper salt and a surfactant are first added to preheated water, and after they are completely dissolved, a base and a reducing agent are added in sequence.

[0010] Further, in step S1, the soluble copper salt includes one or more of copper chloride, copper nitrate, copper acetate, and copper sulfate, preferably copper chloride;

[0011] and / or, the surfactant is sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate or hexadecyltrimethylammonium bromide, preferably SDS;

[0012] And / or, the base is sodium hydroxide or potassium hydroxide;

[0013] And / or, the reducing agent includes one or more of hydroxylamine hydrochloride, ascorbic acid, and glucose, preferably hydroxylamine hydrochloride.

[0014] Further, in step S1, the molar ratio of the soluble copper salt, the surfactant, the alkali and the reducing agent is 1:(5.5-6.5):(3-4):(4-5), preferably 1:6:3.6:4.7; preferably, the concentration of the soluble copper salt in the mixed solution is 5-10 mmol / L, preferably 6-7 mmol / L;

[0015] In step S2, the mass ratio of the cuprous oxide polyhedron to chloroauric acid is 1:(0.05-0.2), preferably 1:0.1.

[0016] Furthermore, in step S1, the temperature of the static reaction is 15-40°C, preferably 25-35°C; and the time is 0.5-2h;

[0017] In step S2, the solvent is ethanol; the concentration of cuprous oxide polyhedrons in the dispersion is 1-2 g / L; and the stirring reaction time is 12-30 h.

[0018] In some specific embodiments, a method for preparing a Cu2O / CuO heterostructure based on Au modification comprises the following steps:

[0019] S1. Dissolve cupric chloride solution, SDS, sodium hydroxide solution, and hydroxylamine hydrochloride solution in 30°C water, and mix them evenly to obtain a mixed solution; after the mixed solution is allowed to stand at 30°C for 1 hour, wash it three times with a mixed solution of ethanol and water, and dry it to obtain cuprous oxide polyhedrons;

[0020] S2. Add cuprous oxide polyhedrons into ethanol to disperse them to obtain a dispersion, add chloroauric acid solution, stir continuously in an ice-water bath for 24 hours, wash three times with a mixture of ethanol and water, and freeze-dry to obtain a heterogeneous structure.

[0021] In step S1, the concentration of the copper chloride solution is 0.1 mol / L, the concentration of the sodium hydroxide solution is 1 mol / L, the concentration of the hydroxylamine hydrochloride solution is 0.1 mol / L, and the mass fraction of SDS in water is 1.95% as a surfactant. In the formed mixed solution, the mass fraction of SDS is 1.16%.

[0022] The mixed solution in step S1 is prepared by first adding copper chloride and SDS, and then adding sodium hydroxide and hydroxylamine hydrochloride after the SDS is completely dissolved, and the color of the solution changes from blue to green.

[0023] After the mixed solution in step S1 is left to stand for a period of time, the color changes from green to orange.

[0024] The volume ratio of the ethanol and water mixture in step S1 is 1:1.

[0025] The concentration of the chloroauric acid solution in step S2 is 50 mmol / L, and the mass ratio of chloroauric acid to cuprous oxide is (0.05-0.2):1, for example, 0.05:1, 0.1:1, 0.15:1 and 0.2:1. Preferably, the mass ratio of chloroauric acid to cuprous oxide is 0.1:1.

[0026] The freeze-drying temperature in step S2 is -10 to -60°C and the time is 24 to 36 hours.

[0027] The dispersion in step S2 is carried out by ultrasonic dispersion, the power of the ultrasonic dispersion is 80 to 100 W, and the ultrasonic time is 0.5 to 1.5 hours.

[0028] In a second aspect, the present invention provides a method for preparing a self-supporting electrochemical immunosensor based on an Au-modified Cu2O / CuO heterostructure, comprising: forming a dispersion in water with the Au-modified Cu2O / CuO heterostructure obtained by any of the preparation methods described above, and then dip-coating it on an electrode; then dispersing ochratoxin A antibody (OTA antibody) on the electrode, incubating, and finally dispersing a BSA solution on the electrode to obtain a self-supporting electrochemical immunosensor.

[0029] Further, the concentration of the Au-modified Cu2O / CuO heterostructure in the dispersion is 0.5-2 mg / mL, preferably 1 mg / mL;

[0030] The concentration of the ochratoxin A antibody is 20-30 μg / mL, preferably 25 μg / mL;

[0031] The volume ratio of the dispersion, ochratoxin A antibody and BSA solution is (1.5-2.5):(2-6):1, preferably 2:4:1;

[0032] The mass fraction of the solute in the BSA solution is 0.05%-0.2%, preferably 0.1%.

[0033] Furthermore, the incubation temperature is 1-5°C and the time is 0.5-2h.

[0034] Furthermore, the electrode is a carbon paper electrode, the thickness of the electrode is 0.1-0.15 mm, when the length of the electrode is 2.5 cm and the width is 0.2 cm, the dipping volume of the dispersion is 18-22 μL, the volume of the ochratoxin A antibody is 35-60 μL, and the volume of the BSA solution is 8-12 μL.

[0035] Preferably, the concentration of the OTA antibody is 25 μg / mL, and the volume is 20-100 μL. Preferably, the antibody volume is 40 μL.

[0036] In a third aspect, the present invention provides a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure, which is prepared by any of the preparation methods described above.

[0037] In a fourth aspect, the present invention provides an application of the above-mentioned self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure, wherein the self-supporting electrochemical immunosensor is used for the specific detection of ochratoxin A in food.

[0038] The specific detection method of ochratoxin A in food by the self-supporting electrochemical immunosensor comprises:

[0039] (1) Immersing the self-supporting electrochemical immunosensor in ochratoxin A standard solutions of different concentrations, performing electrochemical detection using differential pulse voltammetry, and establishing a standard curve of ochratoxin A concentration and current; wherein the scanning range is: -0.6-0.3V.

[0040] (2) Immersing the self-supporting electrochemical immunosensor in a test solution, performing electrochemical detection using differential pulse voltammetry to obtain the current of the test solution, and obtaining the concentration of ochratoxin A in the test solution according to the standard curve.

[0041] In general, the above technical solutions conceived by the present invention have the following technical advantages and beneficial effects compared with the prior art:

[0042] 1. The self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure provided by the present invention realizes charge redistribution and manipulates the electronic structure on the interface by utilizing the difference in reduction potential between gold and copper. Cuprous oxide acts as both a stabilizer and a reducing agent, and can oxidize the surface cuprous oxide into cupric oxide through an electrical replacement process. 3+ Reduction to Au 0 NPs, without the need to add additional reducing agents, the heterostructure constructed in this way can effectively improve the electrocatalytic activity.

[0043] 2. The present invention uses hierarchical assembly technology to synthesize a multilayer immunosensor, using Au-S bonds as labels to enhance the loading of OTA antibodies. After the Cu2O / CuO@AuNPs heterostructure is functionalized with OTA antibodies, it has good catalytic performance for OTA detection, and the self-supporting electrode based on the Au-modified Cu2O / CuO heterostructure obtained by self-assembly has good stability and dispersibility, and has the advantages of flexibility, lightness, and flexibility. It can be used for accurate and sensitive on-site real-time detection of OTA, and is easy to process, with a simple preparation process and low cost.

[0044] 3. The present invention optimizes and controls the relative amounts of cuprous oxide and chloroauric acid to regulate the charge redistribution process of the Au-modified Cu2O / CuO heterostructure, thereby improving the electrocatalytic activity and further improving the sensitivity of sensor detection.

[0045] 4. When the self-supporting electrode prepared by the present invention is used for ultrasensitive detection of ochratoxin A in food, due to the loading of OTA antibodies, ochratoxin A and other foodborne pathogens can be effectively distinguished, which makes it have great application potential in food safety detection.

[0046] 5. The present invention has simple operation process, fast detection and good accuracy. The immunosensor used for OTA specific detection has the advantages of high sensitivity, low detection limit and wide linear range. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are scanning electron microscope images of cuprous oxide polyhedrons prepared in Example 1 of the present invention at different magnifications.

[0048] Figure 2 These are scanning electron microscope images of the Cu2O / CuO@AuNPs heterostructure prepared in Example 1 of the present invention at different magnifications.

[0049] Figure 3 This is a real picture of the self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure prepared in Example 1 of the present invention.

[0050] Figure 4 3. It is the XRD spectrum of the cuprous oxide polyhedron and Cu2O / CuO@AuNPs heterostructure prepared in Example 1 of the present invention.

[0051] Figure 5 It is the full XPS spectrum of the cuprous oxide polyhedron and the Cu2O / CuO@AuNPs heterostructure prepared in Example 1 of the present invention, as well as the peak separation of Cu2p, Au4f and O1s of the Cu2O / CuO@AuNPs heterostructure.

[0052] Figure 6 This is the differential pulse voltammetry current response diagram (left figure) and the response concentration-current standard curve (right figure) of the self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure prepared in Example 1 of the present invention to different concentrations of OTA in 0.1 mol / L phosphate buffered saline (PBS); scanning range: -0.6-0.3V.

[0053] Figure 7 The left figure shows the detection of different concentrations of OTA (0.5ng / mL, 1ng / mL, 3ng / mL) in PBS, raw soybean and corn solutions by standard addition method, and then using Figure 6 (Right) The linear equation: Y = -3.942X + 30.05, the concentration detected by this electrochemical immunosensor is obtained. The recovery rate of OTA detection is 96% to 102%, and the relative standard deviation is low, 1.67% to 4%; the right figure uses the elliptical joint confidence region (EJCR) method to evaluate the accuracy of this method.

[0054] Figure 8 The scanning electron microscope images of the synthesized Cu2O / CuO@AuNPs heterostructures are shown in the left figure. The nanomaterial synthesized in Example 4 by adding an excess of HAuCl4 (50 mM, 441 μL) is shown. The nanomaterial synthesized in Example 5 at room temperature (30°C) is shown in the right figure.

[0055] Fig. 9Electrochemical impedance spectra (left) and corresponding line graph (right) of the self-supporting electrode modified with different volumes (20 μL-100 μL) of OTA antibody.

[0056] Fig.10 This is a schematic diagram of the preparation principle of the self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure provided by the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0058] The present invention provides a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure, such as Fig.10 As shown, the preparation method thereof comprises the following steps:

[0059] (1) Dissolve copper chloride, SDS, sodium hydroxide and hydroxylamine hydrochloride in 30° C. water in sequence and mix them evenly to obtain a mixed solution; the specific operation steps are as follows:

[0060] a. Prepare 0.1 mol / mL copper chloride solution, 1 mol / mL sodium hydroxide solution, 0.1 mol / mL hydroxylamine hydrochloride solution, and weigh 3.9 g SDS powder;

[0061] b. Pour the copper chloride solution and SDS powder into the 30°C water prepared in advance and mix them, and stir continuously to make the solution evenly mixed and the SDS powder completely dissolved;

[0062] c. Add sodium hydroxide solution and hydroxylamine hydrochloride solution to the above mixture and continue stirring to promote uniform mixing;

[0063] d. Place the beaker containing the above solution in a 30°C water bath and let stand for 1-1.5 hours to allow crystal growth;

[0064] The mixed solution in step (1) is allowed to stand for 1-1.5 hours, preferably 1 hour, for crystal growth.

[0065] (2) centrifuging the solution obtained in step (1) at 5000-10000 rpm for 20 minutes, washing three times with a mixture of water and ethanol to remove unreacted reagents and SDS surfactant, and then freeze-drying to obtain cuprous oxide polyhedrons;

[0066] In step (2), the mixed solution is centrifuged at 5000-10000 rpm for 20 minutes, preferably at 8000 rpm.

[0067] In step (2), the solid precipitate is washed with a mixture of water and ethanol, wherein the volume ratio of water to ethanol is 1:1.

[0068] (3) dispersing the cuprous oxide polyhedron powder obtained in step (2) in 30 mL of ethanol solution, adding 50 mmol / mL of chloroauric acid solution to the dispersion, stirring continuously in an ice-water bath, and reacting for 24 hours;

[0069] The mass ratio of chloroauric acid to cuprous chloride in the dispersion in step (3) is 0.05:1, 0.1:1, 0.15:1, 0.2:1, preferably 0.1:1.

[0070] (4) The dispersion obtained in step (3) was centrifuged at 8000 rpm for 20 minutes, washed three times with a mixture of water and ethanol, and then freeze-dried to obtain a Cu2O / CuO@AuNPs heterostructure.

[0071] (5) The Cu2O / CuO@AuNPs heterostructure dispersion was dip-coated on the carbon paper electrode, and then the OTA antibody was dispersed on the electrode, and finally BSA was drop-coated on the electrode.

[0072] In step (5), the Cu2O / CuO@AuNPs heterostructure dispersion is dispersed by ultrasonic dispersion, the ultrasonic dispersion power is 80-100W, the ultrasonic time is 0.5-1.5 hours, preferably the ultrasonic power is 100W, and the time is 1 hour.

[0073] In step (5), the BSA concentration is 0.1% (w / w); the OTA antibody concentration is 25 μg / mL, and the volume is 20-100 μL, preferably 40 μL.

[0074] The self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure prepared according to the above method has a thickness of 0.1-0.15 mm, a length of 2.5 cm and a width of 0.2 cm.

[0075] The self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure prepared by the present invention has good conductivity and flexibility and can be directly used for electrochemical detection. Figure 3The self-supporting electrode is flexible. The self-supporting electrochemical immunosensor based on the Au-modified Cu2O / CuO heterostructure can be applied to electrochemical immunosensing, and the electrochemical immunosensor is preferably used for ochratoxin A, which is highly carcinogenic among foodborne pathogens. The electrochemical biosensor performs sensitive detection through the current response caused by the binding of ochratoxin A antigen and antibody.

[0076] The method for detecting ochratoxin A by a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure includes immersing the self-supporting electrode in ochratoxin A solutions of different concentrations for 30 minutes, performing electrochemical detection by differential pulse voltammetry, and calculating the detection limit by establishing a current standard curve of different concentrations. The concentration of ochratoxin A in the linear range of the standard curve is 0.05-200 ng / mL -1 The specific test method for detecting the concentration of ochratoxin A in raw soybeans and corn is:

[0077] The self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure was immersed in soybean and corn solutions with different concentrations for 30 min, and then electrochemical detection was carried out using differential pulse voltammetry.

[0078] Example 1

[0079] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0080] Take 200mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 22.5mL of 0.1mol / L copper chloride solution, 8mL of 1mol / L sodium hydroxide solution, 105mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 3.9g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue stirring to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote the growth of crystals. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol (volume ratio 1:1, the same below, no more details) until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of cuprous oxide was freeze-dried in a freeze dryer to obtain 96.2 mg of cuprous oxide polyhedral powder, which was collected for use. Figure 1 The scanning electron microscope images of the cuprous oxide polyhedron prepared in this example at different magnifications show that the cuprous oxide has a smooth surface and an average size of 500 nanometers.

[0081] (2) 50 mg of cuprous oxide polyhedrons obtained in step (1) were dispersed in 30 mL of ethanol by ultrasonication for 0.5 h to form a uniform dispersion, with an ultrasonic power of 100 W, and then 290 μL of 50 mmol / L chloroauric acid solution was added, and the mixture was continuously stirred in an ice-water bath for 24 h. The suspension was then centrifuged, and thoroughly washed three times with a mixture of ethanol and water in a volume ratio of 1:1, and freeze-dried at -40°C for 24 h to obtain a Cu2O / CuO@AuNPs nanocomposite material with a diameter of about 500 nm. Figure 2 The following are scanning electron micrographs of the Cu2O / CuO@AuNPs nanocomposites prepared in this example at different magnifications. The figure shows that the Au-modified Cu2O / CuO heterostructure presents a rougher surface, indicating that the cuprous oxide template is etched by HAuCl4 and Au 3+ Reduced to Au 0 And deposited on the surface of Cu2O.

[0082] (3) The Cu2O / CuO@AuNPs nanocomposite material obtained in step (2) was ultrasonically dispersed in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 40 μL of OTA antibody (ochratoxin A antibody (mouse IgG), concentration of 25 μg / mL) was evenly dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immune electrode; and 10 μL of 0.1% BSA solution was dispersed on the surface of the above immune electrode to block any nonspecific active sites, thereby obtaining a self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure (thickness 0.1-0.15 mm, length 2.5 cm, width 0.2 cm).

[0083] In order to prove that an galvanic replacement reaction occurs from step (1) to step (2), the surface of the cuprous oxide polyhedron is oxidized to cupric oxide, and the gold ions are reduced to gold nanoparticles, the present invention performs XRD and XPS analysis on the materials obtained from the two steps, as shown in FIG. Figure 4 and 5 As shown. Figure 4 By comparing the XRD spectra of Cu2O and Cu2O / CuO@AuNPs, we can see the transformation of the crystal structure during the preparation process and the appearance of the corresponding lattice of gold atoms. Figure 5 By comparing the XPS spectra of Cu2O and Cu2O / CuO@AuNPs, it can be seen that the oscillation peaks observed at 962.6, 943.9, and 941.8 eV are characteristic peaks of the copper oxide phase with d9 electronic configuration, proving that the cuprous oxide phase is partially converted into the cupric oxide phase; in the Au4f spectrum, the peaks at 87.9 and 84.3 eV correspond to the Au4f5 / 2 and Au4f 7 / 2 , indicating that Au 3+ and Au 0 exist at the same time.

[0084] Example 2

[0085] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0086] Take 100mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 22.5mL of 0.1mol / L copper chloride solution, 8mL of 1mol / L sodium hydroxide solution, 105mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 3.9g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue stirring to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote the growth of crystals. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of copper oxide was freeze-dried in a freeze dryer to obtain 83.2mg of cuprous oxide polyhedral powder, which was collected for use. Compared with Example 1, it is shown that when the concentration of the mixed solution is too high, the yield is reduced.

[0087] (2) 50 mg of cuprous oxide polyhedrons obtained in step (1) were dispersed in 30 mL of ethanol by ultrasonication for 0.5 h to form a uniform dispersion, with an ultrasonic power of 100 W, and then 290 μL of 50 mmol / L chloroauric acid solution was added, and the mixture was continuously stirred in an ice-water bath for 24 h. The suspension was then centrifuged, thoroughly washed three times with a mixture of ethanol and water in a volume ratio of 1:1, and freeze-dried at -40°C for 24 h to obtain a Cu2O / CuO@AuNPs nanocomposite material with a diameter of about 500 nm.

[0088] (3) Ultrasonic dispersion of the Cu2O / CuO@AuNPs nanocomposite obtained in step (2) in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 40 μL of OTA antibody was dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immunoelectrode; 0.1% BSA solution was prepared, and 10 μL was dispersed on the surface of the above immunoelectrode to block any nonspecific active sites. A self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure was obtained.

[0089] Example 3

[0090] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0091] Take 300mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 33.75mL of 0.1mol / L copper chloride solution, 12mL of 1mol / L sodium hydroxide solution, 157.5mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 5.85g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue stirring to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote crystal growth. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of cuprous oxide was freeze-dried in a freeze dryer to obtain 135.5mg of cuprous oxide polyhedral powder, which was collected for use.

[0092] (2) 50 mg of cuprous oxide polyhedrons obtained in step (1) were dispersed in 30 mL of ethanol by ultrasonication for 0.5 h to form a uniform dispersion, with an ultrasonic power of 100 W, and then 290 μL of 50 mmol / L chloroauric acid solution was added, and the mixture was continuously stirred in an ice-water bath for 24 h. The suspension was then centrifuged, and thoroughly washed three times with a mixture of ethanol and water in a volume ratio of 1:1, and freeze-dried at -40°C for 24 h to obtain a Cu2O / CuO@AuNPs nanocomposite material with a diameter of about 500 nm.

[0093] (3) Ultrasonic dispersion of the Cu2O / CuO@AuNPs nanocomposite obtained in step (2) in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 40 μL of OTA antibody was dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immunoelectrode; 0.1% BSA solution was prepared, and 10 μL was dispersed on the surface of the above immunoelectrode to block any nonspecific active sites. A self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure was obtained.

[0094] Example 4

[0095] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0096] Take 200mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 22.5mL of 0.1mol / L copper chloride solution, 8mL of 1mol / L sodium hydroxide solution, 105mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 3.9g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue stirring to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote crystal growth. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of cuprous oxide was freeze-dried in a freeze dryer and collected for use.

[0097] (2) 50 mg of the cuprous oxide polyhedron obtained in step (1) was dispersed in 30 mL of ethanol by ultrasonication for 0.5 h to form a uniform dispersion, the ultrasonic power was 100 W, and then 441 μL of 50 mmol / L chloroauric acid solution was added, and the mixture was continuously stirred in an ice-water bath for 24 h. The suspension was then centrifuged, thoroughly washed with water and ethanol, and freeze-dried to obtain a Cu2O / CuO@AuNPs nanocomposite material. However, excess gold would cause the gold nanoparticles to agglomerate on the surface of the cuprous oxide, such as Figure 8 (Left), resulting in uneven distribution of gold nanoparticles, affecting the sensitivity of detection.

[0098] (3) Ultrasonic dispersion of the Cu2O / CuO@AuNPs nanocomposite obtained in step (2) in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 40 μL of OTA antibody was dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immunoelectrode; 0.1% BSA solution was prepared, and 10 μL was dispersed on the surface of the above immunoelectrode to block any nonspecific active sites. A self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure was obtained.

[0099] Example 5

[0100] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0101] Take 200mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 22.5mL of 0.1mol / L copper chloride solution, 8mL of 1mol / L sodium hydroxide solution, 105mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 3.9g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue stirring to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote the growth of crystals. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of cuprous oxide was freeze-dried in a freeze dryer to obtain 96mg of cuprous oxide polyhedral powder, which was collected for future use.

[0102] (2) 50 mg of the cuprous oxide polyhedron obtained in step (1) was dispersed in 30 mL of ethanol by ultrasonic treatment for 0.5 h to form a uniform dispersion, with an ultrasonic power of 100 W, and then 290 μL of a 50 mmol / L chloroauric acid solution was added, and the mixture was stirred continuously for 24 h at room temperature (30° C.), and then the suspension was centrifuged, and thoroughly washed with a mixture of ethanol and water in a volume ratio of 1:1 for 3 times, and freeze-dried at -40° C. for 24 h to obtain a Cu2O / CuO@AuNPs nanocomposite material. When reacted at room temperature, the gold nanoparticles agglomerated and unevenly distributed, such as Figure 8 (Right) It can be clearly observed that Au aggregates on the Cu2O surface, which affects the detection sensitivity.

[0103] (3) Ultrasonic dispersion of the Cu2O / CuO@AuNPs nanocomposite obtained in step (2) in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 40 μL of OTA antibody was dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immunoelectrode; 0.1% BSA solution was prepared, and 10 μL was dispersed on the surface of the above immunoelectrode to block any nonspecific active sites. A self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure was obtained.

[0104] Example 6

[0105] (1) Preparation of self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure

[0106] Take 200mL of water and place it in a beaker, preheat it in a water bath at 30℃ for 15 minutes, prepare 22.5mL of 0.1mol / L copper chloride solution, 8mL of 1mol / L sodium hydroxide solution, 105mL of 0.1mol / L hydroxylamine hydrochloride solution, and weigh 3.9g of SDS powder. Pour the prepared copper chloride solution and SDS powder into the preheated water, and stir vigorously to completely dissolve the SDS powder; then add the prepared sodium hydroxide solution and hydroxylamine hydrochloride solution to the mixed solution in turn, continue to stir to mix evenly, and the color of the solution changes from blue to green; stop stirring after mixing evenly, and let it stand at a constant temperature of 30℃ for 1 hour to promote the growth of crystals. The obtained orange solution was centrifuged at 8000rpm for 20min, and then washed with water and ethanol until the unreacted reagents and SDS surfactant were completely removed. Finally, the orange precipitate of cuprous oxide was freeze-dried in a freeze dryer and collected for future use.

[0107] (2) 50 mg of cuprous oxide polyhedrons obtained in step (1) were dispersed in 30 mL of ethanol by ultrasonication for 0.5 h to form a uniform dispersion, with an ultrasonic power of 100 W, and then 290 μL of 50 mmol / L chloroauric acid solution was added, and the mixture was continuously stirred in an ice-water bath for 24 h. The suspension was then centrifuged, and thoroughly washed three times with a mixture of ethanol and water in a volume ratio of 1:1, and freeze-dried at -40°C for 24 h to obtain a Cu2O / CuO@AuNPs nanocomposite material with a diameter of about 500 nm.

[0108] (3) Ultrasonic dispersion of the Cu2O / CuO@AuNPs nanocomposite obtained in step (2) in water to prepare a 1 mg / mL dispersion, and 20 μL of the dispersion was dip-coated on a carbon paper electrode to obtain a CP / Cu2O / CuO@AuNPs electrode; 60 μL of OTA antibody was evenly dispersed on the CP / Cu2O / CuO@AuNPs electrode and incubated at 4°C for 1 h to obtain an immune electrode; a 0.1% BSA solution was prepared, and 10 μL was dispersed on the surface of the above immune electrode to block any nonspecific active sites, thereby obtaining a self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure. Excessive antibody modification may lead to reduced anti-OTA activity, so the antibody volume was selected to be 40 μL, such as Fig. 9 . It can be observed from the figure that when the antibody volume increases to 40μL, the Rct value no longer changes significantly, indicating that the antibody content on the electrode is saturated at this time. If the concentration is too low, it may lead to insufficient adsorption of OTA antibodies and reduce the detection efficiency. However, too much concentration may lead to reduced activity of OTA antibodies. Therefore, 40μL is selected as the optimal antibody volume.

[0109] The testing process of materials is the same

[0110] The 0.2 cm × 2.5 cm self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure prepared in Example 1 was immersed in different concentrations of ochratoxin A standard solution for 30 minutes, wherein the concentration range of the ochratoxin A standard solution was 0.05-200 ng / mL; a differential pulse voltammetry test was performed using an electrochemical workstation using a three-electrode system (Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and a self-supporting electrode based on the Cu2O / CuO@AuNPs heterostructure as a working electrode) to obtain a current response curve, and the standard curve and equation were calculated. Figure 6 As shown in the figure, after adding ochratoxin A, due to the interaction between OTA antigen and OTA antibody on CP / Cu2O / CuO@AuNPs / anti-OTA / BSA immunoelectrode, immune complex (antigen-antibody) is formed on the electrode surface. At -0.1V, the peak current decreases with the increase of OTA concentration. And a good linear relationship is formed in the range of 0.05-200ng / mL. The obtained standard curve equation is Y=-3.942X+30.05, where X represents the logarithm of OTA concentration (in pg / mL) with a base of 10, and the detection limit is 0.0002ng / mL.

[0111] In order to investigate the practical application possibility of the electrode, ochratoxin A in raw soybeans and corn was detected. 20 g of purchased soybean and corn flour were taken respectively, dissolved in 100 mL of ethanol-water (v:v=7:3) mixture, stirred continuously for 3 hours, centrifuged at 8000 r / min for 10 minutes, and the supernatant was taken. 0.5 ng / mL, 1 ng / mL and 3 ng / mL OTA were added by standard addition method. The 0.2 cm×2.5 cm self-supporting electrode based on Cu2O / CuO@AuNPs heterostructure prepared in Example 1 was immersed in three groups of PBS buffer (0.1 M, pH=7) containing 0.5 ng / mL, 1 ng / mL and 3 ng / mL OTA, three groups of soybeans and three groups of corn centrifuges for 30 minutes, and DPV test was carried out by electrochemical workstation using a three-electrode system (Ag / AgCl electrode as reference electrode, platinum wire electrode as counter electrode, and self-supporting electrode based on Cu2O / CuO@AuNPs heterostructure as working electrode). According to the current response signal, the content of ochratoxin A in PBS buffer, soybean and corn centrifuge liquid was calculated according to the standard curve, such as Figure 7(Left). The recovery rate of OTA detection is 96% to 102%, and the relative standard deviation is low, which is 1.67% to 4%. At the same time, the ellipse joint confidence region (EJCR) method is used to evaluate the accuracy of the method. EJCR is achieved by comparing the added OTA concentration with the predicted OTA concentration. Here, the predicted OTA concentration is regressed to the standard addition value by ordinary least squares. The intercept and slope values ​​calculated from the ellipse obtained by EJCR are compared with the expected theoretical values ​​(intercept = 0, slope = 1, also known as the "ideal point"). If the ideal point is within the ellipse, it means that the method has good accuracy. EJCR test results are shown in Figure 7 (Right) All ellipses included the ideal point (intercept = 0, slope = 1), confirming the practicality and accuracy of the developed analytical method for OTA detection in soybean and corn samples.

[0112] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Cu2O / CuO heterostructure based on Au modification, characterized in that: The following steps are involved: S1, dissolving a soluble copper salt, a surfactant, an alkali and a reducing agent in water to prepare a mixed solution, and then standing to react, centrifuging, washing and drying after the reaction is completed to obtain cuprous oxide polyhedrons; S2, forming a dispersion of the cuprous oxide polyhedron in a solvent, then adding chloroauric acid, stirring and reacting in an ice-water bath, and after the reaction is completed, centrifuging, washing, and drying to obtain an Au-modified Cu2O / CuO heterostructure.

2. The method for preparing the Au-modified Cu2O / CuO heterostructure according to claim 1, characterized in that: In step S1, the soluble copper salt includes one or more of copper chloride, copper nitrate, copper acetate, and copper sulfate; And / or, the surfactant is sodium dodecyl sulfate, sodium dodecylbenzene sulfonate or hexadecyltrimethylammonium bromide; And / or, the base is sodium hydroxide or potassium hydroxide; And / or, the reducing agent includes one or more of hydroxylamine hydrochloride, ascorbic acid, and glucose.

3. The method for preparing the Au-modified Cu2O / CuO heterostructure according to claim 1, characterized in that: In step S1, the molar ratio of the soluble copper salt, the surfactant, the alkali and the reducing agent is 1:(5.5-6.5):(3-4):(4-5); preferably, the concentration of the soluble copper salt in the mixed solution is 5-10 mmol / L, preferably 6-7 mmol / L; In step S2, the mass ratio of the cuprous oxide polyhedron to chloroauric acid is 1:(0.05-0.2), preferably 1:0.

1.

4. The method for preparing the Au-modified Cu2O / CuO heterostructure according to claim 1, characterized in that: In step S1, the temperature of the static reaction is 15-40°C, preferably 25-35°C; the time is 0.5-2h; In step S2, the solvent is ethanol; the concentration of cuprous oxide polyhedrons in the dispersion is 1-2 g / L; and the stirring reaction time is 12-30 h.

5. A method for preparing a self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure, characterized in that: include: The Au-modified Cu2O / CuO heterostructure obtained by the preparation method according to any one of claims 1 to 4 is dispersed in water to form a dispersion, which is then dip-coated on an electrode; ochratoxin A antibody is then dispersed on the electrode, incubated, and finally a BSA solution is drop-coated on the electrode to obtain a self-supporting electrochemical immunosensor.

6. The preparation method according to claim 5, characterized in that: The concentration of the Au-modified Cu2O / CuO heterostructure in the dispersion is 0.5-2 mg / mL, preferably 1 mg / mL; The concentration of the ochratoxin A antibody is 20-30 μg / mL, preferably 25 μg / mL; The volume ratio of the dispersion, ochratoxin A antibody and BSA solution is (1.5-2.5):(2-6):1, preferably 2:4:1; The mass fraction of the solute in the BSA solution is 0.05%-0.2%, preferably 0.1%; And / or, the incubation temperature is 1-5°C and the incubation time is 0.5-2h.

7. The preparation method according to claim 5, characterized in that: The electrode is a carbon paper electrode, and the thickness of the electrode is 0.1-0.15 mm. When the length of the electrode is 2.5 cm and the width is 0.2 cm, the dipping volume of the dispersion is 18-22 μL, the volume of the ochratoxin A antibody is 35-60 μL, and the volume of the BSA solution is 8-12 μL.

8. A self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure, characterized in that: The method is prepared by any one of claims 5 to 7.

9. An application of the self-supporting electrochemical immunosensor based on Au-modified Cu2O / CuO heterostructure according to claim 9, characterized in that: The self-supporting electrochemical immunosensor is used for the specific detection of ochratoxin A in food.

10. The use according to claim 9, characterized in that: The specific detection method of ochratoxin A in food by the self-supporting electrochemical immunosensor comprises: (1) immersing the self-supporting electrochemical immunosensor into ochratoxin A standard solutions of different concentrations, performing electrochemical detection by differential pulse voltammetry, and establishing a standard curve of ochratoxin A concentration and current; (2) Immersing the self-supporting electrochemical immunosensor in a test solution, performing electrochemical detection using differential pulse voltammetry to obtain the current of the test solution, and obtaining the concentration of ochratoxin A in the test solution according to the standard curve.

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