CN111517360A

CN111517360A - Nanocomposite based on phosphorus-molybdenum-containing polyoxometallate and preparation method thereof, aptamer sensor and electrode thereof

Info

Publication number: CN111517360A
Application number: CN202010108449.5A
Authority: CN
Inventors: 宋英攀; 杜淼; 张治红; 徐妙然; 何领好; 王明花; 贾巧娟
Original assignee: Zhengzhou University of Light Industry
Current assignee: Zhengzhou University of Light Industry
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2020-08-11
Anticipated expiration: 2040-02-21
Also published as: CN111517360B

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to a phosphorus-molybdenum-containing polyoxometallate-based nano composite material and a preparation method thereof, an aptamer sensor and an electrode thereof. The nanocomposite material comprises carbon, molybdenum disulfide nanosheets and silver-containing nanoparticles; the nano composite material is obtained by calcining silver-doped phosphomolybdic polyoxometallate, wherein the silver-doped phosphomolybdic polyoxometallate is obtained by reacting a silver source, phosphomolybdic acid and thioacetamide. The nano composite material has higher specific surface area and stronger biological affinity, and an electrochemical sensor constructed by the nano composite material has lower detection limit when being used for detecting bisphenol A (BPA), and has high selectivity, good stability and reproducibility, excellent reproducibility and applicability under different environments.

Description

Nanocomposite based on phosphorus-molybdenum-containing polyoxometallate and preparation method thereof, aptamer sensor and electrode thereof

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to a phosphorus-molybdenum-containing polyoxometallate-based nano composite material and a preparation method thereof, an aptamer sensor and an electrode thereof.

Background

Bisphenol a (2, 2-p-phenol propane, BPA) is a phenolic resin with a large yield, is widely applied to the polymer industry, and is commonly used as a monomer for synthesizing polycarbonate plastics, polysulfone resin, polyphenylene oxide resin, epoxy resin, unsaturated polyester resin and the like. BPA is in an extremely wide commercial range, such as for baby bottles, beverage containers, medical equipment, food packaging, thermal paper, and the like, and 8 million tons of BPA are consumed globally in 2016, thereby causing extensive environmental pollution. Numerous studies have shown that bisphenol a has estrogenic activity, leading to reproductive and developmental toxicity; and can affect the behavior and intelligence development of experimental animals and the normal function of the immune system; prolonged exposure to bisphenol a promotes the development of diabetes and obesity in experimental animals. According to the EU regulations, the BPA content of the commercial product should be less than 3 mg/kg^-1. Since low doses of BPA can cause diseases such as endocrine dyscrasia and tumors, from 2011, china banned BPA from the production of baby bottles.

Due to the negative impact of BPA on human health and the environment, there is an urgent need to detect trace amounts of BPA in order to improve food safety, monitor environmental pollution, and improve human health. Various methods are currently available for detecting BPA, such as chromatography, enzyme-linked immunosorbent assays (ELISA), surface enhanced raman scattering, Surface Plasmon Resonance (SPR), quartz crystal microbalance, fluorescence, colorimetric analysis, dual-polarization interferometry, liquid chromatography, and electronic sensing methods. Despite the considerable effort involved, conventional methods still suffer from drawbacks such as time consuming, high cost, cumbersome preparation prior to bioassay, etc.

Various probe molecules, such as enzymes, antibodies, aptamers and the like, can be used as a biological recognition element to realize the high selectivity detection of BPA by the electrochemical biosensor. The aptamer has the advantages of high stability, easiness in modification, convenience in repeated and simple synthesis, low cost and the like compared with an antibody. Therefore, they are increasingly applied to the development of various nucleic acid aptamer sensors. The DNA functionalized solution controlled graphene transistor can be used for detecting BPA by combining with a microfluidic system. Several BPA nucleic acid aptamer sensors have been constructed by different methods, such as inhibition of xanthine oxidase, non-targeted induced bridge assembly and nucleic acid aptamer extension reaction by terminal deoxynucleotidyl transferase, single-stranded DNA-methylene blue complex, and the like. Different detection methods, such as electrochemiluminescence, electrochemical techniques, and plasmon methods, are also used in the development of nucleic acid aptamer sensors for detection of BPA. Clearly, electrochemical techniques are the most commonly used methods for BPA detection. Most electrochemical biosensors are constructed using electrochemical indicators, so that although electrochemical signals can be greatly amplified or sensing performance can be enhanced, the corresponding construction steps are complicated.

Nanomaterials, such as carbon nanomaterials, inorganic nanoparticles, porous organic framework materials, etc., can be used as a platform for electrochemical biosensors to improve the selectivity and sensitivity of the biosensors. A novel Polyoxometallate (POMs) -based nanomaterial that exhibits superior electrochemical sensing performance due to its electronic versatility, such as molybdenum carbide nanoparticles and nitrogen-rich graphene-like carbon layers, which are obtainable from POM precursors containing organic carbon sources, contains electrochemically active and catalytic sites, and is therefore frequently used as an electrocatalyst in the field of clean energy or energy transfer. Also, the POM-based electrochemical biosensor shows a rapid response and good catalytic activity to dopamine, hydrogen peroxide, L-cysteine, ascorbic acid and glucose.

However, the nanocomposites of electrochemical sensors for BPA detection currently focus mainly on nanocomposites comprising nanogold particles, which can simplify the construction steps of existing electrochemical sensors: mei et al (chen hui sweet et al, application of nucleic acid aptamers in environmental analysis, environmental chemistry 2015, 34(1), 89-96) developed a rapid and sensitive method for detecting bisphenol a, specifically: the BPA aptamer is modified on the surface of the gold nanoparticles, the gold nanoparticles cannot aggregate under the condition that bisphenol A does not exist, but when the bisphenol A is added, the nucleic acid aptamer is specifically combined with the bisphenol A, so that the gold nanoparticles are separated from the nucleic acid aptamer, the gold nanoparticles aggregate together, the rapid detection of the bisphenol A is realized by utilizing the change of color, and the detection limit of the obtained BPA is 0.1 ng/mL^-1. Chinese patent publication No. CN109060913B discloses a method for preparing an electrochemical sensor for detecting bisphenol a based on a nanocomposite of gold nanoparticles, nano molybdenum disulfide, and ionic liquid functionalized mercaptographene. The method utilizes the good catalytic performance and the good electron transfer promotion effect of the nanogold, the good selective reactivity of the nano molybdenum disulfide and the good dispersibility and conductivity of the ionic liquid functionalized graphene, the obtained electrochemical sensor has high sensitivity and good anti-interference performance when used for BPA detection, and the lowest concentration of the BPA capable of being detected is 0.05 mu mol.L^-1(about 0.01. mu.g. mL)^-1)。

The detection method still has the defect of low detection limit and cannot meet the detection requirement on trace BPA.

Disclosure of Invention

The invention aims to provide a nanocomposite based on phosphorus-molybdenum-containing polyoxometallate, and aims to solve the problem that the detection limit of the existing nanocomposite is low when the nanocomposite is used for detecting BPA by an electrochemical sensor.

The invention also aims to provide a preparation method of the nanocomposite based on the phosphorus-molybdenum-containing polyoxometallate, so as to solve the problem that the nanocomposite obtained by the existing preparation method has lower detection limit when being used for detecting BPA by an electrochemical sensor.

The third purpose of the invention is to provide an electrode for an aptamer sensor, which has strong biological affinity, excellent biocompatibility and high electrochemical activity, and can enable the aptamer sensor to obtain a lower detection limit when detecting BPA.

A fourth object of the present invention is to provide an aptamer sensor having a low detection limit for BPA detection, and having high selectivity, good stability and reproducibility, superior reproducibility, and applicability under various environments.

In order to realize the purpose, the technical scheme of the phosphorus-molybdenum-containing polyoxometallate-based nanocomposite material comprises the following steps:

a phosphorus molybdenum polyoxometalate-based nanocomposite comprising carbon, molybdenum disulphide nanoplatelets and silver-containing nanoparticles; the nano composite material is obtained by calcining silver-doped phosphomolybdic polyoxometallate, wherein the silver-doped phosphomolybdic polyoxometallate is obtained by reacting a silver source, phosphomolybdic acid and thioacetamide.

Nanocomposite of the invention, denoted as Ag₂O/Ag₂S/MoS₂@ C, which is obtained by calcining silver-doped phosphomolybdic polyoxometallate, wherein the silver-doped phosphomolybdic polyoxometallate simultaneously contains phosphorus, molybdenum and doped silver, so that the material has good electronic universality and electrochemical activity. After calcination, organic carbon in the silver-doped phosphomolybdic polyoxometallate is converted into cracked carbon, so that the nano composite material has higher specific surface area and stronger biological affinity; the formation of silver-containing nanoparticles further increases the specific surface area of the material, and the presence of silver in the form of silver oxide or silver sulfide is advantageous for improving the bioaffinity of the material. The electrochemical sensor constructed by the nano composite material has a lower detection limit when being used for detecting BPA, and has high selectivity, good stability and reproducibility, excellent reproducibility and applicability under different environments.

The silver-containing nano particles are nano silver oxide, or nano silver oxide and nano silver sulfide, or nano silver oxide, nano silver sulfide and nano silver. Different types of silver-containing nanoparticles can form a plurality of nano composite materials with outstanding performance, and can be correspondingly selected according to the requirements on the performance of the nano composite materials.

The technical scheme of the preparation method of the phosphorus-molybdenum-containing polyoxometallate-based nano composite material comprises the following steps:

a preparation method of a nanocomposite based on phosphorus-molybdenum-containing polyoxometallate comprises the following steps: and (2) carrying out solvothermal reaction on mixed liquor consisting of phosphomolybdic acid, thioacetamide, a silver source and a solvent, and calcining a product to obtain the catalyst.

The preparation method of the nanocomposite based on the phosphomolybdic polyoxometallate takes phosphomolybdic acid as a molybdenum source, thioacetamide as a sulfur source and a carbon source, and a silver source, and the silver-doped phosphomolybdic polyoxometallate is obtained after solvothermal reaction, after calcination, organic carbon in the silver-doped phosphomolybdic polyoxometallate is converted into cracked carbon and the silver source to form silver-containing nanoparticles, the preparation method is simple, and the obtained nanocomposite has high electrochemical activity, biological affinity and biocompatibility.

The temperature of the solvothermal reaction is 160-240 ℃. The method has the advantages that the mild reaction temperature is adopted, so that the generated molybdenum disulfide nanosheet has a large specific surface area, and the electron transfer is facilitated. Preferably, the reaction time is 10-15 h.

The calcination temperature is 300-800 ℃. At the calcining temperature, each nano structure in the obtained nano composite material can keep the original excellent performance, and the generated carbon layer has larger specific surface area and stronger biological affinity. The nano composite materials obtained by calcining at 300 ℃, 600 ℃ and 800 ℃ are respectively marked as Ag₂O/Ag₂S/MoS₂@C₃₀₀、Ag₂O/Ag₂S/MoS₂@C₆₀₀、Ag₂O/Ag₂S/MoS₂@C₈₀₀。

In order to further optimize the electrochemical activity and the biological affinity of the obtained nano material, the mass ratio of the phosphomolybdic acid, the thioacetamide and the silver source is 4:10:1-6:10: 1.

The mixed solution is obtained by mixing an aqueous solution of phosphomolybdic acid, an ethanol solution of thioacetamide and an aqueous solution of a silver source. Specifically, the silver source may be commonly used silver nitrate. The mixed solution can be obtained by simple mixing, and the whole preparation process is simplified.

The technical scheme of the electrode for the aptamer sensor comprises the following steps:

the electrode for the aptamer sensor comprises an electrode substrate and an electrode modification material on the surface of the electrode substrate, wherein the electrode modification material is the nanocomposite material based on the phosphorus-molybdenum-containing polyoxometallate.

Because the nanocomposite has excellent electrochemical activity, good biocompatibility and uniform dispersibility in aqueous solution, the constructed electrode has excellent electrochemical activity and biocompatibility and good biological affinity to targeted molecules, and the characteristics can expand electrochemical signals, be used for constructing aptamer sensors and improve the detection sensitivity of the targeted molecules.

The technical scheme of the aptamer sensor is as follows:

an aptamer sensor comprises an electrode substrate, an electrode modification material on the surface of an electrode and a nucleic acid aptamer fixed on the electrode modification material, wherein the electrode modification material is the nanocomposite based on the phosphorus-molybdenum-containing polyoxometallate.

The aptamer sensor can realize trace detection of various target molecules by selectively fixing different aptamers on the electrode modification material. The electrode modification material for constructing the aptamer sensor has excellent electrochemical activity, good biocompatibility and uniform dispersibility in aqueous solution, and the characteristics can enlarge electrochemical signals and remarkably improve the sensing performance of the developed aptamer sensor.

The aptamer sensor of the invention has the following advantages: the nano-molybdenum disulfide nano-sheet can be prepared by simple hydrothermal synthesis and calcination processes without any post-synthesis treatment, (ii) a label-free aptamer chain is used without an electrochemical indicator, (iii) the electrochemical signal can be greatly enhanced by active metal centers (Ag and Mo), (iv) the molybdenum disulfide nano-sheet can be uniformly dispersed under the protection of a carbon coating. The aptamer sensor of the invention establishes a novel POM base platform for the aptamer sensor in the fields of environmental monitoring and food safety.

The aptamer is a nucleic aptamer which specifically recognizes bisphenol A. The aptamer of bisphenol a can be immobilized on the surface of the nanocomposite material through pi-pi stacking, van der waals force, and coordination effect between the metal oxide or metal sulfide and the aptamer. Due to the specific affinity between the aptamers of bisphenol A and bisphenol A, the conformation of the aptamers is greatly changed after bisphenol A binding, thereby causing [ Fe (CN) ]on the surface of the electrode₆]^3-/4-The change in electrochemical signal can be detected and taken as a function of the bisphenol a concentration.

Drawings

FIG. 1 is a schematic diagram of the preparation of the nanocomposite material of the present invention and the construction of an aptamer sensor;

FIG. 2 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₃₀₀FE-SEM picture of (b);

FIG. 3 shows the nano-composite Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀FE-SEM picture of (b);

FIG. 4 shows the nano-composite Ag of the present invention₂O/Ag₂S/MoS₂@C₈₀₀FE-SEM picture of (b);

FIG. 5 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₃₀₀HR-TEM image of;

FIG. 6 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀HR-TEM image of;

FIG. 7 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₈₀₀HR-TEM image of;

FIG. 8 is a PMo of a comparative example of the present invention₁₂FE-SEM image of (1);

FIG. 9 is a PMo of a comparative example of the present invention₁₂HR-TEM image of;

FIG. 10 is a graph of Ag-PMo of a comparative example of the present invention₁₂FE-SEM picture of (b);

FIG. 11 is a graph of Ag-PMo of a comparative example of the present invention₁₂HR-TEM image of (a);

FIG. 12 is an XRD spectrum of a nanocomposite of the invention and a comparative example;

FIG. 13 is a Raman spectrum of a nanocomposite of the present invention and a comparative example;

FIG. 14 is a comparison of XPS survey spectra of nanocomposites of the invention and comparative examples;

FIG. 15 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₃₀₀、Ag₂O/Ag₂S/MoS₂@C₆₀₀、Ag₂O/Ag₂S/MoS₂@C₈₀₀The XPS energy spectrograms of high-resolution Ag3d, Mo 3d and S2p are obtained;

FIG. 16 is a PMo of a comparative example of the present invention₁₂XPS energy spectrograms of high-resolution Mo 3d, S2p, C1S and O1S;

FIG. 17 is a Ag-PMo of comparative example of the present invention₁₂XPS energy spectrograms of high-resolution Ag3d, Mo 3d, S2p, C1S and O1S;

FIG. 18 is a Nyquist plot of the electrochemical impedance spectroscopy test used in the experimental examples of the present invention;

FIG. 19 is an equivalent circuit diagram of an electrochemical impedance spectroscopy test used in an experimental example of the present invention;

FIG. 20 shows a nanocomposite Ag according to the present invention₂O/Ag₂S/MoS₂@C₃₀₀The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 21 shows a nanocomposite Ag according to the present invention₂O/Ag₂S/MoS₂@C₆₀₀The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 22 shows a nanocomposite Ag according to the present invention₂O/Ag₂S/MoS₂@C₈₀₀The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 23 is the present inventionPMo of the open comparative example₁₂The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 24 is a Ag-PMo of comparative example of the present invention₁₂The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 25 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₃₀₀The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 26 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 27 shows a nanocomposite Ag of the present invention₂O/Ag₂S/MoS₂@C₈₀₀The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 28 is a PMo of a comparative example of the present invention₁₂The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 29 is a Ag-PMo of comparative example of the present invention₁₂The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 30 shows PMo-based samples of the present invention, comparative example₁₂、Ag-PMo₁₂、Ag₂O/Ag₂S/MoS₂@C₃₀₀、Ag₂O/Ag₂S/MoS₂@C₆₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀Δ R at each stage when the aptamer sensor of (1) detects BPA_ctA difference in value;

FIG. 31 shows Ag constructed using different concentrations of electrode modification materials in accordance with the present invention₂O/Ag₂S/MoS₂@C₆₀₀Delta R of each stage when detecting BPA based on aptamer sensor_ctA difference in value;

FIG. 32 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Influence of aptamer solutions with different concentrations on BPA detection based on the aptamer sensor;

FIG. 33 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Nucleic acid based aptamer sensors in BPAEIS profile of incubation in solution (50mM) for various times;

FIG. 34 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Nucleic acid based aptamer sensors were incubated in BPA solution (50mM) for varying periods of time with the corresponding R_ctA value;

FIG. 35 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀The basic nucleic acid aptamer sensor is used for BPA (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 pg. mL) with different concentrations^-1) EIS response curve of (a);

FIG. 36 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Delta R of nucleic acid aptamer-based sensor_ctCalibration curves for different concentrations of BPA;

FIG. 37 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Delta R of nucleic acid aptamer-based sensor_ctA linear fit to the logarithm of the BPA concentration;

FIG. 38 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀The reproducibility of BPA is detected by a base nucleic acid aptamer sensor;

FIG. 39 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Detecting the stability of BPA by a base nucleic acid aptamer sensor;

FIG. 40 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀Detecting the specificity of BPA by a base nucleic acid aptamer sensor;

FIG. 41 shows Ag of the present invention₂O/Ag₂S/MoS₂@C₆₀₀The nucleic acid aptamer sensor detects the reproducibility of BPA.

Detailed Description

The present invention is further illustrated by the following specific examples.

AgNO₃Phosphomolybdic acid and ethanol were purchased from national pharmaceutical group chemical reagents, ltd.

Thioacetamide, BPA were purchased from Beijing Soraocaobao group, Inc.

Uric Acid (UA), benzidine (DAB), Phenol (PN), 4-nitrophenol (NPN), Dopamine (DA), Ascorbic Acid (AA), Benzaldehyde (BA), Benzophenone (BP), and resorcinol (DB) were purchased from solibao life science ltd.

Ultrapure water (18.2. omega. cm) was used for all experiments.

The aptamer sequence for BPA is shown below:

5’-CCG CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACA GCT GTT ATAGAC GTC TCC AGC-3’。

preparation of PBS buffer: mixing 0.24g KH₂PO₄、1.44g Na₂HPO₄·12H₂O, 0.20g of KCl and 8.0g of NaCl were dissolved in ultrapure water to prepare a phosphate buffer solution (PBS, 0.1M), and 0.1M HCl solution was added to adjust the pH of the PBS to 7.4, which was used as a biological buffer.

Preparing electrolyte: 1.6g of K₃Fe(CN)₆2.1g of K₄Fe(CN)₆And 7.5g of KCl in 1.0L of PBS to prepare an electrolyte.

Stock solutions of aptamer (100. mu.M) and BPA solutions (0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 pg. mL) at various concentrations were prepared using 0.1M PBS^-1). All solutions were freshly prepared before each experiment and stored at 4 ℃ until use.

First, specific examples of the nanocomposite based on a phosphorus-molybdenum-containing polyoxometalate of the present invention

Example 1

The nanocomposite material of the embodiment is prepared from carbon, molybdenum disulfide nanosheets and nano Ag₂O particles are formed and are obtained by calcining silver-doped phosphomolybdic polyoxometallate, organic carbon in the silver-doped phosphomolybdic polyoxometallate is converted into cracking carbon after calcination, and doped silver is converted into nano Ag₂And O particles, wherein the silver-doped phosphomolybdic polyoxometallate is obtained by carrying out hydrothermal reaction on silver nitrate, phosphomolybdic acid and thioacetamide.

The method comprises the following specific steps:

(1) solution A was prepared by adding 50mg of phosphomolybdic acid to 20mL of ultrapure water, and 1g of AgNO was added₃Dissolving in 10mL of ultrapure water to prepare AgNO₃Solution (0.1 g. mL)^-1) Taking 100 μ L of AgNO₃Adding the solution into the solution A to obtain a solution A', and carrying out ultrasonic treatment for 10min for later use;

(2) adding 100mg thioacetamide into 20mL absolute ethyl alcohol to prepare a solution B, and carrying out ultrasonic treatment for 10min for later use;

(3) gradually adding the solution A' into the solution B, carrying out ultrasonic treatment on the mixed solution for 10min, transferring the mixed solution into a 50mL stainless steel reaction kettle with a polytetrafluoroethylene lining, and heating the solution for 12h at 200 ℃;

(4) after the reaction is finished, cooling the product at room temperature, then centrifuging the product, washing the precipitate for 3 times by using absolute ethyl alcohol, and drying at 60 ℃ in vacuum to obtain the product.

(5) Taking 100mg of the final product obtained in the step (4), placing the final product in a tube furnace, and carrying out N treatment at the temperature of 300 DEG C₂Heating for 2h under atmosphere, with the heating rate of 5 ℃ min^-1After heating, the product is at N₂Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag₂O/Ag₂S/MoS₂@C₃₀₀。

Example 2

The nanocomposite material of the embodiment comprises carbon, molybdenum disulfide nanosheets and nano Ag₂O、Ag₂S particles are obtained by calcining silver-doped phosphomolybdic polyoxometallate, organic carbon in the silver-doped phosphomolybdic polyoxometallate is converted into cracking carbon after calcination, and doped silver is converted into nano Ag₂O、Ag₂And S particles, wherein the silver-doped phosphomolybdic polyoxometallate is obtained by carrying out hydrothermal reaction on silver nitrate, phosphomolybdic acid and thioacetamide.

The method comprises the following specific steps:

(1) solution A was prepared by adding 50mg of phosphomolybdic acid to 20mL of ultrapure water, and 1g of AgNO was added₃Dissolving in 10mL of ultrapure water to prepare AgNO₃Solution (0.1 g. mL)^-1) Taking 100 mu L of AgNO₃Adding the solution into the solution A to obtain a solution A', and carrying out ultrasonic treatment for 10min for later use;

(5) Taking 100mg of the final product obtained in the step (4), placing the final product in a tube furnace, and carrying out N treatment at 600 DEG C₂Heating for 2h under atmosphere, with the heating rate of 5 ℃ min^-1After heating, the product is at N₂Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag₂O/Ag₂S/MoS₂@C₆₀₀。

Example 3

The nanocomposite material of the embodiment comprises carbon, molybdenum disulfide nanosheets and nano Ag₂O、Ag₂S, Ag granule is prepared by calcining silver doped phosphomolybdic polyoxometallate, wherein after calcination, the organic carbon in the silver doped phosphomolybdic polyoxometallate is converted into cracking carbon, and the doped silver is converted into nano Ag₂O、Ag₂S, Ag, wherein the silver doped phosphomolybdic polyoxometallate is obtained by carrying out hydrothermal reaction on silver nitrate, phosphomolybdic acid and thioacetamide.

The method comprises the following specific steps:

(5) Taking 100mg of the final product obtained in the step (4), placing the final product in a tube furnace, and adding N at 800 DEG C₂Heating for 2h under atmosphere, with the heating rate of 5 ℃ min^-1After heating, the product is at N₂Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag₂O/Ag₂S/MoS₂@C₈₀₀。

Second, the specific embodiment of the preparation method of the phosphorus-molybdenum-containing polyoxometallate-based nanocomposite material

Example 4

The preparation method of the nanocomposite material of this embodiment is specifically the preparation method of the nanocomposite material in embodiment 1, and is not described again.

Example 5

The preparation method of the nanocomposite material of this embodiment, specifically the preparation method of the nanocomposite material in embodiment 2, is not described again.

Example 6

The preparation method of the nanocomposite material of this embodiment, specifically the preparation method of the nanocomposite material in embodiment 3, is not described again.

Third, the specific embodiment of the electrode for aptamer sensor of the invention

Example 7

The electrode for the aptamer sensor in the embodiment includes an electrode substrate and an electrode modification material on the surface of the electrode substrate, wherein the electrode modification material is the nanocomposite material in embodiment 1.

In other embodiments, the electrode for the aptamer sensor is made of the nanocomposite material in

embodiment

2 or 3, and the description thereof is omitted.

Fourth, embodiments of the aptamer sensor of the invention

Example 8

The aptamer sensor of the embodiment comprises an electrode substrate, an electrode modification material on the surface of an electrode and a bisphenol a aptamer fixed on the electrode modification material, wherein the electrode modification material is the nanocomposite material in the embodiment 1. The construction process of the aptamer sensor is shown in fig. 1, the aptamer sensor uses a conventional three-electrode system, and the construction steps are as follows:

(1) adding 1.0mg of Ag₂O/Ag₂S/MoS₂@C₃₀₀Dispersing the powder in 1.0mL of ultrapure water, and ultrasonically mixing to obtain uniform Ag₂O/Ag₂S/MoS₂@C₃₀₀A suspension;

(2) 5.0. mu. L1.0 mg/mL^-1Ag of (A)₂O/Ag₂S/MoS₂@C₃₀₀The suspension is dripped on the surface of a pretreated gold electrode AE and dried for 3 hours at room temperature, and the treated electrode is marked as Ag₂O/Ag₂S/MoS₂@C₃₀₀/AE；

(3) Washing Ag with ethanol and ultrapure water respectively₂O/Ag₂S/MoS₂@C₃₀₀AE, and incubating it in 100nM aptamer solution for 30min to ensure aptamer chain in Ag₂O/Ag₂S/MoS₂@C₃₀₀Sufficient anchoring of the/AE surface, the treated electrode is denoted Apt/Ag₂O/Ag₂S/MoS₂@C₃₀₀/AE；

(4) Finally Apt/Ag₂O/Ag₂S/MoS₂@C₃₀₀immersing/AE in BPA solution to obtain BPA/Apt/Ag₂O/Ag₂S/MoS₂@C₃₀₀AE as working electrode, Ag/AgCl (saturated KCl) electrode as reference electrode and platinum sheet as counter electrode.

The pretreatment process of the gold electrode is as follows:

a blank gold electrode (AE) having a diameter of 3mm was used as a working electrode, and was cleaned before use. AE was polished to a mirror-like state with 0.3 μm and 0.05 μm alumina powders and then separately in mixed solutions (v/v, 7:3, H)₂SO₄/H₂O₂) And sonication in ethanol for 15 min. Then, AE was thoroughly washed with ultrapure water and washed in N₂And (4) drying under flowing. AE 0.5M H₂SO₄The potential cycling range is from-0.2 to 1.6V.Finally, AE was rinsed with ultrapure water, again at N₂Dried under reduced flow and stored until use.

In other embodiments, the electrode modification material for constructing the aptamer sensor is the nanocomposite material in

embodiment

2 or 3, and the aptamer may be a specific aptamer capable of being targeted and combined with organic contaminating small molecules, environmental microorganisms, heavy metal ions, and the like, and will not be described again.

Fifth, comparative example

Comparative example 1

In the aptamer sensor of the comparative example, the electrode modification material is phosphorus-molybdenum-containing polyoxometallate PMo₁₂Construction of aptamer sensor the procedure for construction of aptamer sensor is the same as in example 10, PMo₁₂Modified electrode materials with PMo₁₂The AE represents the Apt/PMo of the electrode material after the aptamer of BPA is immobilized₁₂and/AE. Wherein, PMo₁₂The preparation method specifically comprises the following steps:

(1) adding 50mg of phosphomolybdic acid into 20mL of ultrapure water to prepare a solution A, and carrying out ultrasonic treatment for 10min for later use;

(3) gradually adding the solution A into the solution B, carrying out ultrasonic treatment for 10min, transferring the mixed solution into a 50mL stainless steel reaction kettle with a polytetrafluoroethylene lining, and heating for 12h at 200 ℃;

Comparative example 2

In the aptamer sensor of the comparative example, the electrode modification material is Ag doped phosphorus-molybdenum-containing polyoxometallate Ag-PMo₁₂Construction of aptamer sensor the same procedure as in example 10, Ag-PMo₁₂Modified electrode material made of Ag-PMo₁₂The AE represents that the electrode material after the aptamer of BPA is fixed is Apt/Ag-PMo₁₂and/AE. Wherein, Ag-PMo₁₂The preparation method of (2) is different from that in example 4 in that the calcination treatment in the step (5) is not performed, and the others are different from those in example 4The preparation method is the same.

Sixth, Experimental example

The following experimental examples describe the properties of the nanocomposites in examples 1-3 and the nanomaterials in comparative examples 1, 2 in detail.

(first) Material characterization experiment

Experimental example 1: topography testing

Synthesized Ag₂O/Ag₂S/MoS₂@C₃₀₀、Ag₂O/Ag₂S/MoS₂@C₆₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀Nanocomposite and PMo₁₂、Ag-PMo₁₂The surface morphology and microstructure of the nano material are observed by FE-SEM and HR-TEM, FE-SEM images of five materials are respectively shown in figure 2, figure 3, figure 4, figure 8 and figure 10, HR-TEM images of five materials are respectively shown in figure 5, figure 6, figure 7, figure 9 and figure 11, and specifically, JSM-6490LV field emission scanning electron microscope (FE-SEM, Japan) and JEOL JEM-2100 high resolution transmission electron microscope (HR-TEM) are adopted for analyzing the surface morphology of a sample and are provided with a 200kV field emission gun.

FIG. 8 shows, PMo₁₂Spherical structures with different sizes, formed by the accumulation of nanoparticles. At the same time, it is further confirmed by fig. 9 that PMo₁₂The spherical structure of (a) consists of a large number of nanosheets. At PMo₁₂FIG. 9e, a lattice spacing of 0.62nm was observed, corresponding to MoS₂(002) crystal face of (a). This finding shows that the high temperature heat treatment of phosphomolybdic acid and thioacetamide produces a nanocomposite based on phosphomolybdic polyoxometallate, which contains MoS₂Nanosheet (MoS)₂NSs) structure.

FIG. 10 shows Ag-PMo₁₂The SEM images of (a) show a more compact, larger spherical shape, as shown in the TEM image of fig. 11, solid spheres indicate their dense structure. HR-TEM images showed that the dense spheres were surrounded by a thin layer of MoS (FIG. 11d)₂Layer, also showing MoS₂Lattice spacing of NSs (fig. 11 e). No lattice association with Ag was observed, indicating that it was doped with MoS in the ionic state₂In NSs.

FIGS. 2-4 and 5-7 are FE-SEM and HR-TEM images, respectively, of nanocomposites of the present invention. The results show that Ag₂O/Ag₂S/MoS₂@C₃₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀Surface topography and irregular shape PMo of₁₂The spheres are similar. However, in Ag₂O/Ag₂S/MoS₂@C₆₀₀Irregular petal-like structures are found in nanocomposites, which are composed of many nanosheets with a loose nanostructure. Albeit in Ag₂O/Ag₂S/MoS₂@C₃₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀The FE-SEM image of (1) does not obviously observe nano-flake-like nano-structures, but well-structured nano-flakes are found in the HR-TEM image, and Ag₂O/Ag₂S/MoS₂@C₆₀₀The structure of the nanocomposite is similar. Of the three samples, Ag₂O/Ag₂S/MoS₂@C₈₀₀The nanocomposites exhibited a more open structure and thinner nanoplatelets, consistent with the FE-SEM results. All HR-TEM images were observed to correspond to MoS₂The lattice spacing of (002) crystal plane of (a). In Ag₂O/Ag₂S/MoS₂@C₆₀₀Also observed in (1) is a correspondence to Ag₂The lattice spacing of the (200) plane of O was 0.241 nm. In Ag₂O/Ag₂S/MoS₂@C₈₀₀Except for Ag in the HR-TEM image of₂O and MoS₂In addition, a lattice spacing of 0.236nm was observed corresponding to the (111) plane of metallic Ag. It can be seen that the doping is in PMo₁₂Ag in (C)⁺The ions did not undergo conversion upon heat treatment at 300 ℃ but showed Ag at 600 ℃₂Oxidation state of O. Heat treatment at extremely high temperatures, Ag₂O will decompose and reduce to the metallic state.

Experimental example 2: characterization of the Crystal Structure

The crystal structure of the sample was analyzed by XRD technique, specifically, X-ray diffraction (XRD) test using Cu K on Rigaku D/Max-2500X-ray diffractometer_αRadiation (λ 0.15406 nm).

As shown in fig. 12, PMo₁₂The three XRD patterns of the crystal form are MoS at 8.9 degrees, 33.4 degrees and 58.3 degrees₂The diffraction peak of (1). The transmission electron microscope result shows that the phosphomolybdic acid and the thioacetamide generate MoS through hydrothermal reaction₂. But in Ag-PMo due to low crystallization rate₁₂No obvious peak is seen in the product. For Ag₂O/Ag₂S/MoS₂@C₃₀₀A weaker peak appears at 32.6 because of the fact that it is other than belonging to MoS₂In addition to the three peaks, Ag was also present in the sample₂O。Ag₂O/Ag₂S/MoS₂@C₆₀₀The XRD spectrum of the crystal shows MoS at 8.9 degrees, 33.4 degrees and 58.3 degrees₂The characteristic peaks of (A) show Ag at 32.0 °, 32.6 °, 53.3 ° and 56.5 °₂Characteristic peak of O. In addition, two diffraction peaks at 25.4 ° and 45.6 ° correspond to Ag₂S, and the three peaks at 28.9 °, 38.5 ° and 44.1 ° are attributed to metallic Ag. Thus, Ag₂O/Ag₂S/MoS₂@C₆₀₀The nano composite material consists of MoS₂、Ag₂O、Ag₂S and metal Ag. Ag₂O/Ag₂S/MoS₂@C₈₀₀The XRD pattern of the compound shows that the compound is in contact with Ag₂O/Ag₂S/MoS₂@C₆₀₀Similar structure. These results indicate that Ag was doped⁺PMo of₁₂Is converted into embedded Ag₂O、Ag₂MoS of multiple components such as S and Ag₂The nano-sheet and a small amount of graphite carbon layer, which can obviously enhance the electrochemical activity and promote the adsorption of biological molecules.

Experimental example 3: chemical structure characterization-raman spectroscopy

The chemical structure of the sample was studied using raman spectroscopy. Specifically, a Renishaw in Via Raman spectrometer is used to obtain the Raman spectrum of the material under the excitation wavelength of 532nm, and the scanning range is 50-1500cm^-1。

As shown in fig. 13, PMo₁₂The Raman spectrum of (A) shows that the specific peaks are located at 281, 378, 405, 818 and 942cm^-1Here, this is from MoS₂E of (A)¹ _2gAnd A_1gCaused by vibration modes. Ag-PMo₁₂Raman spectroscopy and PMo₁₂Same as above, the description of Ag⁺Addition of ions to PMo₁₂The chemical structure of (a) has no influence. Heat treating at 300 deg.C in Ag₂O/Ag₂S/MoS₂@C₃₀₀In (A) also observed to belong to MoS₂Main peak of (2). This indicates that Ag-PMo is present at this temperature₁₂Does not change much in chemical structure. However, in Ag₂O/Ag₂S/MoS₂@C₆₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀In (1), only attribution to MoS can be observed₂Two peaks (378 and 405 cm)^-1)。

Experimental example 4: chemical Structure characterization-XPS Spectroscopy

XPS spectra were performed on all five samples, see FIG. 14, and specifically, X-ray photoelectron spectroscopy (XPS) data was obtained using an ESCALB 250Xi spectrometer (Manchester, Sammer Miller science, England) and Al K_αCollected from the X-ray source (1486.6eV photons).

FIG. 14 shows that Mo 3d, C1s, N1 s, O1s signals coexist in 5 samples, while Ag-PMo₁₂And their calcination at different temperatures gave derivatives with a clear Ag3d signal.

To characterize the chemical valence and environment of each element in all samples, their high resolution XPS spectra were resolved using XPSPEAK1 software.

FIG. 15 shows Ag₂O/Ag₂S/MoS₂High resolution energy spectrogram of @ C series nanocomposite. In Ag₂O/Ag₂S/MoS₂In the @ C series nanocomposite (FIGS. 15C-e), the high resolution Ag3d XPS spectrum also showed two distinct peaks corresponding to Ag⁺With Ag-PMo₁₂Similarly. For Ag₂O/Ag₂S/MoS₂@C₃₀₀Mo 3d XPS spectrum of the nano composite material obtains similar peak position, but Mo⁵⁺And Mo⁶⁺Relatively high content of ions, and Mo-O_xThe bond content is also higher. In contrast, Ag₂O/Ag₂S/MoS₂@C₆₀₀Mo in (1)⁶⁺The ionic strength is low. In Ag₂O/Ag₂S/MoS₂@C₈₀₀Mo is not found in the nanocomposite⁵⁺And Mo⁶⁺Ionic and Mo-O_xThe bond content is also low, indicating that it is completely decomposed at very high temperatures. Only S appears in S2 pXPS energy spectrum of the series of nano composite materials ^2-2p_3/2And S ^2-2p_1/2Two peaks, indicating the production of Ag₂S or MoS₂. From the above analysis, it can be seen that the Ag-PMo was calcined₁₂Can obtain Ag₂O、Ag₂S and MoS₂The novel nano composite material has an ultrathin nano sheet structure, mixed chemical valence, multiple components and rich oxygen vacancy. And the thioacetamide contains carbon and forms a small carbon layer after high-temperature calcination. These can not only promote electron transfer, but also improve the anchoring of biomolecules, thereby enhancing the electrochemical sensing performance of the corresponding aptamer sensor.

FIG. 16 is PMo₁₂High resolution energy spectrum. FIG. 16a can be divided into two parts, the peaks at the Binding Energies (BEs) of 228.6eV and 231.8eV corresponding to Mo, respectively⁴⁺3d_5/2And Mo ⁴⁺3d_3/2. The other two peaks at BEs higher were 229.5 and 232.9eV, respectively, and were attributed to Mo⁵⁺And Mo⁶⁺. This is illustrated in PMo₁₂Mo in the solution is partially oxidized from Mo⁴⁺State change to Mo⁵⁺And Mo⁶⁺Status. In addition, the peaks at BEs at 226.0eV and 235.4eV are attributed to Mo-S and Mo-O, respectively_xThe function of the bond. PMo₁₂Can be fitted to BEs two peaks at 161.4 and 162.6eV, corresponding to S, respectively^2-S2p of_3/2And S2p_1/2. The peak at BE of 163.7eV corresponds to S₂ ^2-S2p of_3/2. The two peaks at BEs of 168.8eV and 169.9eV are attributed to S, respectively⁶⁺And an S-O bond. It is clear that S2p is composed of mixed valence states, where part of the S is also oxidized to SO₄ ^2-Ions. At PMo₁₂In the high-resolution C1s spectrum (FIG. 16C), two were observed at BEs of 284.3eV and 285.9eV, respectivelyThe major components, corresponding to the C-C bond and C ═ O bond. Three main peaks at 530.6, 531.9 and 533.1eV were obtained on O1s XPS spectra (fig. 16d), corresponding to O vacancies, C ═ O bonds and C — O bonds, respectively. The coexistence of the mixed valence states of Mo 3d and S2p and the occurrence of O vacancy can obviously promote the electron transfer and improve the electrochemical activity.

FIG. 17 shows Ag-PMo₁₂The high resolution spectrum of (2) is used for decomposing high resolution XPS spectra of Ag3d, Mo 3d and S2 p. Clear signals for Ag3d indicate success in PMo₁₂In which Ag is doped⁺. The high resolution Ag3d XPS spectrum is split into two peaks at 367.02 and 374.03eV, which corresponds to Ag₂Ag in S⁺Ionic Ag3d_5/2And Ag3d_3/2. The observation of PMo was observed when Mo 3d and S2p XPS spectra were analyzed₁₂Similar results, except Ag, are shown⁺Presence of Ag-PMo₁₂And original PMo₁₂Are very close in chemical structure.

(II) electrochemical sensing Performance test

All electrochemical tests, including Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV), were performed on solartronalytical electrochemical workstation (uk). In the presence of 0.5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-To obtain an EIS curve (EIS parameter: potential, 0.21V; frequency range, 100kHz to 0.01 Hz; amplitude, 5mV, room temperature). EIS data were analyzed using Zview2 software, where EIS spectra were simulated using equivalent circuits including solution impedance (R)_s) Resistance to charge transfer (R)_ct) Constant Phase Element (CPE) and Warburg impedance (W)_o) See fig. 18 and 19. Parameters of each element in the equivalent circuit are determined using a non-linear least squares fit. Each test was repeated at least three times.

In order to obtain the optimal experimental conditions, parameters such as the amount of the nano composite material, the concentration of the aptamer, the binding time of the BPA and the aptamer sensor and the like are optimized to detect the BPA. In this work, different doses of nanocomposite (0.1, 0.2, 0.5, 1 and 2 mg-mL) were used^-1) Constructing a nucleic acid aptamer sensor and evaluating the sensing performance of the nucleic acid aptamer sensor on BPA. The modified gold is chargedThe electrode is respectively incubated with 10, 20, 50, 100, 200 and 500nM aptamer solutions, and the optimal concentration of the aptamer is optimized to achieve the highest BPA detection efficiency. In addition, selected aptamer sensors were incubated in BPA solution and data were recorded with EIS at different time periods to obtain the effect of binding time on sensing performance. Thereby, optimal experimental conditions can be obtained and used for further testing.

The detection limit of the constructed aptamer sensor is evaluated by using Apt/Ag₂O/Ag₂S/MoS₂@ C/AE with BPA solutions of different concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 pg.mL)^-1) Incubations were performed and data were recorded with EIS, followed by fitting all EIS Nyquist plots using the Zview2 software.

To explore the selectivity of aptamer sensors, Apt/Ag₂O/Ag₂S/MoS₂@ C/AE was immersed in UA, DAB, PN, NPN, DA, AA, BA, BP, DB, and mixtures thereof with BPA, respectively, and detected by EIS. The concentration of the interfering substance was BPA concentration (1ng mL)^-1) 100 times of the total weight of the powder.

By independently developing 5 aptamer sensors, the sensing performances thereof were compared and the reproducibility of the aptamer sensors was examined by EIS. Meanwhile, in order to research the stability of the aptamer sensor, BPA/Apt/Ag₂O/Ag₂S/MoS₂@ C/AE was stored in a refrigerator (4 ℃ C.) for 15 days and tested daily with EIS.

In order to examine the reproducibility of the developed aptamer sensor for detecting BPA, BPA/Apt/Ag was used₂O/Ag₂S/MoS₂@ C/AE was rinsed with 1mM NaOH for 5min at room temperature, then rinsed with a large volume of ultra pure water. Then, the treated electrode was immersed in BPA solution (1 fg. mL)^-1) The test was performed until the EIS response reached the original level and the same procedure was repeated for 7 cycles.

The following are specific experimental procedures and experimental results.

Experimental example 5 construction Process exploration of aptamer sensor based on 5 materials

Application of EIS and CV methods for explorationPMo₁₂、Ag-PMo₁₂Calcining at 300 deg.C, 600 deg.C and 800 deg.C to obtain a series of Ag₂O/Ag₂S/MoS₂The construction process of the nucleic acid aptamer sensor made of the @ C nanocomposite. CV curves for detection of BPA using different aptamer sensors are shown in FIGS. 20-24. Blank AEs showed a distinct redox peak with a peak current of 200.3 μ A and a potential difference between the peaks (. DELTA.E)_p) It was 0.17V. High peak current and narrow Δ E_pIndicating that blank AE has excellent electrochemical activity. After modification with different nanomaterials, the peak current of the modified electrode is significantly reduced, accompanied by Δ E_pIs increased. Among these electrode materials, Ag₂O/Ag₂S/MoS₂@C₆₀₀the/AE showed the highest peak current and the narrowest Δ E_pValues indicating relatively good electrochemical performance. With the original PMo₁₂And Ag-PMo₁₂In contrast, for Ag-PMo at appropriate temperature₁₂The heat treatment is performed to facilitate the acceleration of electron transfer.

When the aptamer chain is fixed on the modified electrode, the peak current is only slightly reduced, and the Delta E is_pThe value increased due to the negatively charged phosphate group on the aptamer chain with [ Fe (CN)₆]^3-/4-Repulsive interactions between the redox probes prevent electron transfer at the electrolyte/electrode interface. Apt/Ag₂O/Ag₂S/MoS₂@C₈₀₀The peak current reduction on/AE was small, indicating that only a small number of aptamer strands were immobilized on the modified electrode surface. In contrast, Apt/Ag-PMo₁₂The peak current of/AE varies greatly, which indicates Apt/Ag-PMo₁₂the/AE has extremely high biological affinity with the aptamer, so that more aptamer is anchored on the surface of the modified electrode.

When BPA in an aqueous solution is detected using the constructed aptamer sensor, the peak current continues to decrease, and Δ E_pThe value increases continuously. As previously described, the immobilized nucleic acid aptamer strand can bind to a BPA molecule and form a nucleic acid aptamer-BPA complex, resulting in a change in the conformation of the nucleic acid aptamer strand. This further interferes with electron transfer, reducing peak current。BPA/Apt/Ag₂O/Ag₂S/MoS₂@C₆₀₀the/AE showed large peak current variation. Although Ag-PMo₁₂The base nucleic acid aptamer sensor shows better nucleic acid aptamer immobilization performance, and the electrochemical signal change caused by the base nucleic acid aptamer sensor in BPA detection is smaller than that of Ag₂O/Ag₂S/MoS₂@C₆₀₀A nucleic acid-based aptamer sensor. This indicates that for Apt/Ag-PMo₁₂AE, the aptamer-BPA complex formed during detection would be from Ag-PMo₁₂The matrix is partially removed.

Using a series of Ag₂O/Ag₂S/MoS₂@ C and PMo₁₂、Ag-PMo₁₂The EIS results from construction of aptamer sensors maintained agreement with the CV curves, see fig. 25-29. Three kinds of Ag₂O/Ag₂S/MoS₂The @ C nanocomposite-based nucleic acid aptamer sensor has a similar electrochemical behavior. All blank AEs showed smaller R of 30.9-33.9 Ω_ctValues indicating that they have significant electrochemical activity, allowing electrons to be readily transferred at the electrode/electrolyte solution interface. Use PMo for blank AEs₁₂、Ag-PMo₁₂And series Ag₂O/Ag₂S/MoS₂After modification of @ C, R_ctThe value becomes larger, and it ranges from 165.7 Ω to 305.6 Ω. This indicates a series of Ag₂O/Ag₂S/MoS₂The @ C nanocomposite has poorer electrochemical activity than blank AE, which hinders electron transfer and increases R_ctThe value is obtained. Compared with the nano porous organic framework reported in the past, Ag₂O/Ag₂S/MoS₂The @ C series nanocomposite shows relatively excellent electrochemical performance, can amplify electrochemical detection signals, and further improves the sensitivity of the existing aptamer sensor. Of the three nanocomposites, Ag₂O/Ag₂S/MoS₂@C₃₀₀R of (A) to (B)_ctThe minimum value is 165.7 omega, which shows that the electrochemical performance is excellent. At relatively low temperatures (300 ℃), Ag-PMo₁₂The nanostructure portion of (a) is decomposed. Due to PMo₁₂Intrinsic properties of, i.e. high electrochemical activity, Ag-PMo₁₂Ultrathin nanoflakes with open electronic transportThe transport channels, therefore, provide more electroactive sites, shorter electron transport and electrolyte diffusion paths. After the aptamer chain is fixed on the modified electrode, R_ctThe value continues to increase to 268.9-788.9 omega. EIS response increased when detecting BPA in aqueous solution, R_ctThe values continue to increase to the range of 526.5-1101.3 omega.

The invention also researches PMo₁₂、Ag-PMo₁₂And series Ag₂O/Ag₂S/MoS₂R for each step of BPA detection by @ C-based nucleic acid aptamer sensor_ctVariation of value (. DELTA.R)_ct) As shown in fig. 30.Δ R_ctMay represent the respective binding amounts. In three kinds of Ag₂O/Ag₂S/MoS₂In @ C nanocomposite, Ag₂O/Ag₂S/MoS₂@C₃₀₀Δ R of (A)_ctThe value was minimal (134.8 Ω), indicating excellent electrochemical activity. Ag₂O/Ag₂S/MoS₂@C₆₀₀And Ag₂O/Ag₂S/MoS₂@C₈₀₀Δ R of (A)_ctThe values are comparable with a small difference, 189.1 and 209.8 Ω, respectively. After aptamer immobilization, Ag₂O/Ag₂S/MoS₂@C₆₀₀Best fixing ability of/AE,. DELTA.R_ctThe value was 222.4 Ω. It has been reported that Ag⁺Can form C-Ag with aptamer⁺-C base pairs, thereby promoting the massive anchoring of aptamer strands on the surface of the modified electrode, which acts on Ag⁺Conversion to Ag₂O and Ag₂S is more pronounced. In contrast, Ag₂O/Ag₂S/MoS₂@C₈₀₀Aptamer anchoring Performance of/AE is the worst, R_ctThe value is only 66.1 omega. TEM and XRD results show that Ag is contained in three nano-composite materials₂O/Ag₂S/MoS₂@C₈₀₀Is the highest, and due to its inherent detection properties for BPA, this highly ordered nanostructure is not conducive to anchoring aptamer chains. BPA/Apt/Ag due to the specific adsorption of BPA to aptamers immobilized on modified AE₂O/Ag₂S/MoS₂@C₆₀₀Delta R of/AE_ctThe value (434.6 Ω) is the maximum. Thus, it is possible to provideWe select Ag with mesopores₂O/Ag₂S/MoS₂@C₆₀₀The nano composite material is used as a platform for constructing a BPA aptamer sensor. This Ag₂O/Ag₂S/MoS₂@C₆₀₀The nano composite material can not only improve electron transfer, but also greatly improve the binding site of the aptamer chain, and shows excellent sensing performance.

Experimental example 6: optimization of experimental parameters

In the process of constructing the electrochemical aptamer sensor, experimental parameters such as the dosage of electrode materials, the concentration of aptamer chains, the pH value of a buffer solution, the binding time of a target analyte and the like have great influence on the detection sensitivity of the sensor. The literature reports that PBS with the pH value of 7.0-7.4 is beneficial to the construction of the aptamer sensor. Therefore, we do not discuss the effect of pH.

Ag in different concentrations₂O/Ag₂S/MoS₂@C₆₀₀Different aptamer sensors were constructed as carriers to study the effect of material usage on BPA detection. FIG. 31 shows the change in EIS response at each step for different aptamer sensors. It can be seen that different amounts of Ag were used₂O/Ag₂S/MoS₂@C₆₀₀Δ R due to AE_ctValue with Ag₂O/Ag₂S/MoS₂@C₆₀₀The amount of (c) increases. This means that the layer thickness of the electrode material can significantly impede the electron transfer, so that a larger R is obtained_ctThe value is obtained. Furthermore, the thick material may provide more anchoring sites for the aptamer strands, with Ag₂O/Ag₂S/MoS₂@C₆₀₀The dosage is 0.1 mg/mL^-1Increased to 1 mg. mL^-1，R_ctThe value also increases. When the dosage is more than 1.0 mg/mL^-1In time, the immobilization of aptamers and detection of BPA reached equilibrium. In addition, too thick material is easily detached from the electrode surface. Therefore, the concentration was selected to be 1.0 mg/mL^-1Ag of (A)₂O/Ag₂S/MoS₂@C₆₀₀The nanocomposite is used for constructing the aptamer sensor and is used for further testing.

To determine the effect of aptamer concentration on sensing performance, several Ag's were used₂O/Ag₂S/MoS₂@C₆₀₀the/AE is respectively cultured in aptamer solutions with different concentrations, and then BPA detection is carried out. As shown in FIG. 32, Δ R due to aptamer fixation and BPA detection when the concentration of the aptamer solution was increased from 10nM to 100nM_ctThe value increases with increasing concentration, and Δ R when the concentration exceeds 100nM_ctThe value tends to be flat. This indicates that both aptamer anchoring and BPA detection are saturated. Therefore, a 100nM aptamer solution was used for construction of aptamer sensors.

The invention also explores the influence of the BPA incubation time on the sensing performance of the aptamer sensor. Mixing Ag with water₂O/Ag₂S/MoS₂@C₆₀₀The base nucleic acid aptamer sensors were tested in PBS (0.1M, pH 7.4) after soaking in BPA (50mM) for various periods of time. As can be seen from FIGS. 33 and 34, EIS response and Δ R increased with the incubation time_ctThe detection of BPA values also increases. BPA detection reached a maximum at 40min, so 40min was used as incubation time. Using Ag₂O/Ag₂S/MoS₂@C₆₀₀The optimal conditions for detecting BPA by the nucleic acid aptamer sensor are as follows: ag₂O/Ag₂S/MoS₂@C₆₀₀The concentration is 1.0 mg/mL^-1(ii) a The concentration of the aptamer is 100 nM; BPA binding time was 40 min.

Experimental example 7: sensitivity test

It is crucial to explore the detection sensitivity of aptamer sensors and can be expressed in terms of limit of detection (LOD). The low LOD indicates that the electrochemical aptamer sensor has higher sensitivity. Under the optimal condition, Apt/Ag₂O/Ag₂S/MoS₂@C₆₀₀AE and BPA solutions of different concentrations (1 fg. mL)^-1-1pg·mL^-1) Incubated together, then incubated using EIS in [ Fe (CN)₆]^3-/4-Tests were performed in solution to evaluate the analytical performance of the developed aptamer sensors.

As shown in FIG. 35, the EIS obtained vs BP with increasing concentration of BPAThe response of the a test increases. High concentrations of BPA solutions can produce more aptamer-BPA complexes, greatly hindering electron transfer at the electrolyte solution/electrode interface. According to 5 independent Ag₂O/Ag₂S/MoS₂@C₆₀₀Delta R of nucleic acid aptamer-based sensor_ctThe values give a calibration curve, see fig. 36. The results show that Δ R_ctThe value increases with increasing BPA concentration, ranging from 1 fg. mL^-1-1pg·mL^-1When the concentration of BPA is more than 1 pg/mL^-1When is Δ R_ctThe values gradually tend to equilibrate. By Δ R_ctThe values as a function of the logarithm of the BPA concentration gave a very good linear relationship, as shown in FIG. 37 for the regression equation Δ R_ct(Ω)＝647.46+172.44log(C_BPA) The correlation coefficient is R²0.9921. According to the IUPAC method, the LOD is 0.2 fg. mL at a signal-to-noise ratio of 3^-1。

Compared to other reported BPA sensors, as shown in Table 1, the Ag-based sensors used₂O/Ag₂S/MoS₂@C₆₀₀The electrochemical sensing strategy of the nanocomposite material has lower LOD and wider dynamic range. This good sensing performance is mainly due to the following factors: (i) ag₂O/Ag₂S/MoS₂@C₆₀₀The nano composite material inherits Ag-PMo₁₂The body has certain inherent characteristics such as stable skeleton structure, stable physical and chemical properties and high electrochemical activity, so that the stability in aqueous solution is improved, the electron transfer is accelerated, and more aptamer fixing points are provided; (ii) the two-dimensional nanosheet assembly structure of the POM-derived nanocomposite has good adsorption capacity on small molecules, so that the stability of the aptamer-BPA compound is improved, and the sensing performance is enhanced; (iii) the working nano composite material has excellent electrochemical activity, can avoid using an electrode indicator, does not need a marking design, thereby reducing the cost and shortening the construction process of the aptamer sensor. Ag constructed by combining the above factors₂O/Ag₂S/MoS₂@C₆₀₀The nucleic acid aptamer sensor has remarkable BPA detection performance.

TABLE 1 comparison of the present invention with other reported BPA detection techniques

Experimental example 8: reproducibility, stability, specificity, reproducibility

As shown in FIG. 38, Ag was tested by detecting BPA using 5 modified electrodes constructed in the same manner₂O/Ag₂S/MoS₂@C₆₀₀Reproducibility of the basic nucleic acid aptamer sensor. The obtained Relative Standard Deviation (RSD) is 4.4%, which indicates that the prepared aptamer sensor has good reproducibility.

As shown in FIG. 39, BPA/Apt/Ag₂O/Ag₂S/MoS₂@C₆₀₀After 15 days of storage at 4 ℃ in a refrigerator,/AE was tested for its long-term stability at the same BPA concentration level. The results show that Δ R_ctThe change of the value is less than 5% (4.7%), which indicates that the aptamer sensor has good stability.

The high specificity is Apt/Ag₂O/Ag₂S/MoS₂@C₆₀₀Another important indicator of the AE aptamer sensor. The specificity was detected using different interferents such as BPA analogues (BA, BP, DB, DAB, PN, NPN) and small biological molecules (DA, AA, UA) present in biological fluids at concentrations 100 times higher than BPA. Delta R to be obtained from pure BPA_ctThe value was set to 100%. In contrast, as shown in FIG. 40, the Δ R of the solutions containing the interferents_ctThe value fluctuates from 0.4% to 13.8%, indicating that the aptamer sensor has high specificity. When all interferents were mixed with BPA, the Δ R of the resulting mixture_ctValues of only pure BPA Δ R_ct106.28% of the value.

Unlike most conventional BPA aptamer sensors, the aptamer sensor can also be regenerated. FIG. 41 shows Apt/Ag after the first seven regeneration cycles₂O/Ag₂S/MoS₂@C₆₀₀The detection of BPA by/AE showed only slight fluctuations, indicating that the sensor had good reproducibility.

(III) analysis of real samples

In view of the developed Ag₂O/Ag₂S/MoS₂@C₆₀₀The nucleic acid aptamer sensor has good sensing performance, and can be further applied to BPA detection in practical samples (such as river water, milk, human serum and the like) to evaluate the practical applicability of the nucleic acid aptamer sensor. Three real river water, milk and human serum samples were pre-treated prior to use. Subsequently, BPA solutions of different concentrations were added to these samples and detected using the developed aptamer sensor. Based on FIG. 37 and the derived formula, the concentration of BPA in the different samples was calculated and the results are summarized in tables 2-4. Compared with the theoretical value, the deduced BPA concentration respectively shows the recovery rates of 96.8-108.7%, 98.9-109.6% and 96.3-109.6% for river water, milk and human serum samples, and the RSD of 0.2-1.1%, 0.3-1.3% and 0.3-0.9%. The result shows that the aptamer sensor has good applicability and can be used for detecting trace BPA in an actual sample.

Table 2 detection of BPA in river water samples by the developed aptamer sensor (n ═ 3)

Table 3 detection of BPA in milk samples by the developed aptamer sensor (n ═ 3)

Table 4 detection of BPA in human serum samples by the developed aptamer sensor (n ═ 3)

The above feature tests of morphology, structure and the like show that the nano composite material is Ag₂O、Ag₂S、MoS₂And a graphite carbon layer. The results of the nano composite material used as an aptamer probe for fixing BPA to construct an entity sensor for carrying out electrochemical sensing performance tests such as EIS and CV and real sample analysis tests show that the synthesized Ag₂O/Ag₂S/MoS₂The @ C nanocomposite inherits Ag-PMo₁₂Such as skeleton structure, stable physical and chemical properties, high electrochemical activity, etc. In contrast, Ag prepared₂O/Ag₂S/MoS₂@C₆₀₀The nano composite material has higher stability in aqueous solution, high electron transfer rate and more aptamer immobilization sites. Thus, Ag₂O/Ag₂S/MoS₂@C₆₀₀The basic nucleic acid aptamer sensor has excellent sensing performance at 1 fg. mL^-1To 1 pg.mL^-1Has an extremely low LOD in the range of (1), i.e., 0.2 fg. multidot.mL^-1. The aptamer sensing strategy has three obvious advantages, namely that the preparation of the platform nano material is feasible, a labeled aptamer chain is not needed, an electrochemical indicator is not used, and an electrochemical signal is enhanced. The novel strategy has great potential in rapid and simple detection of toxic and harmful substances in environment and food.

Claims

1. A nanocomposite based on phosphomolybdic polyoxometallate, characterized in that it comprises carbon, molybdenum disulphide nanosheets and silver-containing nanoparticles; the nano composite material is obtained by calcining silver-doped phosphomolybdic polyoxometallate, wherein the silver-doped phosphomolybdic polyoxometallate is obtained by reacting a silver source, phosphomolybdic acid and thioacetamide.

2. The phosphorus-molybdenum-containing polyoxometallate-based nanocomposite material as claimed in claim 1, wherein the silver-containing nanoparticles are nano silver oxide, or nano silver oxide and nano silver sulfide, or nano silver oxide, nano silver sulfide and nano silver.

3. The method of preparing the phosphorus molybdenum polyoxometalate-based nanocomposite material according to claim 1, comprising the steps of: and (2) carrying out solvothermal reaction on mixed liquor consisting of phosphomolybdic acid, thioacetamide, a silver source and a solvent, and calcining a product to obtain the catalyst.

4. The method as claimed in claim 3, wherein the temperature of the solvothermal reaction is 160-240 ℃.

5. The method as claimed in claim 3, wherein the calcination temperature is 300-800 ℃.

6. The method for preparing the phosphorus-molybdenum-containing polyoxometalate-based nanocomposite material according to claim 3, wherein the mass ratio of the phosphomolybdic acid, the thioacetamide and the silver source is 4:10:1 to 6:10: 1.

7. The method for preparing the phosphomolybdic polyoxometallate-based nanocomposite according to any one of claims 3 to 6, wherein the mixed solution is obtained by mixing an aqueous solution of phosphomolybdic acid, an ethanol solution of thioacetamide and an aqueous solution of a silver source.

8. An electrode for an aptamer sensor, which is characterized by comprising an electrode substrate and an electrode modification material on the surface of the electrode substrate, wherein the electrode modification material is the phosphorus-molybdenum-polyoxometallate-based nanocomposite material in the claim 1 or 2.

9. An aptamer sensor, which is characterized by comprising an electrode substrate, an electrode modification material on the surface of an electrode and a nucleic acid aptamer fixed on the electrode modification material, wherein the electrode modification material is the phosphorus-molybdenum-containing polyoxometallate-based nanocomposite material in claim 1 or 2.

10. The aptamer sensor of claim 9, wherein the aptamer is a nucleic acid aptamer that specifically recognizes bisphenol a.