CN111517360B - Nanocomposite based on phosphorus-molybdenum-containing polyoxometallate and preparation method thereof, aptamer sensor and electrode thereof - Google Patents

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 nano composite 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 large yield, is widely applied to the polymer industry, and is commonly used as a monomer for synthesizing polycarbonate plastics, polysulfone resin, polyphenyl ether resin, epoxy resin, unsaturated polyester resin and the like. Commercial use of BPAThe range is extremely wide, such as used for baby feeding bottles, beverage containers, medical equipment, food packages, thermal sensitive paper and the like, 8 million tons of BPA are consumed globally in 2016, and therefore, the environment is polluted widely. 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 a pressing need for detection of 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 highly selective detection of BPA by an electrochemical biosensor. Compared with an antibody, the aptamer has the advantages of high stability, easiness in modification, convenience in repeated and simple synthesis, low cost and the like. 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 micro-fluidic 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, plasmon methods, and the like, are also used in the development of nucleic acid aptamer sensors for BPA detection. 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 Huitian et al, applications of aptamers in environmental analysis, environmental chemistry 2015, 34 (1), 89-96) developed a rapid and sensitive method for the detection of 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 . The Chinese patent with publication number CN109060913B discloses an electrochemical sensor for bisphenol A detection based on a nano composite material of gold nanoparticles, nano molybdenum disulfide and ionic liquid functionalized mercapto grapheneThe preparation method. 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 Ag ₂ O/Ag ₂ S/MoS ₂ @ 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 corresponding selection can be carried out according to the requirements on the performance of the nano composite materials.

The technical scheme of the preparation method of the nano composite material based on the polyoxometallate containing phosphorus and molybdenum 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.

According to the preparation method of the phosphorus-molybdenum-containing polyoxometallate-based nanocomposite, phosphomolybdic acid is used as a molybdenum source, thioacetamide is used as a sulfur source and a carbon source, a silver source is added, the silver-doped phosphorus-molybdenum polyoxometallate is obtained after solvothermal reaction, after calcination, organic carbon in the silver-doped phosphorus-molybdenum polyoxometallate is converted into cracking carbon and the silver source to form silver-containing nanoparticles, the preparation method is simple, and the obtained nanocomposite is high in 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 10-15h.

The calcining 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ 。

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

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 is as follows:

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 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 electrode surface ₆ ] ^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 and the construction of an aptamer sensor according to the invention;

FIG. 2 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ FE-SEM picture of (b);

FIG. 3 shows the nano-composite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ FE-SEM picture of (b);

FIG. 4 shows the nano-composite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ FE-SEM picture of (b);

FIG. 5 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ HR-TEM image of (a);

FIG. 6 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ HR-TEM image of;

FIG. 7 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ HR-TEM image of (a);

FIG. 8 is a PMo of a comparative example of the invention ₁₂ FE-SEM image of (1);

FIG. 9 is a PMo of a comparative example of the invention ₁₂ HR-TEM image of;

FIG. 10 is a graph of Ag-PMo of a comparative example of the present invention ₁₂ FE-SEM picture of (b);

FIG. 11 is a graph of Ag-PMo of a comparative example of the present invention ₁₂ HR-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 invention ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The XPS energy spectrograms of high-resolution Ag3d, mo 3d and S2 p are obtained;

FIG. 16 is a PMo of a comparative example of the present invention ₁₂ XPS energy spectrograms of high-resolution Mo 3d, S2 p, C1S and O1S;

FIG. 17 is a comparison of the present inventionExample Ag-PMo ₁₂ XPS energy spectrograms of high-resolution Ag3d, mo 3d, S2 p, 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 the experimental examples of the present invention;

FIG. 20 shows a nanocomposite Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 21 shows a nanocomposite Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 22 shows a nanocomposite Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 23 is a PMo of a comparative example of the present invention ₁₂ The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 24 is a Ag-PMo of comparative example of the present invention ₁₂ The CV curve of the aptamer sensor of (1) when detecting BPA;

FIG. 25 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 26 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nucleic acid aptamer sensor of (1) has an EIS spectrogram in the detection of BPA;

FIG. 27 shows a nanocomposite Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The 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 invention ₁₂ The 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 invention ₁₂ The 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 example ₁₂ 、Ag-PMo ₁₂ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Δ R at each stage when the aptamer sensor of (1) detects BPA _ct A difference in value;

FIG. 31 shows Ag constructed using different concentrations of electrode modification materials in accordance with the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Delta R of each stage when detecting BPA based on aptamer sensor _ct A difference in value;

FIG. 32 shows Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Influence of aptamer solutions with different concentrations on BPA detection based on the aptamer sensor;

FIG. 33 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ EIS profiles of the base nucleic acid aptamer sensors incubated in BPA solution (50 mM) for various times;

FIG. 34 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Nucleic acid aptamer sensor incubation in BPA solution (50 mM) for varying periods of time corresponding R _ct A value;

FIG. 35 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The 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 invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Delta R of nucleic acid aptamer-based sensor _ct Calibration curves for different concentrations of BPA;

FIG. 37 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Delta R of nucleic acid aptamer-based sensor _ct A linear fit to the logarithm of the BPA concentration;

FIG. 38 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The reproducibility of BPA is detected by a base nucleic acid aptamer sensor;

FIG. 39 shows Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Detecting the stability of BPA by a base nucleic acid aptamer sensor;

FIG. 40 shows Ag of the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Detecting the specificity of BPA by a base nucleic acid aptamer sensor;

FIG. 41 shows Ag according to the present invention ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nucleic acid aptamer sensor detects the reproducibility of BPA.

Detailed Description

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

AgNO ₃ Phosphomolybdic acid and ethanol were purchased from national pharmaceutical group chemical reagents, ltd.

Thioacetamide, BPA was purchased from Beijing Peking Sorpao 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 ATA GAC GTC TCC AGC-3’。

preparation of PBS buffer: mixing 0.24g KH ₂ PO ₄ 、1.44g Na ₂ HPO ₄ ·12H ₂ O,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, and this was used as a biological buffer.

Preparing electrolyte: 1.6g of K ₃ Fe(CN) ₆ 2.1g of K ₄ Fe(CN) ₆ And 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.

1. Specific examples of the phosphorus molybdenum polyoxometallate-based nanocomposites of the present invention

Example 1

The nanocomposite material of the embodiment is prepared from carbon, molybdenum disulfide nanosheets and nano Ag ₂ O 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 Ag ₂ And 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 added ₃ Dissolving in 10mL of ultrapure water to prepare AgNO ₃ Solution (0.1 g. ML) ^-1 ) Taking 100 mu L of AgNO ₃ Adding 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, then 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 C ₂ Heating for 2h under atmosphere, with the temperature rise rate of 5 ℃ min ^-1 After heating, the product is at N ₂ Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ 。

Example 2

This exampleThe nano composite material comprises carbon, molybdenum disulfide nano sheets and nano Ag ₂ O、Ag ₂ S 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 Ag ₂ O、Ag ₂ And 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 added ₃ Dissolving in 10mL of ultrapure water to obtain AgNO ₃ Solution (0.1 g. ML) ^-1 ) Taking 100 mu L of AgNO ₃ Adding 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, then 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 600 DEG C ₂ Heating for 2h under atmosphere, with the heating rate of 5 ℃ min ^-1 After heating, the product is at N ₂ Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ 。

Example 3

The nanocomposite material of the embodiment comprises carbon, molybdenum disulfide nanosheets and nano Ag ₂ O、Ag ₂ S, ag granule is prepared 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 Ag ₂ O、Ag ₂ S, ag 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 added ₃ Dissolving in 10mL of ultrapure water to prepare AgNO ₃ Solution (0.1 g. ML) ^-1 ) Taking 100 mu L of AgNO ₃ Adding 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 adding N at 800 DEG C ₂ Heating for 2h under atmosphere, with the temperature rise rate of 5 ℃ min ^-1 After heating, the product is at N ₂ Cooling to room temperature in the atmosphere, and collecting to obtain the nanocomposite material of the embodiment, which is marked as Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ 。

2. Specific examples of the preparation method of the phosphorus-molybdenum-containing polyoxometallate-based nanocomposite

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

3. Specific embodiments 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.

4. Specific embodiments of aptamer sensors 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ Dispersing the powder in 1.0mL of ultrapure water, and ultrasonically mixing to obtain uniform Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ A suspension;

(2) 5.0. Mu. L1.0 mg/mL ^-1 Ag of (A) ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ The suspension is dripped on the surface of a pretreated gold electrode AE, the surface is dried for 3 hours at room temperature, and the treated electrode is marked as Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ /AE；

(3) Washing Ag with ethanol and ultrapure water respectively ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ AE, and incubating it in 100nM aptamer solution for 30min to ensure aptamer chain in Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ Adequate anchoring of the/AE surface, the treated electrode is denoted Apt/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ /AE；

(4) Finally Apt/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ immersing/AE in BPA solution to obtain BPA/Apt/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ AE 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 mixed in a mixed solution (v/v, 7, 3,h ₂ SO ₄ /H ₂ O ₂ ) And sonication in ethanol for 15min. Subsequently, AE was rinsed thoroughly with ultrapure water and washed in N ₂ And (4) drying under flowing. AE is 0.5M H ₂ SO ₄ The potential cycling range is from-0.2 to 1.6V. Finally, AE was rinsed with ultrapure water, again at N ₂ Dried 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 combined with organic pollution small molecules, environmental microorganisms, heavy metal ions and the like in a targeted manner, which is not described in detail herein.

5. Comparative example

Comparative example 1

In the aptamer sensor of the comparative example, the electrode modification material is phosphorus-molybdenum polyoxometallate PMo ₁₂ Construction of aptamer sensor the procedure for construction of aptamer sensor is the same as in example 10, PMo ₁₂ Modified electrode materials with PMo ₁₂ The AE represents the Apt/PMo of the electrode material after the aptamer of BPA is immobilized ₁₂ and/AE. Wherein, PMo ₁₂ The 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;

(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 for 10min, transferring the mixed solution into a 50mL stainless steel reaction kettle with a polytetrafluoroethylene lining, and heating 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.

Comparative example 2

In the aptamer sensor of the comparative example, the electrode modification material is Ag doped phosphorus-molybdenum-containing polyoxometallate Ag-PMo ₁₂ Construction of aptamer sensor the same procedure as in example 10, ag-PMo ₁₂ Modified electrode material made of Ag-PMo ₁₂ The AE represents that the electrode material after the aptamer immobilization of BPA is Apt/Ag-PMo ₁₂ and/AE. Wherein, ag-PMo ₁₂ The production method of (4) is different from that in example 4 in that the calcination treatment of step (5) is not performed, and the other steps are the same as those in example 4.

6. Examples of the experiments

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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ 、Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Nanocomposite and PMo ₁₂ 、Ag-PMo ₁₂ The 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, PMo ₁₂ Spherical structures with different sizes are formed by the accumulation of nano-particles. At the same time, it is further confirmed by fig. 9 that PMo ₁₂ The spherical structure of (a) consists of a large number of nanosheets. At PMo ₁₂ FIG. 9e, a lattice spacing of 0.62nm was observed, corresponding to MoS ₂ (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 MoS ₂ Nanosheet (MoS) ₂ NSs) structure.

FIG. 10 shows Ag-PMo ₁₂ The 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. 11 d) ₂ Layer, also showing MoS ₂ Lattice spacing of NSs (fig. 11 e). No lattice association with Ag was observed, indicating that it was doped with MoS in the ionic state ₂ In 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Surface topography and irregular shape PMo of ₁₂ The spheres are similar. However, in Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Irregular petal-like structures are found in nanocomposites, which are composed of many nanosheets with a loose nanostructure. Albeit in Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The structure of the nanocomposite is similar. Of the three samples, ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The nanocomposites exhibited a more open structure and thinner nanoplatelets, comparable to FE-SEMThe results were consistent. All HR-TEM images were observed to correspond to MoS ₂ The lattice spacing of the (002) crystal plane of (b). In Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Also observed in (D) is a correspondence to Ag ₂ The lattice spacing of the (200) crystal plane of O was 0.241nm. In Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Except for Ag in the HR-TEM image of ₂ O and MoS ₂ In 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 PMo ₁₂ Ag in (C) ⁺ The ions did not undergo conversion upon heat treatment at 300 ℃ but showed Ag at 600 ℃ ₂ Oxidation state of O. Heat treatment at extremely high temperatures, ag ₂ O 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 using, in particular, a Cu K X-ray diffraction (XRD) test on a Rigaku D/Max-2500X X-ray diffractometer _α Radiation (λ =0.15406 nm).

As shown in fig. 12, PMo ₁₂ The three XRD patterns of the crystal form are MoS at 8.9 degrees, 33.4 degrees and 58.3 degrees ₂ The diffraction peak of (1). The transmission electron microscope result shows that the phosphomolybdic acid and the thioacetamide generate MoS through hydrothermal reaction ₂ . But in Ag-PMo due to low crystallization rate ₁₂ No obvious peak is seen in the product. For Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ A weaker peak appears at 32.6 because of the fact that it is other than belonging to MoS ₂ In addition to the three peaks, ag was also present in the sample ₂ O。Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The XRD spectrum of the crystal shows MoS at 8.9 degrees, 33.4 degrees and 58.3 degrees ₂ The characteristic peaks of (A) show Ag at 32.0 °, 32.6 °, 53.3 ° and 56.5 ° ₂ Characteristic peak of O. In addition, two diffraction peaks at 25.4 ° and 45.6 ° correspond to Ag ₂ S, and the three peaks at 28.9 °, 38.5 ° and 44.1 ° are attributed to metallic Ag. Thus, ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nano composite material consists of MoS ₂ 、Ag ₂ O、Ag ₂ S and metallic Ag, etcThe components are mixed. Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The XRD pattern of the compound shows that the compound is in contact with Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Similar structure. These results indicate that Ag was doped ⁺ PMo of ₁₂ Is converted into embedded Ag ₂ O、Ag ₂ MoS of multiple components such as S and Ag ₂ The 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, PMo ₁₂ The Raman spectrum of (A) shows that the specific peaks are located at 281, 378, 405, 818 and 942cm ^-1 Here, this is from MoS ₂ E of (A) ¹ _2g And A _1g Caused by the vibration modes. Ag-PMo ₁₂ Raman spectroscopy and PMo ₁₂ Same as above, the description of Ag ⁺ Addition of ions to PMo ₁₂ Has no influence on the chemical structure of (2). Heat treating at 300 deg.C in Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ In (A) also observed to belong to MoS ₂ Main peak of (2). This indicates that Ag-PMo is present at this temperature ₁₂ Does not change much in chemical structure. However, in Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ In (1), only attribution to MoS can be observed ₂ Two 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 _α Collected from the X-ray source (1486.6 eV photons).

FIG. 14 shows the coexistence of Mo 3d, C1s, N1 s, and O1s signalsIn 5 samples, and Ag-PMo ₁₂ And 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 Ag ₂ O/Ag ₂ S/MoS ₂ High resolution energy spectrogram of @ C series nanocomposite. In Ag ₂ O/Ag ₂ S/MoS ₂ In the @ C series nanocomposite (FIGS. 15C-e), the high resolution Ag3d XPS spectrum also showed two distinct peaks corresponding to Ag ⁺ With Ag-PMo ₁₂ Similarly. For Ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ Mo 3d XPS spectrum of the nano composite material obtains similar peak position, but Mo ⁵⁺ And Mo ⁶⁺ Relatively high content of ions, and Mo-O _x The bond content is also higher. In contrast, ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Mo in (1) ⁶⁺ The ionic strength is low. In Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Mo is not found in the nanocomposite ⁵⁺ And Mo ⁶⁺ Ions and Mo-O _x The bond content is also low, indicating that it is completely decomposed at very high temperatures. Only S appears in S2 p XPS energy spectrum of the series of nano composite materials ^2- 2p _3/2 And S ^2- 2p _1/2 Two peaks, indicating the production of Ag ₂ S or MoS ₂ . From the above analysis, it is found that the Ag-PMo is calcined ₁₂ Can obtain Ag ₂ O、Ag ₂ S and MoS ₂ The 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 PMo ₁₂ High resolution spectrum of (2). FIG. 16a can be divided into two parts, with peaks at a Binding Energy (BEs) of 228.6eV and 231.8eV, respectivelyCorresponds to Mo ⁴⁺ 3d _5/2 And Mo ⁴⁺ 3d _3/2 . The other two peaks at 5363 higher of BEs are 229.5 and 232.9eV, respectively, and belong to Mo ⁵⁺ And Mo ⁶⁺ . This is illustrated in PMo ₁₂ Mo in the solution is partially oxidized from Mo ⁴⁺ State change to Mo ⁵⁺ And Mo ⁶⁺ Status. In addition, the peaks at BEs 226.0eV and 235.4eV are attributed to Mo-S and Mo-O, respectively _x The function of the bond. PMo ₁₂ Can be fitted to BEs at 161.4 and 162.6eV, corresponding to S2 p XPS spectra (FIG. 16 b), respectively ^2- S2 p of _3/2 And S2 p _1/2 . The peak at BE 163.7eV corresponds to S ₂ ^2- S2 p of _3/2 . The two peaks at BEs of 168.8eV and 169.9eV are attributed to S, respectively ⁶⁺ And an S-O bond. It is clear that S2 p is composed of mixed valencies, in which part of the S is also oxidized to SO ₄ ^2- Ions. At PMo ₁₂ In the high-resolution C1s spectrum of (fig. 16C), two main components corresponding to C-C bonds and C = O bonds were observed at BEs of 284.3eV and 285.9eV, respectively. Three main peaks at 530.6, 531.9 and 533.1eV were obtained on the O1s XPS spectra (fig. 16 d), corresponding to O vacancies, C = O bonds and C-O bonds, respectively. The coexistence of the mixed valence states of Mo 3d and S2 p and the occurrence of O vacancy can obviously promote the electron transfer and improve the electrochemical activity.

FIG. 17 shows Ag-PMo ₁₂ The 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 at PMo ₁₂ In which Ag is doped ⁺ . The high resolution Ag3d XPS spectrum is split into two peaks at 367.02 and 374.03eV, which corresponds to Ag ₂ Ag in S ⁺ Ionic Ag3d _5/2 And Ag3d _3/2 . The observation of PMo in the analysis of Mo 3d and S2 p XPS spectra ₁₂ Similar results, except Ag, indicate ⁺ Presence of Ag-PMo ₁₂ And original PMo ₁₂ Are very close in chemical structure.

(II) electrochemical sensing Performance test

All electrochemical tests, including Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV), were performed in Solartron ANALYTIC electrochemical workstation (UK). In the presence of 0.5mM [ 2 ], [ Fe (CN) ], containing 0.1M KCl ₆ ] ^3-/4- To obtain an EIS curve (EIS parameter: potential, 0.21V; frequency range, 100kHz to 0.01Hz; 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. And respectively incubating the modified gold electrode with 10, 20, 50, 100, 200 and 500nM aptamer solutions, and optimizing the optimal concentration of the aptamer 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 time of binding 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/Ag ₂ O/Ag ₂ S/MoS ₂ @ 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/Ag ₂ O/Ag ₂ S/MoS ₂ @ 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 is BPA concentration (1 ng 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/Ag ₂ O/Ag ₂ S/MoS ₂ @ C/AE was stored in a refrigerator (4 ℃) 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 used ₂ O/Ag ₂ S/MoS ₂ @ 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

PMo-based exploration by using EIS and CV methods ₁₂ 、Ag-PMo ₁₂ Calcining at 300 deg.C, 600 deg.C and 800 deg.C to obtain a series of Ag ₂ O/Ag ₂ S/MoS ₂ The construction process of the 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 _p Indicating 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 _p Is increased. Among these electrode materials, ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ the/AE showed the highest peak current and the narrowest Δ E _p Values indicating relatively good electrochemical performance. With the original PMo ₁₂ And Ag-PMo ₁₂ In contrast, for Ag-PMo at appropriate temperature ₁₂ The heat treatment is performed to facilitate the acceleration of electron transfer.

When the aptamer chain is fixed on the modified electrode, only a slight decrease of peak current is caused, and delta E _p Value increaseThis is due to the negatively charged phosphate group on the aptamer chain and [ Fe (CN) ] ₆ ] ^3-/4- Repulsive interactions between the redox probes prevent electron transfer at the electrolyte/electrode interface. Apt/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ The 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-PMo ₁₂ The peak current of/AE varies greatly, which indicates Apt/Ag-PMo ₁₂ the/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 _p The 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/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ the/AE showed large peak current variation. Although Ag-PMo ₁₂ The base nucleic acid aptamer sensor shows better nucleic acid aptamer immobilization performance, and electrochemical signal change caused when the base nucleic acid aptamer sensor detects BPA is smaller than Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ A nucleic acid-based aptamer sensor. This indicates that for Apt/Ag-PMo ₁₂ AE, the aptamer-BPA complex formed during detection would be from Ag-PMo ₁₂ The matrix is partially removed.

Using a series of Ag ₂ O/Ag ₂ S/MoS ₂ @ C and PMo ₁₂ 、Ag-PMo ₁₂ The EIS results from construction of aptamer sensors maintained agreement with the CV curves, see fig. 25-29. Three kinds of Ag ₂ O/Ag ₂ S/MoS ₂ The @ C nanocomposite-based nucleic acid aptamer sensor has a similar electrochemical behavior. All blank AEs showed smaller R of 30.9-33.9 Ω _ct Values indicating that they have significant electrochemical activity, making it extremely easy for electrons to transfer at the electrode/electrolyte solution interface. Use PMo for blank AEs ₁₂ 、Ag-PMo ₁₂ And series Ag ₂ O/Ag ₂ S/MoS ₂ After modification of @ C, R _ct The value becomes larger, and it ranges from 165.7 Ω to 305.6 Ω. This indicates a series of Ag ₂ O/Ag ₂ S/MoS ₂ The @ C nanocomposite has poor electrochemical activity compared to blank AE, which hinders electron transfer, and increases R _ct The value is obtained. Compared with the nano porous organic framework reported in the past, ag ₂ O/Ag ₂ S/MoS ₂ The @ 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, ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ R of (A) to (B) _ct The minimum value is 165.7 omega, which shows that the electrochemical performance is excellent. At relatively low temperatures (300 ℃), ag-PMo ₁₂ The nanostructure portion of (a) is decomposed. Due to PMo ₁₂ Intrinsic properties of, i.e. high electrochemical activity, ag-PMo ₁₂ Ultrathin nanoflakes have open electron transport channels, thus providing more electroactive sites, shorter electron transport and electrolyte diffusion paths. After the aptamer chain is fixed on the modified electrode, R _ct The value continues to increase to 268.9-788.9 Ω. EIS response increased when detecting BPA in aqueous solution, R _ct The values continue to increase to the range 526.5-1101.3 Ω.

The invention also researches PMo ₁₂ 、Ag-PMo ₁₂ And series Ag ₂ O/Ag ₂ S/MoS ₂ R for each step of BPA detection with an @ C-based aptamer sensor _ct Variation of value (. DELTA.R) _ct ) As shown in fig. 30.Δ R _ct May represent the respective binding amounts. In three kinds of Ag ₂ O/Ag ₂ S/MoS ₂ In @ C nanocomposite, ag ₂ O/Ag ₂ S/MoS ₂ @C ₃₀₀ Δ R of (A) _ct The value was minimal (134.8 Ω), indicating excellent electrochemical activity. Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ And Ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Δ R of (A) _ct The values are comparable but not very different, 189.1 and 209.8 Ω, respectively. Nucleic acidsAfter aptamer immobilization, ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Best fixing ability of/AE,. DELTA.R _ct The value is 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 Ag ₂ O and Ag ₂ S is more pronounced. In contrast, ag ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Aptamer anchoring Performance of/AE is the worst, R _ct The value is only 66.1 omega. TEM and XRD results show that Ag is contained in three nano-composite materials ₂ O/Ag ₂ S/MoS ₂ @C ₈₀₀ Is 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 AE ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Delta R of/AE _ct The value (434.6 Ω) is the largest. Therefore, we have selected Ag with mesopores ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nano composite material is used as a platform for constructing a BPA aptamer sensor. This Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The 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 concentrations ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Different aptamer sensors were constructed as carriers to study the effect of material usage on BPA detection. FIG. 31 shows different nucleiChange in EIS response at each step of the acid aptamer sensor. It can be seen that different amounts of Ag were used ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Δ R due to AE _ct Value with Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Increases with increasing amounts. This means that the layer thickness of the electrode material can significantly impede the electron transfer, so that a larger R is obtained _ct The value is obtained. In addition, the thick material may provide more anchoring sites for the aptamer strands, along with Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The dosage is 0.1 mg/mL ^-1 Increased to 1 mg. ML ^-1 ，R _ct The value also increases. When the dosage is more than 1.0 mg/mL ^-1 In time, the immobilization of aptamers and detection of BPA reached equilibrium. In addition, too thick a material is easily peeled off from the electrode surface. Therefore, the concentration was selected to be 1.0 mg/mL ^-1 Ag of (A) ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nanocomposite is used for constructing the aptamer sensor and is used for further testing.

To determine the effect of aptamer concentration on sensory properties, several ags were used ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ the/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 _ct The value increases with increasing concentration, and Δ R when the concentration exceeds 100nM _ct The 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 water ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The base nucleic acid aptamer sensors were tested in PBS (0.1M, pH 7.4) after various periods of soaking in BPA (50 mM). As can be seen from FIGS. 33 and 34, EIS response and Δ R were observed as the incubation time was increased _ct The detection of BPA also increases. BPA detection reaches the maximum value at 40min, so 40min is adopted asThe incubation time is indicated. Using Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The optimal conditions for detecting BPA by the nucleic acid aptamer sensor are as follows: ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The concentration is 1.0 mg/mL ^-1 (ii) a The concentration of the aptamer is 100nM; BPA binding time was 40min.

Experimental example 7: sensitivity test

It is crucial to explore the detection sensitivity of aptamer sensors, which 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/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ AE and BPA solutions of different concentrations (1 fg. ML) ^-1 -1pg·mL ^-1 ) Incubated together, and 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 resulting EIS response to BPA detection increased with increasing BPA concentration. 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Delta R of nucleic acid aptamer based sensor _ct The values give a calibration curve, see fig. 36. The results show that Δ R _ct The value increases with increasing BPA concentration, ranging from 1 fg. ML ^-1 -1pg·mL ^-1 When the concentration of BPA is more than 1 pg/mL ^-1 When is Δ R _ct The values gradually tend to equilibrate. By Δ R _ct The values as a function of the logarithm of the BPA concentration gave a very good linear relationship, as shown in FIG. 37, with a regression equation of Δ R _ct (Ω)＝647.46+172.44log(C _BPA ) The correlation coefficient is R ² =0.9921. 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 used ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The electrochemical sensing strategy of the nano composite material has lower LOD and is moreWide dynamic range. This good sensing performance is mainly due to the following factors: (i) Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nano composite material inherits Ag-PMo ₁₂ The 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 nucleic acid 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 factors ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The 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 manner ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Reproducibility 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/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ After 15 days of storage at 4 ℃ in a refrigerator, at the same BPA concentration levelThe long-term stability was tested. The results show that Δ R _ct The change of the value is less than 5% (4.7%), which indicates that the aptamer sensor has good stability.

The high specificity is Apt/Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ Another 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 _ct The value was set to 100%. In contrast, as shown in FIG. 40, the Δ R of the interferent-containing solution _ct The 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 _ct Values of only pure BPA Δ R _ct Value 106.28%.

Unlike most conventional BPA aptamer sensors, the aptamer sensor can also be regenerated. FIG. 41 shows Apt/Ag after the first seven regeneration cycles ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The 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 Ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The 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 various 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 results showed that the aptamerThe 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 Ag ₂ O、Ag ₂ S、MoS ₂ And 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 Ag ₂ O/Ag ₂ S/MoS ₂ The @ C nanocomposite inherits Ag-PMo ₁₂ Such as skeleton structure, stable physical and chemical properties and higher electrochemical activity. In contrast, ag prepared ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The nano composite material has higher stability in aqueous solution, high electron transfer rate and more aptamer immobilization sites. Thus, ag ₂ O/Ag ₂ S/MoS ₂ @C ₆₀₀ The basic nucleic acid aptamer sensor has excellent sensing performance at 1 fg. ML ^-1 To 1 pg.mL ^-1 Has 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 (7)

1. A preparation method of a nanocomposite based on phosphorus-molybdenum-containing polyoxometallate is characterized by comprising the following steps: 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 composite material; the silver source is silver nitrate; the product is silver-doped phosphomolybdic polyoxometallate; the calcination is in N ₂ Carried out under an atmosphere;

the temperature of the solvothermal reaction is 160-240 ℃; the calcining temperature is 300-800 ℃.

2. The preparation method of the phosphomolybdic acid polyoxometallate-based nanocomposite according to claim 1, wherein the mass ratio of phosphomolybdic acid, thioacetamide and silver source is 4.

3. The method for preparing the phosphomolybdic acid polyoxometallate-based nanocomposite according to claim 1 or 2, 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; the silver source is silver nitrate.

4. A nanocomposite material produced by the production method according to any one of claims 1 to 3, wherein the nanocomposite material comprises carbon, molybdenum disulfide nanosheets and silver-containing nanoparticles; the silver-containing nano particles are nano silver oxide or nano silver oxide, nano silver sulfide and nano silver.

5. 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 claim 4.

6. An aptamer sensor 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 disclosed by claim 4.

7. The aptamer sensor according to claim 6, wherein the aptamer is a nucleic acid aptamer that specifically recognizes bisphenol A.

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