Copper-based compound / graphene oxide nano-enzyme as well as preparation method and application thereof

Abstract

The invention is suitable for the technical field of environment and food safety detection, and provides a copper-based compound / graphene oxide nano-enzyme and a preparation method and application thereof.The preparation method comprises the following steps that cysteamine hydrochloride and copper chloride dihydrate are dissolved in deionized water, stirring is conducted under nitrogen protection, the pH is adjusted, then the mixed solution is transferred into an oil bath to be continuously stirred, and the copper-based compound / graphene oxide nano-enzyme is obtained; the preparation method comprises the following steps: preparing a two-dimensional copper-based compound, centrifuging, collecting precipitate, washing and drying to obtain the two-dimensional copper-based compound, dispersing the two-dimensional copper-based compound and graphene oxide in ultrapure water to prepare dispersion liquid, mixing the two-dimensional copper-based compound dispersion liquid and graphene oxide dispersion liquid, stirring at room temperature, and centrifugally drying to obtain the composite nano-enzyme. According to the method, the contradiction that the nano-enzyme activity pH is not matched with the optimal reaction pH of the formaldehyde is solved, the detection sensitivity and the anti-interference capability are remarkably improved, and an efficient technical scheme is provided for rapid and quantitative detection of the formaldehyde in multiple scenes such as food and environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental and food safety detection, and in particular relates to a copper-based compound / graphene oxide nanozyme and a preparation method and application thereof. Background Art

[0002] Formaldehyde, a highly toxic volatile organic compound, is widely present in indoor air pollution, industrial emissions and illegal food additives, posing a serious threat to human health. In the food industry, formaldehyde is often used by unscrupulous businesses for preservation of aquatic products, dairy products, etc. Its concealment and high harmfulness make the development of efficient and sensitive detection technology an urgent need for food safety supervision.

[0003] At present, formaldehyde detection mainly adopts chromatography (such as high performance liquid chromatography, gas chromatography) and electrochemical methods, but these methods rely on expensive instruments, professional operations and complex pretreatment, which can hardly meet the needs of rapid on-site detection; although the colorimetric method has the advantages of simple operation and intuitive results, the traditional color development system (such as acetylacetone method, chromotropic acid method) has the disadvantages of low sensitivity, harsh reaction conditions (high temperature or strong acid environment is required), and high reagent toxicity. In recent years, colorimetric sensing technology based on nanozymes has attracted attention due to its high catalytic efficiency and stability. However, the catalytic activity of nanozymes usually depends on acidic environment (~pH4.0), while formaldehyde has strong reaction activity under neutral to weak alkaline conditions, resulting in pH adaptability contradictions in the detection system, which limits the reaction efficiency and detection sensitivity; in addition, existing nanozymes (such as Fe 3 O 4 , precious metal nanoparticles) generally face problems such as complex preparation process and insufficient exposure of catalytic active sites, and their activity is relatively low at room temperature, making it difficult to meet the actual sample detection needs.

[0004] In view of the above technical bottlenecks, the present invention proposes to construct a two-dimensional copper-based compound / graphene oxide (Cu-Cy / GO) composite nanozyme. This material exhibits excellent peroxidase-like activity at neutral pH and room temperature through the interfacial synergistic effect of two-dimensional copper-based compounds and graphene oxide, breaking through the dependence of traditional nanozymes on acidic environments. At the same time, formaldehyde molecules can specifically inhibit the catalytic activity of Cu-Cy / GO, and achieve accurate correlation between color development signals and formaldehyde concentrations through competitive mechanisms. Compared with the prior art, the present invention solves the contradiction between the mismatch between the active pH of nanozymes and the optimal reaction pH of formaldehyde, significantly improves the detection sensitivity (detection limit as low as 0.4μg / L) and anti-interference ability, and simplifies the sample pre-treatment process, providing an efficient technical solution for rapid quantitative detection of formaldehyde in multiple scenarios such as food and environment. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a method for preparing a copper-based compound / graphene oxide nanozyme, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for preparing a copper-based compound / graphene oxide nanozyme comprises the following steps:

[0007] Synthesis of a two-dimensional copper-based compound (Cu-Cy): Dissolve cysteamine hydrochloride and cupric chloride dihydrate in deionized water, stir under nitrogen protection, adjust the pH, transfer the mixture to an oil bath, continue stirring, collect the precipitate by centrifugation, wash, and dry to obtain a two-dimensional copper-based compound;

[0008] Construction of Cu-Cy / GO composite nanozyme: The two-dimensional copper-based compound and graphene oxide are separately dispersed in ultrapure water to prepare a dispersion, and then the two-dimensional copper-based compound dispersion is mixed with the graphene oxide dispersion. After stirring at room temperature, centrifugal drying is performed to obtain a composite nanozyme in which the copper-based compound is loaded on the surface of graphene oxide.

[0009] Preferably, in the step of synthesizing the two-dimensional copper-based compound, the molar ratio of cysteamine hydrochloride to copper chloride is 1:1-1.5:1.

[0010] Preferably, in the step of synthesizing the two-dimensional copper-based compound, the pH is 7.0-8.0.

[0011] Preferably, in the step of synthesizing the two-dimensional copper-based compound, the temperature of the oil bath is 85-95°C.

[0012] Preferably, in the step of constructing the composite nanozyme, the concentration of the two-dimensional copper-based compound dispersion is 1.0-1.5 mg / mL, the concentration of the graphene oxide dispersion is 0.1-0.15 mg / mL, and the volume ratio of the two-dimensional copper-based compound dispersion to the graphene oxide dispersion is 1:5-3:1.

[0013] Another object of an embodiment of the present invention is to provide a copper-based compound / graphene oxide nanozyme, which is prepared using the above-mentioned preparation method.

[0014] Another object of an embodiment of the present invention is to provide an application of a copper-based compound / graphene oxide nanozyme in formaldehyde detection.

[0015] Preferably, the formaldehyde detection comprises the following steps:

[0016] Standard curve establishment: In a pH 7.0 buffer system, the copper-based compound / graphene oxide nanozyme, hydrogen peroxide, tetramethylbenzidine (TMB) and formaldehyde standard solution were added in sequence, and the absorbance difference at 652 nm (ΔA = A 0-A), establish the quantitative relationship between ΔA and formaldehyde concentration;

[0017] Actual sample testing: Add the test solution according to the above system and calculate the formaldehyde content according to the standard curve.

[0018] Preferably, the concentration of the copper-based compound / graphene oxide nanozyme is 0.015-0.03 mg / mL, the concentration of hydrogen peroxide is 0.5-2 mmol / L, and the concentration of tetramethylbenzidine is 0.1-1 mmol / L.

[0019] The copper-based compound / graphene oxide nanozyme provided in the embodiment of the present invention has a synergistic effect of the interface electrons of the two-dimensional sheet structure of Cu-Cy and graphene oxide, so that the nanozyme still maintains efficient peroxidase-like activity under neutral conditions (pH 7.0) and room temperature, catalyzing H 2 O 2 The OH free radicals are generated to oxidize TMB for color development, which solves the contradiction between the acidic environment required by traditional nanozymes and the neutral conditions suitable for formaldehyde detection, and the reaction does not require heating.

[0020] Formaldehyde competitively captures OH to inhibit the oxidation reaction of TMB, resulting in a change in the colorimetric signal. The colorimetric signal change (ΔA) is negatively correlated with the formaldehyde concentration, with a linear range of 4-10 6 μg / L, with a detection limit of 0.4μg / L, which is more than 10 times more sensitive than the existing nanozyme method;

[0021] There is no need to use toxic colorimetric reagents (such as chromic acid), and the reaction conditions are mild (room temperature, neutral pH). The entire detection process takes only 15 minutes, supports colorimetric analysis, and is suitable for on-site detection of food (aquatic products, dairy products) and environmental samples (air absorption liquid, wastewater). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope photograph of Cu-Cy prepared in Example 1 of the present invention;

[0023] Figure 2 The Cu 2p high-resolution X-ray photoelectron spectrum of Cu-Cy prepared in Example 1 of the present invention;

[0024] Figure 3 Zeta potential data diagram of GO, Cu-Cy and Cu-Cy / GO nanozymes prepared in Example 1 of the present invention;

[0025] Figure 4 This is a transmission electron microscopy photograph of the Cu-Cy / GO nanozyme prepared in Example 1 of the present invention;

[0026] Figure 5This is a graph verifying the peroxidase activity of the Cu-Cy / GO nanozyme prepared in Example 1 of the present invention;

[0027] Figure 6 This is a graph showing the change in peroxidase activity of the Cu-Cy / GO nanozyme prepared in Example 1 of the present invention as a function of pH;

[0028] Figure 7 This is a graph showing the change in peroxidase activity of the Cu-Cy / GO nanozyme prepared in Example 1 of the present invention as a function of temperature;

[0029] Figure 8 The UV-visible absorption spectrum (A) and the linear regression equation diagram (B) of the formaldehyde working curve provided in Example 2 of the present invention;

[0030] Fig. 9 This is a graph of experimental data for selectivity and anti-interference evaluation of formaldehyde detection provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0033] Example 1: A copper-based compound / graphene oxide Cu-Cy / GO nanozyme, the preparation method of which comprises the following steps:

[0034] Step 1, synthesis of a two-dimensional copper-based compound (Cu-Cy): cysteamine hydrochloride (278 mg) and cupric chloride dihydrate (205 mg) were dissolved in 50 mL of deionized water, stirred at 1000 rpm for 15 minutes under nitrogen protection, and then the pH was adjusted to 7.5 with 1 mol / L NaOH; the mixed solution was transferred to a 90° C. oil bath and stirred for 15 minutes, centrifuged (8000 rpm, 10 minutes) to collect the precipitate, washed 3 times with ultrapure water, and vacuum dried to obtain a two-dimensional sheet-like copper-based compound (Cu-Cy);

[0035] Step 2, construction of Cu-Cy / GO composite nanozyme: Cu-Cy was dispersed in ultrapure water to prepare a 1.0 mg / mL dispersion, and graphene oxide (GO) was prepared to prepare a 0.1 mg / mL dispersion. 5 mL of GO dispersion was mixed with 10 mL of Cu-Cy dispersion (volume ratio 1:2), stirred at 1000 rpm for 30 minutes at room temperature, centrifuged (12000 rpm, 15 minutes) and freeze-dried to obtain Cu-Cy / GO nanozyme.

[0036] The Cu-Cy, GO, and Cu-Cy / GO nanozymes prepared in Example 1 were tested and the scanning electron microscopy (SEM) results were as follows: Figure 1 As shown in Figure 2, it can be seen that Cu-Cy presents a two-dimensional sheet structure with a size of 2.6±0.3μm×0.8±0.2μm. Its two-dimensional structure significantly increases the exposed area of ​​the active sites; X-ray photoelectron spectroscopy (XPS) is shown in Figure 2 Figure 2 As shown, it can be seen that the binding energy in the Cu 2p spectrum is 932.6 eV (2p 3 / 2 ) and 952.2eV(2p 1 / 2 ) at the peak, confirming that copper in Cu-Cy is in a monovalent state (Cu + ) form, which is the valence state of H 2 O 2 The activation of Figure 3 As shown in the figure, the surface potential of Cu-Cy is +8.1mV, while the surface potential of GO is -31.9mV. The two self-assemble through strong electrostatic interaction to form a stable Cu-Cy / GO composite structure, avoiding nanosheet agglomeration and maintaining high catalytic activity; transmission electron microscopy (TEM) Figure 4 As shown, it is further confirmed that Cu-Cy is anchored on the surface of graphene oxide (GO) to form a stable heterogeneous interface structure.

[0037] The catalytic performance of the Cu-Cy / GO nanozyme prepared in Example 1 was verified:

[0038] (1) Peroxidase activity test:

[0039] The following groups were added to pH 7.0 Tris-HCl buffer: Group 1: TMB (0.5 mmol / L) + H 2 O 2 (1mmol / L); Group 2: Group 1 + Cu-Cy / GO (0.03mg / mL); Control group: Group 1 + pure Cu-Cy (control group 1) or GO (control group 2) (concentrations are the same as those in group 2); After 15 minutes of reaction, the solution in group 2 showed obvious blue (λ max =652nm), the absorbance was 0.37, while the absorbance of group 1 and the control group was <0.1 (e.g. Figure 5 It is shown that Cu-Cy / GO has significant catalytic activity under neutral conditions, and the activity is derived from the synergistic effect of Cu-Cy and GO;

[0040] (2) pH and temperature adaptability analysis:

[0041] pH effect: In the pH range of 3-9, the catalytic activity of Cu-Cy / GO reached its peak at pH 7.0 (absorbance 1.93), and 25% activity was retained at pH 9 (Fig. Figure 6 As shown), significantly better than Fe 3 O 4 Nanozymes (optimal at pH 3.5, activity <5% at pH 7.0) were prepared under the following experimental conditions: Cu-Cy / GO: 0.03 mg / mL, TMB: 1 mmol / L, H 2 O 2 : 2mmol / L, reaction time: 15 minutes, temperature: room temperature (~20-25°C);

[0042] Temperature effect: The catalytic activity is optimized in the range of 10-30℃ (e.g. Figure 7 As shown in Figure 2, natural enzymes and other nanozymes usually have optimal activity at around 40°C, while their activity is significantly reduced at room temperature. The experimental conditions are: Cu-Cy / GO: 0.03 mg / mL, TMB: 1 mmol / L, H 2 O 2 : 2mmol / L, reaction time: 15 minutes, pH 7.

[0043] Example 2: Sensitivity and selectivity of Cu-Cy / GO nanozyme for formaldehyde detection. The experiment includes the following steps:

[0044] (1) Standard curve establishment: 0.03 mg / mL Cu-Cy / GO and 1 mmol / L H 2 O 2 , 0.5mmol / LTMB and 0-10 6 μg / L formaldehyde standard solution, and measure the absorbance difference at 652nm after 15 minutes of reaction (ΔA=A 0 -A); the linear relationship of the standard curve is ΔA = -0.0337 + 0.6154lgC (R 2 =0.998), linear range 4-10 6 μg / L (such as Figure 8 As shown), A 0 and A are Cu-Cy / GO+H 2 O 2 +The absorbance of the TMB system before and after the addition of formaldehyde, C is the formaldehyde concentration, and the detection limit is 0.424 μg / L according to the 3σ method;

[0045] (2) Anti-interference ability test: 5 μg / L formaldehyde system, add 5 times the concentration of interference (Na + , K + , Ca2+ , ethanol, etc.), the detection signal change rate is less than 5% (such as Fig. 9 This indicates that the method has strong anti-interference ability to complex matrices.

[0046] Example 3, actual sample detection and spike recovery verification:

[0047] (1) Determination of formaldehyde in beer samples:

[0048] Sample treatment: Take 2 mL of beer sample and add 2 mL of deionized water to obtain the sample solution to be tested;

[0049] Sample detection: Take 100 μL of the sample solution to be tested, add the following reagents in sequence: 50 μL of 0.6 mg / mL Cu-Cy / GO nanozyme, 100 μL of 10 mmol / L hydrogen peroxide, 100 μL of 5 mmol / L TMB, dilute to 1 mL with Tris-HCl buffer solution at pH 7, shake well and let stand for 15 min; measure the absorbance A at 652 nm, bring it into the formaldehyde detection standard curve in Example 3, and calculate the formaldehyde content in the beer sample to be 1.4 mg / L;

[0050] Beer sample spike test: add three different concentrations of formaldehyde standard solutions to the sample solution to be tested; each concentration is measured three times in parallel, and the spike recovery rate is calculated, and the relative standard deviation RSD is calculated. The results are shown in Table 1:

[0051] Table 1 Recovery and RSD of beer sample spiked (n=3)

[0052]

[0053] (All samples were diluted 20 times in the detection system)

[0054] The measured formaldehyde spike recovery rate was 98.00% to 101.00%, and the RSD was 1.39% to 3.11%, indicating that the nanozyme provided in the embodiment of the present invention has good accuracy and precision in formaldehyde detection;

[0055] (2) Determination of formaldehyde in frozen shrimp samples:

[0056] Actual sample processing: Weigh 2g of frozen shrimp sample and place it in a 15mL glass bottle, add 2mL of deionized water containing 100mmol / L HCl, and place it at room temperature for 2h to obtain the sample solution to be tested;

[0057] Actual sample detection: Take 100 μL of the sample solution to be tested, add the following reagents in sequence: 50 μL of 0.6 mg / mL Cu-Cy / GO nanozyme, 100 μL of 10 mmol / L hydrogen peroxide, 100 μL of 5 mmol / L TMB, dilute to 1 mL with pH 7 Tris-HCl buffer solution, shake well and let stand for 15 min; measure the absorbance A at 652 nm, bring it into the formaldehyde detection standard curve in Example 3, and calculate the formaldehyde content in the frozen shrimp sample to be 2.24 mg / L;

[0058] Actual sample spike detection: Three different concentrations of formaldehyde standard solutions were added to the sample solution to be tested; each concentration was measured three times in parallel, the spike recovery rate was calculated, and the relative standard deviation RSD was calculated. The results are shown in Table 2:

[0059] Table 2 Recovery and RSD of spiked frozen shrimp samples (n=3)

[0060]

[0061] (All samples were diluted 20 times in the detection system)

[0062] The recoveries of formaldehyde were measured to be between 96.00% and 104.33%, with RSDs between 1.58% and 3.71%;

[0063] (3) Determination of formaldehyde in frozen chicken samples:

[0064] Actual sample processing: Weigh 2g of frozen chicken sample and place it in a 15mL glass bottle, add 2mL of deionized water containing 100mmol / L HCl, and place it at room temperature for 2h to obtain the sample solution to be tested;

[0065] Actual sample detection: Take 100 μL of the sample solution to be tested, add the following reagents in sequence: 50 μL of 0.6 mg / mL Cu-Cy / GO nanozyme, 100 μL of 10 mmol / L hydrogen peroxide, 100 μL of 5 mmol / L TMB, dilute to 1 mL with pH 7 Tris-HCl buffer solution, shake well and let stand for 15 min; measure the absorbance A at 652 nm, bring it into the formaldehyde detection standard curve in Example 3, and calculate the formaldehyde content in the frozen chicken sample to be 1.70 mg / L;

[0066] Actual sample spike detection: Three different concentrations of formaldehyde standard solutions were added to the sample solution to be tested; each concentration was measured three times in parallel, the spike recovery rate was calculated, and the relative standard deviation RSD was calculated. The results are shown in Table 3:

[0067] Table 3 Recovery and RSD of spiked frozen chicken samples (n=3)

[0068]

[0069] (All samples were diluted 20 times in the detection system)

[0070] The recoveries of formaldehyde added to the samples were found to be between 98.00% and 102.50%, and the RSDs were between 1.97% and 3.25%.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a copper-based compound / graphene oxide nanozyme, characterized in that: The following steps are involved: Synthesis of two-dimensional copper-based compounds: Dissolve cysteamine hydrochloride and copper chloride dihydrate in deionized water, stir under nitrogen protection, adjust pH, transfer the mixed solution to an oil bath, continue stirring, collect the precipitate by centrifugation, wash and dry to obtain a two-dimensional copper-based compound; Construction of composite nanozyme: The two-dimensional copper-based compound and graphene oxide are separately dispersed in ultrapure water to prepare a dispersion, and then the two-dimensional copper-based compound dispersion is mixed with the graphene oxide dispersion. After stirring at room temperature, the mixture is centrifuged and dried to obtain a composite nanozyme in which the copper-based compound is loaded on the surface of graphene oxide.

2. The method for preparing the copper-based compound / graphene oxide nanozyme according to claim 1, characterized in that: In the step of synthesizing the two-dimensional copper-based compound, the molar ratio of cysteamine hydrochloride to copper chloride is 1:1-1.5:

1.

3. The method for preparing the copper-based compound / graphene oxide nanozyme according to claim 1, characterized in that: In the step of synthesizing the two-dimensional copper-based compound, the pH is 7.0-8.

0.

4. The method for preparing the copper-based compound / graphene oxide nanozyme according to claim 1, characterized in that: In the step of synthesizing the two-dimensional copper-based compound, the temperature of the oil bath is 85-95°C.

5. The method for preparing the copper-based compound / graphene oxide nanozyme according to claim 1, characterized in that: In the step of constructing the composite nanozyme, the concentration of the two-dimensional copper-based compound dispersion is 1.0-1.5 mg / mL, the concentration of the graphene oxide dispersion is 0.1-0.15 mg / mL, and the volume ratio of the two-dimensional copper-based compound dispersion to the graphene oxide dispersion is 1:5-3:

1.

6. A copper-based compound / graphene oxide nanozyme, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 5.

7. An application of the copper-based compound / graphene oxide nanozyme as claimed in claim 6 in formaldehyde detection.

8. The use according to claim 7, characterized in that: The formaldehyde detection comprises the following steps: Establishment of standard curve: In a pH 7.0 buffer system, the copper-based compound / graphene oxide nanozyme, hydrogen peroxide, tetramethylbenzidine and formaldehyde standard solution were added in sequence, and after the reaction, the absorbance difference ΔA at 652 nm was measured to establish a quantitative relationship between ΔA and formaldehyde concentration; Actual sample testing: Add the test solution according to the above system and calculate the formaldehyde content according to the standard curve.

9. The use according to claim 8, characterized in that: The concentration of the copper-based compound / graphene oxide nanozyme is 0.015-0.03 mg / mL, the concentration of hydrogen peroxide is 0.5-2 mmol / L, the concentration of tetramethylbenzidine is 0.1-1 mmol / L, and formaldehyde inhibits the oxidation reaction of tetramethylbenzidine by competitively capturing ·OH, thereby generating a change in the colorimetric signal.

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