Nanometer enzyme activity tetracycline specific sensor as well as preparation method and application thereof

Abstract

The invention relates to the field of pesticide and veterinary drug residue and food safety detection, and discloses a preparation method and application of a nano-enzyme activity tetracycline specific sensor. The method comprises the following steps: at 20-35 DEG C, carrying out first mixing on an Ag-coated NH2-MIL-101 (Al) probe solution and an AgNPs solution according to a dosage volume ratio of 1: (1.8-2.3) for 8-12 hours, and then carrying out centrifugal washing to obtain a nano-enzyme activity tetracycline specific sensor; the method provided by the invention can be used for preparing the nano-enzyme activity tetracycline specific sensor with stable catalytic activity and Raman enhancement performance at the same time, can realize high-sensitivity, high-specificity and high-accuracy dual-mode qualitative and quantitative detection of tetracycline through the action with tetracycline, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of pesticide and veterinary drug residues and food safety detection, and in particular to a nanoenzyme active tetracycline specific sensor and a preparation method and application thereof. Background Art

[0002] Antibiotics are currently the main method for treating bacterial infections in animal husbandry and clinical medicine around the world and have been widely used. However, tetracycline can accumulate in the body through the food chain, and even low doses may have harmful effects on human health. Therefore, it is necessary to establish a sensitive, accurate and efficient tetracycline analysis technology. However, traditional detection methods have high instrument costs, complex pretreatment steps and long time consumption. At present, most rapid detection methods for tetracycline use single reading technology, and slight changes in the detection environment, instruments and operations can easily affect the reliability of the results. Therefore, compared with single-mode detection methods, dual-mode detection has more advantages.

[0003] The inventor's prior art document "Food Chemistry" No. 2024, Volume 435, Page 137586, "A fluorescence and SERS dual-mode sensing on tetracycline antibiotics based on Ag@NH2-MIL-101(Al) nanoprobe" reported a tetracycline antibiotic fluorescence and SERS dual-mode sensing based on Ag@NH2-MIL-101(Al) nanoprobe. The article describes in detail the application of Ag@NH2-MIL-101(Al) nanoprobe in fluorescence and SERS dual-mode detection (see Junjie Qi, et al. (2024). Food Chemistry, 435, Article 137586). However, this technology has the problem that the SERS signal is easily interfered by the fluorescence signal and the SERS signal is weak.

[0004] Based on this, it is of great significance to develop a tetracycline-specific sensor with the characteristics of rapidity, sensitivity and low detection limit, as well as a method for detecting tetracycline using the same. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems in the prior art of detecting tetracycline antibiotics based on Ag@NH2-MIL-101(Al) nanoprobe that the SERS signal is easily interfered by the fluorescence signal and the SERS signal is weak.

[0006] In order to achieve the above object, the first aspect of the present invention provides a method for preparing a nanoenzyme activity tetracycline specific sensor, the method comprising:

[0007] At 20-35°C, the Ag@NH2-MIL-101(Al) probe solution and the AgNPs solution were first mixed at a volume ratio of 1:1.8-2.3 for 8-12 hours, and then centrifuged and washed to obtain a nanozyme active tetracycline specific sensor;

[0008] The AgNPs solution is a product synthesized by a citric acid method;

[0009] The Ag@NH2-MIL-101(Al) probe solution is a product obtained by a first contact reaction between NH2-MIL-101(Al) and silver nitrate and trisodium citrate.

[0010] The second aspect of the present invention provides a nanozyme activity tetracycline-specific sensor prepared by the method described in the first aspect.

[0011] The third aspect of the present invention provides the use of the nanoenzyme active tetracycline-specific sensor described in the second aspect in detecting the presence of tetracycline and / or detecting the content of tetracycline, which is carried out by colorimetric-surface enhanced Raman dual-mode signal detection.

[0012] Compared with the prior art, the method provided by the present invention has at least the following beneficial effects:

[0013] (1) The nanozyme-active tetracycline-specific sensor (Ag@NH2-MIL-101(Al)-Ag) provided by the present invention has both stable catalytic activity and Raman enhancement performance, and can achieve high sensitivity, high specificity and high accuracy of dual-mode qualitative and quantitative detection of tetracycline by acting with tetracycline. The nanozyme-active tetracycline-specific sensor not only has significant Raman enhancement performance, but also has excellent peroxidase-like activity, and can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized 3,3',5,5'-tetramethylbenzidine (ox-TMB). Since the presence of tetracycline inhibits the oxidation of TMB by Ag@NH2-MIL-101(Al)-Ag to generate ox-TMB, TMB itself can be used as a chromogen reagent and Raman label molecule, and the tetracycline residues in food can be indirectly qualitatively and quantitatively detected by colorimetric-surface enhanced Raman dual mode, and the nanozyme-active tetracycline-specific sensor has low cost and great application potential;

[0014] (2) In the application provided by the present invention, the detection of as low as 2.12×10 -3 ppm of tetracycline, and can be 7.63×10 -3 A linear detection model was built in the range of -1.53ppm, and the detection range was as low as 1.11×10 -5ppm of tetracycline, and can be 2.11×10 -5 -2.11×10 -2 A linear detection model was constructed in the ppm range, thus providing a new and feasible method for trace detection of tetracycline in complex systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A scanning electron microscope image of NH2-MIL-101 (Al) in a preferred embodiment provided by the present invention;

[0016] Figure 2 A scanning electron microscope image of a nanoenzyme-active tetracycline-specific sensor prepared in a preferred embodiment of the present invention;

[0017] Figure 3 A comparison diagram of the effect of tetracycline on the color change of the reaction system in a preferred embodiment provided by the present invention;

[0018] Figure 4 A surface enhanced Raman spectra of a blank group and a tetracycline sample solution group containing tetracycline in a preferred embodiment provided by the present invention.

[0019] Figure 5 The surface enhanced Raman spectra of the blank group and the tetracycline sample solution group containing tetracycline in comparative application example 2;

[0020] Figure 6 This is a comparison chart of the effect of tetracycline on the color change of the reaction system in Application Example 3. DETAILED DESCRIPTION

[0021] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] As mentioned above, the first aspect of the present invention provides a method for preparing a nanoenzyme activity tetracycline-specific sensor, the method comprising:

[0023] At 20-35°C, the Ag@NH2-MIL-101(Al) probe solution and the AgNPs solution were first mixed at a volume ratio of 1:1.8-2.3 for 8-12 hours, and then centrifuged and washed to obtain a nanozyme active tetracycline specific sensor;

[0024] The AgNPs solution is a product synthesized by a citric acid method;

[0025] The Ag@NH2-MIL-101(Al) probe solution is a product obtained by a first contact reaction between NH2-MIL-101(Al) and silver nitrate and trisodium citrate.

[0026] Preferably, the first mixing is performed under shaking conditions with a rotation speed of 600-1000 rpm.

[0027] According to a preferred embodiment, the Ag@NH2-MIL-101(Al) probe solution is a product prepared by the first contacting method comprising the following steps:

[0028] S1. Add 0.50-0.53 g of aluminum chloride hexahydrate and 0.54-0.58 g of 2-aminoterephthalic acid to 15 mL of solvent, dissolve and mix by ultrasonication, transfer to a polytetrafluoroethylene-lined autoclave, and then heat at 130° C. for 70-74 h. After cooling to 20-28° C., wash with anhydrous ethanol and water three times respectively, and then vacuum dry at 60° C. for 11-13 h to obtain NH2-MIL-101 (Al);

[0029] S2. Add 10-15 mg of the NH2-MIL-101 (Al) to 10 mL of water, fully suspend under ultrasonic conditions, then add 5 mL of a 12-17 mM silver nitrate aqueous solution, stir the resulting mixed solution at 45 ° C for 2.5-3 h, then heat to intense boiling, then quickly add 200-230 μL of trisodium citrate with a mass fraction of 10-12 wt%, and continue stirring under boiling conditions for 15-25 min to obtain the Ag@NH2-MIL-101 (Al) probe solution without dilution.

[0030] Preferably, the solvent is N,N-dimethylformamide.

[0031] Preferably, the AgNPs solution is a product obtained by subjecting a trisodium citrate solution to a second contact reaction with a silver nitrate solution, comprising the following steps:

[0032] 1.8-2 mL of 1 wt% trisodium citrate solution was added to 100 mL of boiling 1 mmol / L silver nitrate solution, and then boiled for 60-70 min at a stirring speed of 600-800 rpm, and concentrated 10 times to obtain the AgNPs solution.

[0033] As mentioned above, the second aspect of the present invention provides a nanozyme activity tetracycline-specific sensor prepared by the method described in the first aspect.

[0034] As mentioned above, the third aspect of the present invention provides the use of the nanoenzyme active tetracycline-specific sensor described in the second aspect in detecting the presence of tetracycline and / or detecting the content of tetracycline, which is carried out by colorimetric-surface enhanced Raman dual-mode signal detection.

[0035] Preferably, when the nanoenzyme activity tetracycline-specific sensor is used to detect the presence of tetracycline, the application comprises the following steps:

[0036] The test solution I was dripped onto the nanoenzyme activity tetracycline specific sensor, and its ultraviolet absorption spectrum and Raman spectrum were collected, with the absorbance at 652±3nm and 1601±5cm -1 Tetracycline is detected by Raman peak at 652±3nm. If there is a UV absorption peak at 1601±5cm -1 If a surface enhanced Raman peak appears at , it means that tetracycline is not present in the test liquid I; otherwise, it means that tetracycline is present in the test liquid I.

[0037] Preferably, when the nanoenzyme activity tetracycline-specific sensor is used to detect tetracycline content, the application comprises the following steps:

[0038] (1) 5-10 tetracycline samples of known concentration were added to the nanoenzyme active tetracycline specific sensor, and then 15 μL of 10 mmol / L 3,3',5,5'-tetramethylbenzidine and 2.5 μL of hydrogen peroxide solution were added to react for 5 min, and then the UV absorption spectrum and Raman spectrum were collected respectively, and then the absorbance at 652±3 nm and 1601±5 cm -1 A linear relationship is established between the Raman peak intensity at and the concentration of tetracycline, which is the detection model;

[0039] (2) Then, the tetracycline content in the test solution II is determined according to the detection model.

[0040] Preferably, the concentration of the hydrogen peroxide solution is 30 wt %.

[0041] Preferably, the parameters for collecting the Raman spectrum include at least: a laser wavelength of 785 nm, an exposure time of 4.55 milliseconds, and a scanning number of 1000 times.

[0042] The present invention is described in detail below by way of examples. Unless otherwise specified, the raw materials used are all common commercially available products.

[0043] Unless otherwise specified, the "room temperature" or "normal temperature" mentioned in the present invention means a temperature of 25±5°C.

[0044] Preparation Example

[0045] Preparation of Ag@NH2-MIL-101(Al) probe solution:

[0046] S1. Add 0.51 g aluminum chloride hexahydrate and 0.56 g 2-aminoterephthalic acid to 15 mL of solvent, dissolve and mix by ultrasonication, transfer to a polytetrafluoroethylene-lined autoclave, and then heat at 130 ° C for 72 h. After cooling to room temperature, wash with anhydrous ethanol and water three times respectively, and then vacuum dry at 60 ° C for 12 h to obtain NH2-MIL-101 (Al). Scan the electron microscope for the results. See Figure 1 ;

[0047] S2. Add 15 mg of the NH2-MIL-101 (Al) to 10 mL of water, fully suspend under ultrasonic conditions, then add 5 mL of a 15 mM silver nitrate aqueous solution, stir the resulting mixed solution at 45 ° C for 2.5 h, and then heat to intense boiling. Subsequently, quickly add 220 μL of trisodium citrate with a mass fraction of 12 wt%, and continue stirring under boiling conditions for 20 min. The Ag@NH2-MIL-101 (Al) probe solution is obtained without dilution.

[0048] Synthesis of AgNPs solution by citric acid method:

[0049] 2 mL of 1 wt% trisodium citrate solution was added to 100 mL of boiling 1 mmol / L silver nitrate solution, and then boiled at a stirring speed of 600 rpm for 60 min. The resulting solution was concentrated 10 times to obtain an AgNPs solution.

[0050] Example 1

[0051] This example is used to illustrate a preferred method for preparing a nanozyme activity tetracycline specific sensor provided by the present invention, which is performed according to the following steps:

[0052] At 25°C, the Ag@NH2-MIL-101(Al) probe solution obtained in the preparation example and the AgNPs solution were first mixed by oscillation at a volume ratio of 1:2 for 10 h at a speed of 800 rpm, and then centrifuged and washed to obtain a nanoenzyme active tetracycline specific sensor named P1. The nanoenzyme was subjected to electron microscopy scanning. The results are shown in Figure 2 .

[0053] Comparative Example 1

[0054] In this comparative example, the Ag@NH2-MIL-101(Al) probe solution obtained in the preparation example was directly used as a nanozyme active tetracycline-specific sensor and named DP1.

[0055] Comparative Example 2

[0056] This comparative example was carried out using a similar method to Example 1, except that the volume ratio of the Ag@NH2-MIL-101 (Al) probe solution to the AgNPs solution was 1:2.5; and finally a nanozyme active tetracycline-specific sensor was obtained, named DP2.

[0057] Application Example 1

[0058] This application example is used to illustrate the application of a preferred nanoenzyme activity tetracycline-specific sensor provided by the present invention in detecting the presence of tetracycline. The application is performed according to the following steps:

[0059] The test solution A without tetracycline and the test solution B containing tetracycline were respectively dripped onto the nanozyme active tetracycline specific sensor P1, numbered as group A and group B, and then the ultraviolet absorption spectra and Raman spectra of the two groups were collected respectively. The parameters for collecting Raman spectra included: the laser wavelength was 785nm, the exposure time was 4.55 milliseconds, and the number of scans was 1000 times; the absorbance at 652nm and 1601cm -1 Tetracycline is detected by the Raman peak at;

[0060] The results showed that group A had an ultraviolet absorption peak at 652nm and a peak at 1601cm -1 A surface enhanced Raman peak appears at ; this result indicates that the test solution A does not contain tetracycline, which is consistent with the actual situation of group A;

[0061] However, there was no UV absorption peak at 652±3nm in group B, and the peak at 1601±5cm -1 No surface enhanced Raman peak appeared at , indicating that the test solution B contained tetracycline, and this result was consistent with the actual situation of group B.

[0062] Application Example 2

[0063] This application example is used to illustrate the application of a preferred nanoenzyme activity tetracycline specific sensor provided by the present invention in detecting tetracycline content. The application adopts a colorimetric-surface enhanced Raman dual-mode signal detection method and is performed according to the following steps:

[0064] (1) Seven tetracycline samples of known concentrations were added to the nanozyme active tetracycline specific sensor P1, and then 15 μL of 3,3',5,5'-tetramethylbenzidine with a concentration of 10 mmol / L and 2.5 μL of hydrogen peroxide solution were added to react for 5 min. At the same time, a control group with a tetracycline sample concentration of 0 was set up, and the ultraviolet absorption spectra and Raman spectra of all groups were collected. The parameters for collecting Raman spectra included: laser wavelength of 785 nm, exposure time of 4.55 milliseconds, and scanning times of 1000 times; the absorbance at 652 nm and 1601 cm -1 A linear relationship is established between the Raman peak intensity at and the concentration of tetracycline, which is the detection model (including the colorimetric model and the surface enhanced Raman model);

[0065] (2) Based on the detection model, the tetracycline contents in three groups of test solutions with concentrations ranging from 0.08 to 1.53 ppm were measured respectively. The results showed that the deviation between the detection results and the actual tetracycline contents in the test solutions was only between 0.25 and 5.01.

[0066] in, Figure 3 A comparison of the effect of tetracycline on the color change of the reaction system is shown in Figure 3 In the figure, the mixture in the left cup is a photo of the system after the tetracycline sample solution containing tetracycline reacted with 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution, and the mixture in the right cup is a photo of the system after the control group reacted with 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide solution. It can be seen that the color of the mixture in the left cup is obviously darker than that on the right, which confirms that the presence of tetracycline can inhibit the color change of TMB, and as the concentration of tetracycline continues to decrease, the absorbance value gradually increases and approaches the blank.

[0067] The detection limit of tetracycline in this colorimetric model is 2.12×10 -3 ppm; and when the tetracycline concentration is 7.63×10 -3 In the range of -1.53ppm, the absorbance value at 652nm showed a good linear relationship with the tetracycline concentration, and the correlation coefficient (R2) of the regression model could reach 0.99.

[0068] Figure 4 The surface enhanced Raman spectra of the blank group (no tetracycline added) and the tetracycline sample solution group containing tetracycline (tetracycline added) are shown. Figure 4 It can be seen that the blank group is at 1601cm -1 The characteristic peak intensity at is significantly stronger than that of the sample group containing tetracycline.

[0069] The results also show that as the concentration of tetracycline increases, the intensity of the characteristic peak gradually weakens. The calculation shows that the qualitative detection limit of tetracycline by this surface enhanced Raman model is 1.11×10 -5 ppm, and when the tetracycline concentration is 2.11×10 -5 -2.11×10 -2 In the ppm range, 1601 cm -1 There is a linear relationship between the peak intensity value and the tetracycline concentration. The correlation coefficient (R 2 ) can reach 0.99.

[0070] Comparative application example 1

[0071] This comparative application example is carried out in a similar manner to that of application example 2, except that in step (1), 1391 cm -1 A linear relationship was established between the Raman peak intensity at and the concentration of tetracycline. It was finally found that the tetracycline prediction energy of the surface enhanced Raman spectroscopy model obtained under this condition was low, and there was a problem of strong interference from the fluorescence background.

[0072] Comparative Application Example 2

[0073] This comparative application example is carried out using a method similar to that of application example 2, except that in step (1), the nanozyme active tetracycline-specific sensor used is DP1;

[0074] Figure 5 The surface enhanced Raman spectra of the blank group (DP1 sensor) and the tetracycline sample solution group containing tetracycline (tetracycline added) are shown, wherein the concentration of the tetracycline sample solution group containing tetracycline is Figure 4 The same as in Figure 5 It can be seen that when the Ag@NH2-MIL-101(Al) probe solution obtained in the preparation example is directly used as the nanozyme active tetracycline-specific sensor, there is a problem of weak surface enhanced Raman spectroscopy signal, and the final detection model cannot accurately predict the tetracycline concentration under spectral measurement, and it is difficult to respond to tetracycline solutions at low concentrations.

[0075] Comparative Application Example 3

[0076] This comparative application example is carried out using a method similar to that of application example 2, except that in step (1), the nanozyme active tetracycline-specific sensor used is DP2;

[0077] Figure 6 A comparison of the effect of tetracycline on the color change of the reaction system is shown in Figure 6 The mixture in the left cup is a tetracycline sample solution containing tetracycline (with Figure 3The final results show that when the amount of AgNPs solution is too large, it cannot be fully loaded onto Ag@NH2-MIL-101(Al), and the excess AgNPs will cause the color development of the test solution to be insensitive, and the final detection model cannot accurately detect the content of tetracycline at low concentrations.

[0078] It can be seen from the above results that the method provided by the present invention can prepare a nanozyme active tetracycline-specific sensor with stable catalytic activity and Raman enhancement performance, which can achieve high-sensitivity, high-specificity and high-accuracy dual-mode qualitative and quantitative detection of tetracycline through the action with tetracycline, and has good application prospects.

[0079] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for preparing a nanozyme activity tetracycline-specific sensor, characterized in that: The method includes: At 20-35°C, the Ag@NH2-MIL-101(Al) probe solution and the AgNPs solution were first mixed at a volume ratio of 1:1.8-2.3 for 8-12 hours, and then centrifuged and washed to obtain a nanozyme active tetracycline specific sensor; The AgNPs solution is a product synthesized by a citric acid method; The Ag@NH2-MIL-101(Al) probe solution is a product obtained by a first contact reaction between NH2-MIL-101(Al) and silver nitrate and trisodium citrate.

2. The method according to claim 1, wherein: The first mixing is performed under shaking conditions with a rotation speed of 600-1000 rpm.

3. The method according to claim 1 or 2, wherein: The AgNPs solution is a product obtained by a second contact reaction between a trisodium citrate solution and a silver nitrate solution, and comprises the following steps: 1.8-2 mL of 1 wt% trisodium citrate solution was added to 100 mL of boiling 1 mmol / L silver nitrate solution, and then boiled for 60-70 min at a stirring speed of 600-800 rpm, and concentrated 10 times to obtain the AgNPs solution.

4. A nanozyme-active tetracycline-specific sensor prepared by the method described in any one of claims 1 to 3.

5. The use of the nanoenzyme activity tetracycline specific sensor according to claim 4 in detecting the presence of tetracycline and / or detecting the content of tetracycline, characterized in that: This application is carried out using a colorimetric-surface enhanced Raman dual-mode signal detection method.

6. The use according to claim 5, wherein: When the nanoenzyme-active tetracycline-specific sensor is used to detect the presence of tetracycline, the application comprises the following steps: The test solution I was dripped onto the nanoenzyme activity tetracycline specific sensor, and its ultraviolet absorption spectrum and Raman spectrum were collected, with the absorbance at 652±3nm and 1601±5cm -1 Tetracycline is detected at the Raman peak. If a UV absorption peak appears at 652±3nm and a peak at 1601±5cm -1 If a surface enhanced Raman peak appears at , it means that tetracycline is not present in the test liquid I; otherwise, it means that tetracycline is present in the test liquid I.

7. The use according to claim 5, wherein: When the nanoenzyme-active tetracycline-specific sensor is used to detect tetracycline content, the application includes the following steps: (1) 5-10 tetracycline samples of known concentration were added to the nanoenzyme active tetracycline specific sensor, and then 15 μL of 10 mmol / L 3,3',5,5'-tetramethylbenzidine and 2.5 μL of hydrogen peroxide solution were added to react for 5 min, and then the UV absorption spectrum and Raman spectrum were collected respectively, and then the absorbance at 652±3 nm and 1601±5 cm -1 A linear relationship is established between the Raman peak intensity at and the concentration of tetracycline, which is the detection model; (2) Then, the tetracycline content in the test solution II is determined according to the detection model.

8. The use according to claim 7, wherein: The concentration of the hydrogen peroxide solution is 30 wt %.

9. The use according to claim 6 or 7, wherein: The parameters for collecting the Raman spectrum include at least: a laser wavelength of 785 nm, an exposure time of 4.55 milliseconds, and a scanning number of 1000 times.

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