An Electrochemical Detection Method for Tetrodotoxin Based on Ag@Cuprous Oxide Nanomaterials
Through the electrochemical detection method of Ag@ cuprous oxide nanomaterials combined with tetrodotoxin aptamers, the problem of time-consuming and expensive traditional detection methods is solved, and the rapid and accurate tetrodotoxin detection is achieved, with a detection limit of 3.5pg/mL, which is suitable for food safety and health testing.
Patent Information
- Application Number
- CN202211296064.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-21
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2042-10-21
AI Technical Summary
The traditional twofish toxin detection method is time-consuming, expensive and requires professional instruments, which is difficult to achieve on-site detection, and the detection sensitivity and accuracy are insufficient.
An electrochemical detection method is used to bind Ag@ cuprous oxide nanomaterials with tetrodotoxin aptamers, and a stable nanoprobe is formed through Ag-SH covalent bonds, modified on the electrode surface, and quantitative detection is performed using DPV signals.
It realizes fast and accurate twobushi toxin detection, with a detection limit of 3.5pg/mL, improving the reproducibility and sensitivity of the detection, and is suitable for food safety and health testing.
Smart Images
Figure CN115791915B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of electrochemical analysis, and particularly relates to a method for electrochemically detecting tetrodotoxin based on Ag@cuprous oxide nanomaterials. Background Art
[0002] Pufferfish and certain marine animals contain the potent and deadly neurotoxin tetrodotoxin (TTX). Consumption of food accumulated with TTX by humans and other animals can cause neuromuscular paralysis, respiratory failure, and even death, even at low doses. Therefore, the development of sensitive and accurate TTX detection techniques is crucial for food safety and human health.
[0003] Traditional methods for detecting TTX include chromatography, colorimetry, fluorescence, surface-enhanced Raman scattering, and electrochemical analysis. These traditional detection techniques are usually time-consuming, expensive, and often require professionals or complex instruments, which greatly limits their wide application and makes on-site detection difficult. Electrochemical methods have the advantages of sensitive detection, easy operation, and low cost, and have been widely used in the analysis and sensing of TTX. Therefore, it is particularly important to construct a new electrochemical-based TTX detection method. Summary of the Invention
[0004] To solve the above technical problems, the present invention provides a method for electrochemically detecting TTX based on Ag@cuprous oxide nanomaterials. A nanoprobe with good and stable electrochemical signals is the basis for realizing electrochemical detection. As an electroactive beacon, Ag@Cu2O has a stable oxidation peak, which can effectively reduce the interference caused by environmental factors to the detection. Therefore, a dual-signal detection method is used to quantitatively detect TTX, improving the reproducibility of the measurement results and achieving the purpose of accurate determination.
[0005] The first object of the present invention is to provide a method for electrochemically detecting TTX based on Ag@cuprous oxide nanomaterials, comprising the following steps:
[0006] S1: Mix an Ag NPs solution with a surfactant and a copper salt, then add a reducing agent and stir to react to obtain Ag@Cu2O nanomaterials;
[0007] S2: Connect the Ag@Cu2O nanomaterials in step S1 with a tetrodotoxin aptamer, and then mix with MXenes (two-dimensional carbides and nitrides) to form an assembly; the interaction between the thiol group in the tetrodotoxin aptamer and Ag is connected by an Ag-SH covalent bond;
[0008] S3: Modify the assembly on the surface of the electrode, drop the test solution onto the surface of the electrode, and then drop Nafion solution to obtain a modified electrode; the modification method: polish the glassy carbon electrode (GCE) with aluminum oxide polishing powder with a particle size of 0.05 μm, and clean it with ethanol and ultrapure water respectively. Then drop 10 μL of Ag@Cu2O nanomaterial on the surface of the MGCE and dry it at room temperature;
[0009] S4: Detect the DPV signal intensity of the modified electrode to achieve quantitative or qualitative electrochemical detection of tetrodotoxin.
[0010] In one embodiment of the present invention, in step S1, the mass ratio of Ag NPs to the surfactant in the Ag NPs solution is 1:3 to 1:5.
[0011] In one embodiment of the present invention, in step S1, the surfactant is selected from polyvinylpyrrolidone and / or cetyltrimethylammonium bromide. The surfactant can protect the colloid and prevent the aggregation of nanomaterials.
[0012] In one embodiment of the present invention, in step S1, the reducing agent is selected from one or more of sodium borohydride, ascorbic acid, and N2H4·H2O.
[0013] In one embodiment of the present invention, in step S1, the mass-volume concentration ratio of the copper salt to the reducing agent is 1:1 to 1:2; the stirring reaction time is 2 min to 10 min.
[0014] In one embodiment of the present invention, in step S1, the copper salt is selected from one or more of copper nitrate, copper sulfate, and copper chloride.
[0015] In one embodiment of the present invention, in step S2, the nucleotide sequence of the tetrodotoxin aptamer is SH-TCAAATTTTCGTCTACTCAATCTTTCTGTCTTATC.
[0016] In one embodiment of the present invention, in step S2, the Ag@Cu2O nanomaterial is first incubated with the tetrodotoxin aptamer for 10 h to 14 h, and then mixed with MXenes for 10 h to 14 h to form an assembly.
[0017] In one embodiment of the present invention, in step S3, the electrode is selected from a glassy carbon electrode, a titanium electrode, or a Cu electrode.
[0018] In one embodiment of the present invention, in step S3, the concentration of tetrodotoxin in the test solution is 10 pg / mL to 10 μg / mL.
[0019] In one embodiment of the present invention, in step S3, the mass concentration of the Nafion solution is 0.5% to 1%. After the Nafion solution is dried, a polymer membrane is formed, and the Au-Ag@MnO2 nanomaterial is fixed on the electrode surface to prevent the material from falling off during the electrochemical detection process. Specific steps: 5 μL of 0.5 wt% Nafion solution is dropped on the surface of the GCE electrode and dried at room temperature.
[0020] In one embodiment of the present invention, in step S4, the method for preparing the standard curve in quantitative detection is as follows: A series of test solutions containing tetrodotoxin with known concentrations are dropped on the surface of the electrode after being modified by the assembly, and then the DPV signal intensity value of the electrode is detected. Taking the logarithm of the concentration of tetrodotoxin as the abscissa and the DPV signal intensity value of the Ag@Cu2O nanomaterial as the ordinate, the standard curve for quantitative detection is obtained.
[0021] In one embodiment of the present invention, the concentration of the tetrodotoxin-containing solution with a known concentration is 10 pg / mL to 10 μg / mL.
[0022] The above technical solutions of the present invention have the following advantages compared with the prior art:
[0023] The method provided by the present invention can detect the concentration of TTX in the sample through an electrochemical method, making the detection more convenient. A linear relationship between the electrochemical oxidation current intensity and the logarithm of the TTX concentration is established, improving the accuracy of the detection. The TTX electrochemical detection method of the Ag@Cu2O nanomaterial in the present invention is expected to detect the concentration of TTX in terms of life and health, and has a very broad application prospect. In the detection method described in the present invention, the detection limit of Cu is 3.5 pg / mL, the detection limit of Ag is 4.4 pg / mL, and the quantification limit is 10 pg / mL. Description of the Drawings
[0024] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention and in combination with the drawings, where
[0025] Figure 1 is the transmission electron microscope image of the Ag@Cu2O nanomaterial in the second embodiment of the present invention.
[0026] Figure 2 is the DPV signal and standard curve of Ag@Cu2O in the presence of different concentrations of TTX in the second embodiment of the present invention.
[0027] Figure 3 is the specific detection result of the test example of the present invention. Detailed Description of the Invention
[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.
[0029] Example 1
[0030] 1. Preparation of Ag@Cu2O-MXenes nanomaterials:
[0031] First, Ag NPs were synthesized by the citrate oxidation method. 8 mg of AgNO3 was added to 80 mL of ultrapure water and heated to boiling. 1.6 mL of 1% (w / v) sodium citrate solution was quickly added, and boiling was maintained for 30 min. After centrifugation, the solution was redispersed in 40 mL of water. 4 mL of Ag NPs was dissolved in 16 mL of ultrapure water, 0.1 g of polyvinylpyrrolidone K30 and 5 mL of 1 wt% Cu(NO3)2 were added, 120 μL of 1 wt% N2H4·H2O solution was quickly added, and the mixture was stirred for 2 min. After centrifugation, Ag@Cu2O NPs were redispersed in 1 mL of water. The Ag@Cu2O NPs solution was mixed with the TTX aptamer for 10 h, then mixed with MXenes NSs and allowed to stand for 10 h to obtain an assembly. Finally, the assembly was centrifuged and dissolved in 1 mL of water.
[0032] 2. Establishment of the TTX electrochemical analysis method
[0033] 5 μL of the assembly was modified on a glassy carbon electrode (GCE). Then, mixed solutions of different concentrations of TTX (10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL) standard samples were respectively dropped onto the surface of the modified electrode. Nafion solution was dropped, and a modified electrode was obtained. Then, 5 μL of 0.5% Nafion solution was dropped. After drying at room temperature, its DPV signal was measured using an electrochemical workstation to obtain a standard curve with the logarithm of the TTX concentration as the abscissa and the oxidation current of the Ag@Cu2O NPs nanomaterial as the ordinate.
[0034] Example 2
[0035] 1. Preparation of Ag@Cu2O-MXenes nanomaterials:
[0036] First, Ag NPs were synthesized by the citrate oxidation method. 9 mg of AgNO3 was added to 90 mL of ultrapure water and heated to boiling. 1.8 mL of 1% (w / v) sodium citrate solution was quickly added, and boiling was maintained for 40 min. After centrifugation, the solution was redispersed in 45 mL of water. 8 mL of Ag NPs was dissolved in 32 mL of ultrapure water, 0.2 g of polyvinylpyrrolidone K30 and 10 mL of 1 wt% Cu(NO3)2 were added. 160 μL of 1.5 wt% N2H4·H2O solution was quickly added, and the mixture was stirred for 6 min. After centrifugation, Ag@Cu2O NPs were redispersed in 1 mL of water. After mixing the Ag@Cu2O NPs solution with the TTX aptamer for 12 h, it was then mixed with MXenes NSs and allowed to stand for 12 h to obtain an assembly. Finally, the assembly was centrifuged and dissolved in 1 mL of water.
[0037] 2. Establishment of the TTX electrochemical analysis method
[0038] 5 μL of the assembly was modified on a glassy carbon electrode (GCE). Then, mixed solutions of different concentrations of TTX (10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL) standard samples were respectively dropped onto the surface of the modified electrode. Nafion solution was dropped, and a modified electrode was obtained. Then, 5 μL of 0.75% Nafion solution was dropped. After air-drying at room temperature, its DPV signal was measured using an electrochemical workstation to obtain a standard curve with the logarithm of the TTX concentration as the abscissa and the oxidation current of the Ag@Cu2O NPs nanomaterial as the ordinate. The experimental results are shown in Figure 2 , as can be seen from the figure, in the presence of TTX, the two signals of the electrochemical sensor linearly increase with the increase in the logarithm of the TTX concentration. This sensor can be used for the detection application of quantitative analysis of TTX.
[0039] Example 3
[0040] 1. Preparation of Ag@Cu2O-MXenes nanomaterials:
[0041] First, Ag NPs were synthesized by the citrate oxidation method. 10 mg of AgNO3 was added to 100 mL of ultrapure water and heated to boiling. Then, 2 mL of 1% (w / v) sodium citrate solution was quickly added, and boiling was maintained for 50 min. After centrifugation, the solution was redispersed in 50 mL of water. 12 mL of Ag NPs was dissolved in 48 mL of ultrapure water, 0.3 g of polyvinylpyrrolidone K30 and 15 mL of 1 wt% Cu(NO3)2 were added. Then, 180 μL of 2 wt% N2H4·H2O solution was quickly added, and the mixture was stirred for 10 min. After centrifugation, Ag@Cu2O NPs were redispersed in 1 mL of water. After mixing the Ag@Cu2O NPs solution with the TTX aptamer for 14 h, it was then mixed with MXenes NSs and allowed to stand for 14 h to obtain the assembly. Finally, the assembly was centrifuged and dissolved in 1 mL of water.
[0042] 2. Establishment of the TTX electrochemical analysis method
[0043] 5 μL of the assembly was modified on a glassy carbon electrode (GCE). Then, mixed solutions of different concentrations of TTX (10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL) standard samples were respectively dropped onto the surface of the modified electrode. Nafion solution was dropped, and a modified electrode was obtained. Then, 5 μL of 1% Nafion solution was dropped. After drying at room temperature, its DPV signal was measured using an electrochemical workstation to obtain a standard curve with the logarithm of the TTX concentration as the abscissa and the oxidation current of the Ag@Cu2O NPs nanomaterial as the ordinate.
[0044] Specific detection
[0045] To further evaluate the selectivity of the constructed electrochemical sensor for detecting TTX, the Ag@Cu2O nanomaterial was modified on a glassy carbon electrode, and a 1 μg / mL TTX solution and interfering solutions with a concentration of 10 μg / mL (CaCl2, MgCl2, Lysine (Lys), ascorbic acid (AA), Folate (Fol), Arginine (Arg), and phenylalanine (Phe)) were respectively measured. The results are shown in Figure 3 , and it can be seen from Figure 3 that the sensor has strong selectivity for TTX.
[0046] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.
Claims
1. An electrochemical detection method for tetrodotoxin based on Ag@Cu2O nanomaterials, characterized in that, It includes the following steps: S1: After mixing the Ag NPs solution with a surfactant and a copper salt, a reducing agent is added and a stirring reaction is carried out to obtain Ag@Cu2O nanomaterials. The mass ratio of Ag NPs to the surfactant in the Ag NPs solution is 1:3 - 1:5, and the mass-volume concentration ratio of the copper salt to the reducing agent is 1:1 - 1:2; S2: The Ag@Cu2O nanomaterials in step S1 are connected with tetrodotoxin aptamer, and then mixed with MXenes to form an assembly. The nucleotide sequence of the tetrodotoxin aptamer is -SH-TCAAATTTTCGTCTACTCAATCTTTCTGTCTTATC; S3: The assembly is modified on the electrode surface, and the test solution is dropped onto the surface of the electrode, and then a Nafion solution is dropped to obtain a modified electrode; S4: The DPV signal intensity of the modified electrode is detected by a dual-signal detection method to achieve quantitative or qualitative electrochemical detection of tetrodotoxin.
2. The electrochemical detection method according to claim 1, wherein In step S1, the stirring reaction time is 2 min - 10 min.
3. The electrochemical detection method according to claim 1, characterized in that, In step S1, the copper salt is selected from one or more of copper nitrate, copper sulfate and copper chloride.
4. The electrochemical detection method according to claim 1, wherein In step S1, the reducing agent is selected from one or more of sodium borohydride, ascorbic acid and N2H4·H2O.
5. The electrochemical detection method according to claim 1, wherein In step S1, the surfactant is selected from polyvinylpyrrolidone and / or cetyltrimethylammonium bromide.
6. The electrochemical detection method according to claim 1, wherein In step S3, the electrode is selected from a glassy carbon electrode, a titanium electrode or a Cu electrode.
7. The electrochemical detection method according to claim 1, characterized in that, In step S3, the concentration of tetrodotoxin in the test solution is 10 pg / mL - 10 μg / mL.
8. The electrochemical detection method according to claim 1, characterized in that, In step S4, the preparation method of the standard curve in quantitative detection is: A series of test solutions containing tetrodotoxin with known concentrations are dropped onto the surface of the electrode after being modified with the assembly, and then the DPV signal intensity value of the electrode is detected. Taking the logarithm of the tetrodotoxin concentration as the abscissa and the DPV signal intensity value of the Ag@Cu2O nanomaterials as the ordinate, the standard curve for quantitative detection is obtained.