A t-2 toxin ratio type electrochemical aptamer sensor and a preparation method and application thereof

Abstract

The application discloses a T-2 toxin ratio type electrochemical aptamer sensor and a preparation method and application thereof. First, an electrode coating material (Ce-In)-MOF@MWCNTs for detecting a T-2 toxin electrochemical aptamer sensor and a probe Fe3O4@COF@Au@MB-Apt are prepared, and a T-2 toxin ratio type electrochemical aptamer sensor is constructed based on the same. The sensor is based on the synergistic effect of MOF materials and COF materials, the electrode is modified by using a (Ce-In)-MOF@MWCNTs composite material, and Fe3O4@COF is used to improve the electrochemical signal of MB. The sensor has the advantages of good selectivity to T-2 toxin, strong anti-non-target toxin interference ability, high detection sensitivity, stable performance, reusability, simple pretreatment of a detection sample, portability of equipment, suitability for on-site detection and high application value.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology. More specifically, it relates to the preparation and application of a T-2 toxin ratio electrochemical aptamer sensor. Background Technology

[0002] Food supply safety is paramount, but the threat from certain filamentous fungi is ever-present. These fungi produce mycotoxins during their growth, some of which the World Health Organization (WHO) has listed as among the most dangerous food contaminants. Notably, T-2 toxin is a common type A trichothecene toxin produced by *Fusarium solani* under cold, moist storage conditions. This toxin poses a significant risk to stored crops, especially in cold climates. The potential health effects of T-2 toxin should not be underestimated. It has been linked to a range of harmful health effects, including anorexia, vomiting, growth retardation, and various neurological, hematopoietic, gastrointestinal, and reproductive disorders in humans and animals. To address this threat, the Joint FAO / WHO Expert Committee on Food Additives (JECFA) has developed stringent guidelines, recommending a maximum acceptable daily intake of only 7.6 ng / kg for T-2 toxin.

[0003] Currently, various methods exist for detecting T-2 toxin, including advanced techniques such as ultra-high performance liquid chromatography (UPLC), fluorescence detection, gas chromatography-electron capture detection (GC-ECD), high performance liquid chromatography-mass spectrometry (HPLC-MS), and enzyme-linked immunosorbent assay (ELISA). However, existing technologies face challenges such as complex sample preparation, cost, and time limitations. Therefore, there is an urgent need for more direct, efficient, and cost-effective detection methods. Electrochemical aptamer sensors have attracted increasing attention due to their high sensitivity, strong specificity, low cost, ease of use, and rapid analysis, playing a vital role in trace analysis, drug analysis, and life science research.

[0004] Aptamer sensors, compared to traditional electrochemical sensors, exhibit higher specificity, primarily due to the introduction of nucleic acid aptamers (short single-stranded DNA or RNA oligonucleotides). These aptamers have attracted significant attention due to their high affinity for targets, high specificity, and ease of decoration. Aptamers undergo conformational changes in the presence of target molecules, leading to alterations in the current signal of the biosensor. This characteristic makes aptamers an effective tool for improving the specificity of biosensors. Considering that sensors prepared from single-signal molecules are susceptible to environmental factors such as pH and temperature, which can affect accuracy and precision, ratiometric strategies utilize the ratio of two signals from two different molecules to provide built-in correction for interference, significantly improving the repeatability and precision of electrochemical aptamer sensors.

[0005] However, the sensitivity and stability of ratiometric electrochemical aptamer sensors are still affected by many factors, such as the different analytes, the performance of the working electrode, and the probe signal. Currently, there are no reports of ratiometric electrochemical aptamer sensors used for the detection of T-2 toxin. Summary of the Invention

[0006] To overcome the shortcomings of existing electrochemical aptamer sensors for detecting T-2 toxin, this invention combines nucleic acid aptamers with electrochemical sensors and introduces a ratiometric strategy, improving and innovating the substrate electrode coating material and probe. This provides a method for preparing and applying a T-2 toxin ratiometric electrochemical aptamer sensor.

[0007] The first objective of this invention is to provide an electrode coating material for electrochemical aptamer sensors.

[0008] A second objective of this invention is to provide a probe for a T-2 toxin electrochemical aptamer sensor.

[0009] A third objective of this invention is to provide a T-2 toxin ratio electrochemical aptamer sensor.

[0010] A fourth objective of this invention is to provide the application of the above-described sensor in the qualitative or quantitative detection of T-2 toxin.

[0011] The fifth objective of this invention is to provide a detection method for the aforementioned sensor.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] First, the present invention provides an electrode coating material for an electrochemical aptamer sensor, wherein the electrode coating material is (Ce-In)-MOF@MWCNTs, and the preparation method is as follows: 2-aminoterephthalic acid, 1,3,5-pyromellitic acid, cerium nitrate, indium nitrate, and multi-walled carbon nanotubes (MWCNTs) are dissolved in an ethanol aqueous solution and mixed, and then heated, condensed, refluxed, washed, and dried to obtain (Ce-In)-MOF@MWCNTs.

[0014] Specifically, the (Ce-In)-MOF@MWCNTs are prepared by first dissolving 2-aminoterephthalic acid, 1,3,5-pyromellitic acid, cerium nitrate, and indium nitrate in an ethanol aqueous solution, then adding MWCNTs, ultrasonically mixing, and then refluxing, washing (preferably with an ethanol aqueous solution), and drying the mixture to obtain (Ce-In)-MOF@MWCNTs.

[0015] In use, (Ce-In)-MOF@MWCNTs are dissolved in deionized water and ultrasonically dispersed to form a uniformly dispersed solution before use. Preferably, the concentration of (Ce-In)-MOF@MWCNTs used is 1 mg / mL.

[0016] Preferably, the concentrations of 2-aminoterephthalic acid, 1,3,5-triphenyl benzoic acid, cerium nitrate, and indium nitrate are all 0.5–1.5 mM, and the concentration of MWCNTs is 26–29 mM.

[0017] Preferably, the ethanol aqueous solution is a 40%-60% ethanol aqueous solution (preferably a 50% ethanol aqueous solution).

[0018] Preferably, the heating, condensation, and reflux conditions are reflux at 110℃-130℃ for 4-6 hours (preferably reflux at 120℃ for 5 hours).

[0019] Preferably, the drying conditions are overnight drying at 50–70°C (preferably overnight drying at 60°C).

[0020] In addition, this invention provides a probe for an electrochemical aptamer sensor for detecting T-2 toxin, wherein the probe is Fe3O4@COF@Au@MB-Apt, and its preparation method is as follows:

[0021] (1) 2,5-Dimethoxytetraphenyldialdehyde, 1,3,5-tris(4-aminophenyl)benzene, 1,4-dioxane and butanol were mixed to form a solution. Fe3O4 was dispersed into the solution by ultrasonication. Then, an aqueous acetic acid solution was added to carry out the reaction.

[0022] (2) After the reaction is complete, add an aqueous acetic acid solution and heat the reaction. After the reaction is complete, collect the product, wash it with acetone and tetrahydrofuran, and then dry it to obtain Fe3O4@COF;

[0023] (3) Fe3O4@COF was suspended in tetrahydrofuran, and then HAuCl4 solution was added first to react, followed by NaBH4 solution. After washing and drying, Fe3O4@COF@Au was obtained.

[0024] (4) Fe3O4@COF@Au was dissolved in methylene blue (MB) solution for treatment. After treatment, a nucleic acid aptamer activated by tris(2-carboxyethyl)phosphine (TECP) was added for reaction to obtain Fe3O4@COF@Au@MB-Apt.

[0025] Specifically, in step (1), the concentration of the acetic acid aqueous solution is 12M, and the amount added is 0.03 to 0.08 mL.

[0026] Preferably, the reaction conditions in step (1) are 23-27°C for 1.5-2.5 hours.

[0027] Preferably, in step (1), the ratio of the amounts of 2,5-dimethoxy-terephthalaldehyde, 1,3,5-tris(4-aminophenyl)benzene, 1,4-dioxane, butanol, and Fe3O4 used is as follows, calculated according to the standard: 8-10 mg of 2,5-dimethoxy-terephthalaldehyde, 9.5-11.5 mg of 1,3,5-tris(4-aminophenyl)benzene, 1.8-2.2 mL of 1,4-dioxane, 1.8-2.2 mL of butanol, and 11-13 mg of Fe3O4.

[0028] Preferably, the concentration of the acetic acid aqueous solution in step (2) is 12M, and the amount added is 0.4 to 0.5 mL.

[0029] Preferably, the heating reaction in step (2) is carried out at 60-80°C for 44-52 hours.

[0030] Preferably, the drying conditions in step (2) are vacuum drying at room temperature for 8 to 16 hours.

[0031] Preferably, in step (3), the ratio of Fe3O4@COF to tetrahydrofuran is 2-4 mg: 1 mL.

[0032] Preferably, in step (3), the concentration of HAuCl4 solution is 0.05-0.15%, and the amount used is 0.5-1.5 mL; the concentration of NaBH4 solution is 0.4-0.6 M (more preferably 0.53 M), and the amount used is 1.8-2.2 mL.

[0033] Preferably, the reactions in step (3) are all stirred at room temperature, and the drying conditions are vacuum drying overnight at 50-70°C.

[0034] Preferably, in step (4), the ratio of Fe3O4@COF@Au, methylene blue solution, and nucleic acid aptamer used is as follows: Fe3O4@COF@Au 8-12 mg; the concentration of methylene blue solution is 0.5-1.5 mM, and the amount used is 5 mL; the concentration of nucleic acid aptamer is 8-12 μM, and the amount used is 25-35 μL.

[0035] Preferably, the treatment in step (4) refers to: shaking at room temperature for 22-26 hours, then washing 2-4 times, and finally reconstituted with TMbuff solution. Preferably, the standard amount of TMbuff solution used for reconstitution is: Fe3O4@COF@Au@MB-Apt prepared according to the standard ratio of Fe3O4@COF@Au, methylene blue solution, and nucleic acid aptamer used in the preferred step (4) above, reconstituted with 4-6 mL of TMbuff solution.

[0036] Preferably, the reaction conditions in step (4) are 2℃-8℃ for 12h (preferably 4℃ for 12h).

[0037] The present invention also provides a T-2 toxin ratio electrochemical aptamer sensor, the sensor comprising component 1 and component 2;

[0038] The construction method of component 1 includes the following steps:

[0039] S1. (Ce-In)-MOF@MWCNTs are modified onto a glassy carbon electrode to obtain a functionalized nanomaterial electrode;

[0040] S2. In-situ electrodeposition of modified gold nanoparticles (AuNPs) on the functionalized nanomaterial electrode obtained in S1;

[0041] S3. Add cDNA, 6-mercaptohexanol (MCH), and Fe3O4@COF@Au@MB-Apt probe sequentially to the electrode obtained in S2 to obtain component 1;

[0042] The component 2 is a ferrocene-modified nucleic acid aptamer (Apt-Fc), wherein the nucleic acid aptamer sequence is GTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAA.

[0043] The cDNA sequence is AAAATTCACCTCTCGCATGTGTAA.

[0044] Specifically, in step S1, the concentration of (Ce-In)-MOF@MWCNTs used is 0.8–1.2 mg / mL, and the amount used is 4–6 μL.

[0045] Preferably, the glassy carbon electrode needs to undergo pretreatment, which involves polishing the glassy carbon electrode on chamois leather with Al2O3 powder, rinsing the electrode with ultrapure water, ultrasonically cleaning it in ethanol and water respectively, and then drying it.

[0046] Preferably, in step S2, electrodeposition is performed using the it method, with a deposition potential of -0.1 to -0.3V (preferably -0.2V) and a time of 50 to 70s (preferably 60s).

[0047] Preferably, the amount of Fe3O4@COF@Au@MB-Apt probe used in step S3 is 4 to 6 μL.

[0048] As an alternative implementation, the construction method of component 1 of the T-2 toxin ratio electrochemical aptamer sensor is as follows:

[0049] 1) The glassy carbon electrode is pretreated. During the pretreatment process, the glassy carbon electrode is polished by polishing it with Al2O3 powder on chamois leather. Further, the pretreated glassy carbon electrode is ultrasonically cleaned. Ultrasonic cleaning involves rinsing with ultrapure water after polishing, followed by ultrasonic cleaning with ethanol and water for 20–40 seconds each, and then drying.

[0050] 2) Add the dispersion of (Ce-In)-MOF@MWCNTs dropwise to the electrode surface and dry it;

[0051] 3) The electrode modified with (Ce-In)-MOF@MWCNTs was electrodeposited in situ in chloroauric acid solution to modify AuNPs, rinsed with ultrapure water and dried;

[0052] 4) Spread cDNA onto the electrode modified with AuNPs and incubate at 2–8°C for 10–14 h;

[0053] 5) After cDNA incubation, clean the electrode with TEbuff, dry it, add MCH, and incubate at room temperature for 0.8–1.2 h;

[0054] 6) After MCH incubation is complete, it is also cleaned with TEbuff, dried, and then Fe3O4@COF@Au@MB-Apt is added dropwise. It is then incubated at 35-39℃ for 1.5-2.5h.

[0055] 7) After Fe3O4@COF@Au@MB-Apt incubation is completed, it is also cleaned with TEbuff and dried to complete the construction of component 1.

[0056] The application of the above-mentioned T-2 toxin ratio electrochemical aptamer sensor in the qualitative or quantitative detection of T-2 toxin should also be within the scope of protection of this invention.

[0057] Finally, the present invention provides a method for detecting T-2 toxin in the above-mentioned T-2 toxin ratio electrochemical aptamer sensor. The detection method is as follows: first, the test solution is added to component 1 of the T-2 toxin ratio electrochemical aptamer sensor, and then component 2 is added.

[0058] Specifically, after adding 5-10 μL of the test solution to component 1 of the T-2 toxin ratio electrochemical aptamer sensor, it needs to be incubated at 35-39℃ for 1.5-2.5 h. After incubation, it is cleaned, and then component 2 is added and incubated at 35-39℃ for 1.5-2.5 h.

[0059] Specifically, the quantitative detection method is as follows: using Ag / AgCl as the reference electrode, Pt electrode as the counter electrode, and the prepared modified electrode as the working electrode, connected to an electrochemical workstation, differential pulse voltammetry is performed in the test solution TEbuff, with a detection range of -0.5V to 0.5V. Quantitative analysis is performed by comparing the changes in the current ratio of signal molecules MB and Fc, and the concentration of T-2 toxin in the sample is calculated according to the IMB / IFc concentration standard working curve.

[0060] In addition, when using the sensor of the present invention to detect T-2 toxin, the pretreatment method for solid samples (such as corn, rice, dried tangerine peel, etc.) is as follows: grind the solid sample into a powder to be tested, add 70% acetonitrile aqueous solution to the powder to be tested, sonicate (preferably sonicate for 1 hour), then centrifuge (preferably 5500 rpm), and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution.

[0061] Preferably, the ratio of the powder to be tested to a 70% acetonitrile aqueous solution is 1g:2-6mL (preferably 1g:4mL).

[0062] The present invention has the following beneficial effects:

[0063] This invention develops a ratiometric electrochemical aptamer sensor based on the synergistic effect of MOF and COF materials. The electrode is modified with the prepared (Ce-In)-MOF@MWCNTs composite material, and Fe3O4@COF is used to enhance the electrochemical signal of MB. The nucleic acid aptamer, namely the probe Fe3O4@COF@Au@MB-Apt, is introduced, which greatly improves the specificity of detection. The final sensor has the advantages of strong selective response and high sensitivity to T-2 toxin detection.

[0064] Moreover, the sensor of this invention for detecting T-2 toxin is simple to sample pretreatment, fast to detect, and portable for on-site testing. It simplifies operation, provides rapid response, high detection accuracy, and strong anti-interference capabilities. Furthermore, the sensor is simple to manufacture, has stable performance, and is inexpensive to prepare. This is of great significance for the rapid and sensitive analysis and detection of T-2 toxin. Attached Figure Description

[0065] Figure 1 This is a schematic diagram illustrating the construction and detection process of a ratiometric electrochemical aptamer sensor for T-2 toxin.

[0066] Figure 2 The standard operating curves for the ratiometric electrochemical aptamer sensor of T-2 toxin.

[0067] Figure 3 Figure 1 shows the stability, reproducibility, and interference test results of the ratiometric electrochemical aptamer sensor for T-2 toxin.

[0068] Figure 4 The graph shows the results of differential pulse voltammetry (DPV) tests on the sensors prepared in Example 1 and Comparative Example 1. Detailed Implementation

[0069] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0070] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0071] Component 2 (Apt-Fc) used in the following examples was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0072] The multi-walled carbon nanotubes (MWCNTs) used in the following examples were purchased from Beijing Deco Island Gold Technology Co., Ltd., model (CNT106), with a molar mass of 12.01 g / mol.

[0073] The Fe3O4 (catalog number: I104312) used in the following examples was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0074] Example 1: Preparation of T-2 toxin ratio electrochemical aptamer sensor

[0075] The construction and detection process of the T-2 toxin ratio electrochemical aptamer sensor is as follows: Figure 1 As shown.

[0076] (I) Preparation of Component 1

[0077] 1. Pretreatment of glassy carbon electrode

[0078] The glassy carbon electrode was polished on chamois leather with Al2O3 powder of 0.5 μm and 0.05 μm in sequence, rinsed with ultrapure water, and then ultrasonically cleaned in ethanol and water for 30 seconds each.

[0079] 2. Preparation of electrode coating material (Ce-In)-MOF@MWCNTs

[0080] Weigh out 2-aminoterephthalic acid, 1,3,5-triphenyl phthalic acid, cerium nitrate, and indium nitrate, and dissolve them in 150 mL of 50% ethanol aqueous solution to a final concentration of 1 mM. Then add MWCNTs to bring the final concentration to 27.7 mM. Sonicate the mixture for half an hour, place the mixture in a round-bottom flask, and reflux at 120°C for 5 hours. After the reaction is complete, wash with 50% ethanol aqueous solution, dry at 60°C overnight, weigh out 1 mg of the powder, dissolve it in 1 mL of deionized water, and sonicate for half an hour to form a uniformly dispersed solution.

[0081] 3. Preparation of Fe3O4@COF@Au@MB-Apt signal probe

[0082] (1) Add the following components to a round-bottom flask: 2,5-dimethoxytetraphenyl phthalaldehyde (DMTP, 8.7 mg), 1,3,5-tris(4-aminophenyl)benzene (TAPB, 10.5 mg), 1,4-dioxane (2 mL) and butanol (2 mL); disperse Fe3O4 (12 mg) into the above solution by ultrasonication, and then add 0.05 mL of 12M acetic acid aqueous solution, and react the mixture at 25 °C for 2 hours;

[0083] (2) Then add 0.45 mL of 12M acetic acid aqueous solution to the mixture described in S1, heat the mixture to 70°C and react for 48 hours. After the product is cooled to room temperature, collect it with a magnet, wash it three times with acetone and tetrahydrofuran (THF) in sequence, and then vacuum dry it at room temperature for 12 hours to obtain Fe3O4@COF.

[0084] (3) 20 mg of Fe3O4@COF was suspended in 6 mL of THF, and then 1 mL of 0.1% HAuCl4 solution was added. After stirring the mixture at room temperature for 2 hours, 2 mL of 0.53 M NaBH4 aqueous solution was quickly added, and the reaction was continued to be stirred overnight at room temperature. The product was collected with a magnet, washed three times each with water and ethanol, and finally dried under vacuum at 60 °C overnight to obtain Fe3O4@COF@Au.

[0085] (4) Weigh 10 mg Fe3O4@COF@Au and dissolve it in 5 mL of 1 mM MB solution. Shake at room temperature for 24 h, then wash three times with water. Finally, redissolve with 5 mL of TMbuff solution, add 30 μL of 10 μM Apt activated by TECP, react at 4 °C for 12 h, wash three times with TEbuff at 8000 rpm, and finally redissolve with 500 μL of TMbuff to obtain Fe3O4@COF@Au@MB-Apt.

[0086] 4. Fabrication of component 1 of the T-2 toxin ratio electrochemical aptamer sensor

[0087] 5 μL of 1 mg / mL (Ce-In)-MOF@MWCNTs dispersion was dropped onto the pretreated glassy carbon electrode and dried under an infrared lamp. AuNPs were then deposited in situ in 1% chloroauric acid solution at -0.2 V. After drying, 5 μL of cDNA (1 μM) solution was dropped onto the electrode and incubated at 4 °C for 12 h. After incubation, the electrode was washed with TE buff, dried, and 5 μL of 1 μM MCH was added. The electrode was incubated at room temperature for 1 h. After washing with TE buff, dried, and 5 μL of Fe3O4@COF@Au@MB-Apt dispersion was dropped onto the electrode and incubated at 37 °C for 2 h. After washing with TE buff and drying, component 1 of the electrochemical aptamer sensor was completed.

[0088] (II) Component 2

[0089] Component 2 (Apt-Fc) is a ferrocene-modified nucleic acid aptamer, wherein the nucleic acid aptamer sequence is GTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAA.

[0090] Component 2 (Apt-Fc) was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0091] Example 2: Preparation of T-2 toxin ratio electrochemical aptamer sensor

[0092] (I) Preparation of Component 1

[0093] 1. Pretreatment of glassy carbon electrode

[0094] The glassy carbon electrode was polished on chamois leather with Al2O3 powder of 0.5 μm and 0.05 μm in sequence, rinsed with ultrapure water, and then ultrasonically cleaned in ethanol and water for 30 seconds each.

[0095] 2. Preparation of electrode coating material (Ce-In)-MOF@MWCNTs

[0096] Weigh out 2-aminoterephthalic acid, 1,3,5-triphenyl phthalic acid, cerium nitrate, and indium nitrate, and dissolve them in 150 mL of 40% ethanol aqueous solution to a final concentration of 0.5 mM. Then add MWCNTs to bring the final concentration to 26 mM. Sonicate the mixture for half an hour, place the mixture in a round-bottom flask, and reflux at 110 °C for 4 hours. After the reaction is complete, wash with 40% ethanol aqueous solution, dry at 50 °C overnight, weigh out 1 mg of the powder, dissolve it in 1 mL of deionized water, and sonicate for half an hour to form a uniformly dispersed solution.

[0097] 3. Preparation of Fe3O4@COF@Au@MB-Apt signal probe

[0098] (1) Add the following components to a round-bottom flask: 2,5-dimethoxytetraphenyl phthalaldehyde (DMTP, 8 mg), 1,3,5-tris(4-aminophenyl)benzene (TAPB, 9.5 mg), 1,4-dioxane (1.8 mL) and butanol (1.8 mL); disperse Fe3O4 (11 mg) into the above solution by ultrasonication, and then add 0.03 mL of 12M acetic acid aqueous solution, and react the mixture at 23°C for 2.5 hours;

[0099] (2) Then add 0.4 mL of 12M acetic acid aqueous solution to the mixture described in S1, heat the mixture to 60°C and react for 52 hours. After the product is cooled to room temperature, collect it with a magnet, wash it three times with acetone and tetrahydrofuran (THF) in sequence, and then dry it under vacuum at room temperature for 8 hours to obtain Fe3O4@COF.

[0100] (3) 12 mg of Fe3O4@COF was suspended in 6 mL of THF, and then 0.5 mL of 0.05% HAuCl4 solution was added. After stirring the mixture at room temperature for 2 hours, 1.8 mL of 0.4 M NaBH4 aqueous solution was quickly added, and the reaction was continued to be stirred overnight at room temperature. The product was collected with a magnet, washed three times each with water and ethanol, and finally dried under vacuum at 50 °C overnight to obtain Fe3O4@COF@Au.

[0101] (4) Weigh 8 mg Fe3O4@COF@Au and dissolve it in 5 mL of 0.5 mM MB solution. Shake at room temperature for 22 h. Then wash twice with water and finally redissolve with 4 mL of TMbuff solution. Add 25 μL of 12 μM Apt activated by TECP and react at 2 °C for 12 h. Wash three times with TEbuff at 8000 rpm and finally redissolve with 500 μL of TMbuff to obtain Fe3O4@COF@Au@MB-Apt.

[0102] 4. Fabrication of component 1 of the T-2 toxin ratio electrochemical aptamer sensor

[0103] 6 μL of 0.8 mg / mL (Ce-In)-MOF@MWCNTs dispersion was dropped onto the pretreated glassy carbon electrode and dried under an infrared lamp. Then, AuNPs were deposited in situ in 1% chloroauric acid solution at a potential of -0.2 V. After drying, 5 μL of cDNA (1 μM) solution was dropped onto the electrode and incubated at 8 °C for 10 h. After incubation, the electrode was washed with TE buff, dried, and 5 μL of 1 μM MCH was added. The electrode was incubated at room temperature for 0.8 h. After washing with TE buff, dried, and 5 μL of Fe3O4@COF@Au@MB-Apt dispersion was dropped onto the electrode and incubated at 35 °C for 2.5 h. After washing with TE buff and drying, component 1 of the electrochemical aptamer sensor was completed.

[0104] (ii) Component 2 (Apt-Fc) is the same as in Example 1.

[0105] Example 3: Preparation of T-2 toxin ratio electrochemical aptamer sensor

[0106] (I) Preparation of Component 1

[0107] 1. Pretreatment of glassy carbon electrode

[0108] The glassy carbon electrode was polished on chamois leather with Al2O3 powder of 0.5 μm and 0.05 μm in sequence, rinsed with ultrapure water, and then ultrasonically cleaned in ethanol and water for 30 seconds each.

[0109] 2. Preparation of electrode coating material (Ce-In)-MOF@MWCNTs

[0110] Weigh out 2-aminoterephthalic acid, 1,3,5-triphenyl phthalic acid, cerium nitrate, and indium nitrate, and dissolve them in 150 mL of 60% ethanol aqueous solution to a final concentration of 1.5 mM. Then add MWCNTs to bring the final concentration to 29 mM. Sonicate the mixture for half an hour, place the mixture in a round-bottom flask, and reflux at 130°C for 6 hours. After the reaction is complete, wash with 60% ethanol aqueous solution, dry at 70°C overnight, weigh out 1 mg of the powder, dissolve it in 1 mL of deionized water, and sonicate for half an hour to form a uniformly dispersed solution.

[0111] 3. Preparation of Fe3O4@COF@Au@MB-Apt signal probe

[0112] (1) Add the following components to a round-bottom flask: 2,5-dimethoxytetraphenyl phthalaldehyde (DMTP, 10 mg), 1,3,5-tris(4-aminophenyl)benzene (TAPB, 11.5 mg), 1,4-dioxane (2.2 mL) and butanol (2.2 mL); disperse Fe3O4 (13 mg) into the above solution by ultrasonication, and then add 0.08 mL of 12M acetic acid aqueous solution, and react the mixture at 27°C for 1.5 hours;

[0113] (2) Then add 0.5 mL of 12M acetic acid aqueous solution to the mixture described in S1, heat the mixture to 80°C and react for 44 hours. After the product is cooled to room temperature, collect it with a magnet, wash it three times with acetone and tetrahydrofuran (THF) in sequence, and then vacuum dry it at room temperature for 16 hours to obtain Fe3O4@COF.

[0114] (3) 24 mg of Fe3O4@COF was suspended in 6 mL of THF, and then 1.5 mL of 0.15% HAuCl4 solution was added. After stirring the mixture at room temperature for 2 hours, 2.2 mL of 0.6 M NaBH4 aqueous solution was quickly added, and the reaction was continued to be stirred overnight at room temperature. The product was collected with a magnet, washed three times each with water and ethanol, and finally dried under vacuum at 70 °C overnight to obtain Fe3O4@COF@Au.

[0115] (4) Weigh 12 mg Fe3O4@COF@Au and dissolve it in 5 mL of 1.5 mM MB solution. Shake at room temperature for 26 h. Then wash with water 4 times. Finally, redissolve with 6 mL of TMbuff solution. Add 35 μL of 8 μM Apt activated by TECP. React at 8 °C for 12 h. Wash three times with TEbuff at 8000 rpm. Finally, redissolve with 500 μL of TMbuff to obtain Fe3O4@COF@Au@MB-Apt.

[0116] 4. Fabrication of component 1 of the T-2 toxin ratio electrochemical aptamer sensor

[0117] 4 μL of 1.2 mg / mL (Ce-In)-MOF@MWCNTs dispersion was dropped onto the pretreated glassy carbon electrode and dried under an infrared lamp. Then, AuNPs were deposited in situ in 1% chloroauric acid solution at a potential of -0.2 V. After drying, 5 μL of cDNA (1 μM) solution was dropped onto the electrode and incubated at 2 °C for 14 h. After incubation, the electrode was washed with TE buff, dried, and 5 μL of 1 μM MCH was added. The electrode was incubated at room temperature for 1.2 h. After washing with TE buff, dried, and 5 μL of Fe3O4@COF@Au@MB-Apt dispersion was dropped onto the electrode and incubated at 39 °C for 1.5 h. After washing with TE buff and drying, component 1 of the electrochemical aptamer sensor was completed.

[0118] (ii) Component 2 (Apt-Fc) is the same as in Example 1.

[0119] Example 4: Detection method, plotting of working curve, and determination of detection limit

[0120] The responsiveness of the T-2 toxin ratiometric electrochemical aptamer sensor was tested using differential pulse voltammetry (DPV) to determine the linear range and detection limit. In this embodiment, T-2 toxin solutions with different concentration gradients were used as test solutions. The concentration gradient was set as follows: 5.0 × 10⁻⁶. -3 pg / mL, 5.0×10 -2 pg / mL, 5.0×10 -1 pg / mL, 5.0 pg / mL, 5.0 × 10 1 pg / mL, 5.0×10 2 pg / mL 5.0×10 3 pg / mL.

[0121] (I) Detection Method

[0122] The sensor component 1 prepared in Example 1 was immersed in TEbuff solution for DPV measurement. Ag / AgCl was used as the reference electrode, Pt electrode as the counter electrode, and the prepared electrochemical sensor as the working electrode. Connected to an electrochemical workstation, 7 μL of the test solution was added to component 1 of the T-2 toxin ratio electrochemical aptamer sensor and incubated at 37°C for 2 h. Then, 7 μL of component 2 (Apt-Fc) with a concentration of 1 μM was added and incubated at 37°C for 2 h. Then, the electrode was modified according to different target concentrations. MB / I Fc Plot a working curve showing the relationship between the change in ratio and the change in target analyte concentration.

[0123] (II) Test Results

[0124] The standard operating curve determination results of the T-2 toxin ratiometric electrochemical aptamer sensor are as follows: Figure 2 As shown in the figure. The results indicate that the prepared sensor performs well in the concentration range of 5.0 × 10⁻⁶. -3 pg / mL -5.0×10 3 pg / mL showed a good linear response to T-2 toxin, with the linear equation being IMB / IFc = -0.2593lg CT-2 + 1.797(R). 2 =0.9979). The limit of detection (LOD) for T-2 toxin by the sensor is calculated using the formula. LOD (defined as C) L The formula for calculating ) is C L =10 (Xb+kSD-1.797) / (-0.259) .

[0125] Among them, C L This represents the smallest theoretically detectable measurement signal. X b X is the average of ten blank measurements, while SD represents the standard deviation of the blank measurements. After ten blank measurements, X... bThe value is 2.8007, kSD is 0.2643, and X... b After substituting kSD into the equation, C L It can be determined to be 10. (2.8007+0.2643-1.797) / (-0.259) The calculation result is 1.265 × 10 -5 pg / mL. Therefore, the limit of detection (LOD) for detecting T-2 toxin using this sensor is 1.265 × 10 pg / mL. -5 The pg / mL indicates that the sensor of this invention has extremely high sensitivity to T-2 toxin.

[0126] Example 5: Sensor stability, reproducibility, and interference experiments

[0127] Five identical sensors were prepared under the same preparation conditions as in Example 1. The prepared sensors were used to detect 5 pg / mL of T-2 toxin. The detection method was the same as in Example 4. Each group was measured in parallel 3 times, for a total of 15 data points.

[0128] The constructed sensor component 1 was stored in a refrigerator. At regular intervals, it was taken out and measured with 5 pg / mL of T-2 toxin added to component 2. All measurements were completed after 13 days, and the results are as follows: Figure 3 As shown in Figure A, the results indicate that the IMB / IFc ratio on day 13 remained at 97.7% of that on day 1, demonstrating the good stability of the sensor constructed in Example 1.

[0129] To investigate whether common fungal toxins interfere with the sensor of this invention, aflatoxin B1 (AFB1), vomitoxin (DON), patulin (PAT), and F-2 toxin (ZEN) were selected as interference sources for detection experiments. The experimental results are as follows: Figure 3 As shown in Figure B, the experimental results show that, within the error range, a 10-fold increase in the amount of interfering substance does not interfere with the determination of T-2 toxin, proving that the sensor constructed in Example 1 has good anti-interference ability.

[0130] from Figure 3 As shown in Figure C, the relative standard deviation of the current response values ​​of the five identical sensors is 2.7% (n=3), indicating that the sensor constructed in Example 1 has good reproducibility.

[0131] Example 6: Detection of T-2 toxin in rice, corn, and dried tangerine peel

[0132] To evaluate whether the sensor constructed in Example 1 has the potential for practical application, the sensor was used to detect T-2 toxin in real samples. The real samples selected in this example were corn, rice, and dried tangerine peel.

[0133] First, the actual sample was processed as follows: After grinding the sample, 2 grams of sample were weighed and placed in a 50 mL centrifuge tube. Then, 1 mL of T-2 standard solution (50.0 ng / mL) was added to the sample. Subsequently, 8 mL of 70% acetonitrile aqueous solution was added, and the sample was sonicated (ultrasonic frequency: 28–40 kHz) for 1 hour. Then, the sample was centrifuged at 5500 rpm, and the supernatant was filtered through a 0.22 μm filter membrane to purify the sample and remove impurities that might affect the experimental results. Finally, the sample supernatant was diluted to different concentrations with TE buffer and stored at 4°C when not in use. The sensor constructed in Example 1 was used to quantitatively detect the sample, using the same detection method as in Example 4. Each sample was detected three times, and the results are shown in Table 1. The results show that the sensor of the present invention can quantitatively detect T-2 toxin in the test sample with high sensitivity and high accuracy.

[0134] Table 1

[0135]

[0136] Comparative Example 1

[0137] The component 1 of the T-2 toxin ratio electrochemical aptamer sensor was prepared according to the method of Example 1, except that the (Ce-In)-MOF@MWCNTs material was replaced with (Ce-In)Ox@MWCNTs.

[0138] The preparation method of (Ce-In)Ox@MWCNTs material is as follows: the obtained (Ce-In)-MOF@MWCNTs is calcined at 500℃ for 2 hours under N2 atmosphere at a heating rate of 5℃ / min to prepare (Ce-In)Ox@MWCNTs.

[0139] The sensor prepared in Example 1 and the sensor prepared in this comparative example were subjected to differential pulse voltammetry (DPV) tests. The specific detection method is the same as in Example 4. The test results are as follows: Figure 4 As shown, the results indicate that the electrode modified with (Ce-In)-MOF@MWCNTs exhibits a higher peak current compared to (Ce-In)Ox@MWCNTs, demonstrating that the electrochemical performance of the sensor prepared in Example 1 is significantly better than that of the sensor prepared in this comparative example.

[0140] Comparative Example 2

[0141] The component 1 of the T-2 toxin ratio electrochemical aptamer sensor was prepared according to the method of Example 1, except that the Fe3O4@COF@Au@MB-Apt probe was replaced with the COF@Au-Apt probe. Then, in the preparation process of sensor component 1, after the COF@Au-Apt probe was incubated, 7 μL of MB solution (20 μM) was dropped onto the surface of the modified electrode and incubated at 37°C for 1 hour.

[0142] The preparation method of the COF@Au-Apt probe differs from the preparation method of the Fe3O4@COF@Au@MB-Apt signal probe in Example 1 in that: Fe3O4 is not added in step (1), and the treatment of the MB solution in step (4) is skipped. The COF@Au obtained in step (3) is directly reconstituted with TMbuff solution, and then TECP-activated Apt is added for reaction. The remaining preparation parameters and conditions are the same as those in the preparation method of the Fe3O4@COF@Au@MB-Apt signal probe in Example 1.

[0143] Testing showed that the sensor prepared in Example 1 had significantly better detection performance than the sensor prepared in the comparative example. The sensor prepared in Example 1, due to the addition of Fe3O4 to the Fe3O4@COF@Au@MB-Apt probe, could better load MB, resulting in more significant signal amplification and a lower limit of detection (LOD) compared to COF alone. Furthermore, the construction process of sensor component 1 was simplified, eliminating the need for separate MB solution coating onto the electrode.

[0144] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An electrode coating material for an electrochemical aptamer sensor, characterized by, The electrode coating material is (Ce-In)-MOF@MWCNTs, and the preparation method is as follows: 2-amino terephthalic acid, 1,3,5-benzenetricarboxylic acid, cerium nitrate, indium nitrate and multi-walled carbon nanotubes are dissolved in an ethanol aqueous solution mixture, heated and condensed by reflux, washed, and dried to obtain (Ce-In)-MOF@MWCNTs; the use concentration of 2-amino terephthalic acid, 1,3,5-benzenetricarboxylic acid, cerium nitrate and indium nitrate is 0.5-1.5 mM, and the use concentration of MWCNTs is 26-29 mM.

2. A T-2 toxin ratio-type electrochemical aptamer sensor, characterized by, The assembly 1 and the assembly 2 are included; The construction method of the assembly 1 comprises the following steps: S1. The electrode coating material of claim 1 is modified to a glassy carbon electrode to obtain a functionalized nanomaterial electrode; S2. Gold nanoparticles are in-situ electrodeposited on the functionalized nanomaterial electrode obtained in S1; S3. The cDNA, 6-mercaptohexanol and Fe3O4@COF@Au@MB-Apt probe are sequentially added dropwise on the electrode obtained in S2 to obtain the assembly 1; The Fe3O4@COF@Au@MB-Apt probe is prepared by the following method: (1) 2,5-dimethoxyterephthaldehyde, 1,3,5-tris(4-aminophenyl)benzene, 1,4-dioxane and butanol are mixed to form a solution, Fe3O4 is dispersed in the solution by ultrasonic, and then acetic acid is added for reaction; (2) After the reaction is completed, acetic acid aqueous solution is added for heating reaction, and then the product is collected, washed with acetone and tetrahydrofuran, and dried to obtain Fe3O4@COF; (3) Fe3O4@COF is suspended in tetrahydrofuran, then HAuCl4 solution is added for reaction, and then NaBH4 solution is added for reaction, and after washing and drying, Fe3O4@COF@Au is obtained; (4) Fe3O4@COF@Au is dissolved in methylene blue solution for treatment, and then tris(2-carboxyethyl)phosphine-activated aptamer is added for reaction to obtain Fe3O4@COF@Au@MB-Apt; The assembly 2 is an aptamer modified with ferrocene, and the sequence of the aptamer is GTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAA.

3. The T-2 toxin ratio-type electrochemical aptamer sensor according to claim 2, wherein, In step S1, the use concentration of (Ce-In)-MOF@MWCNTs is 0.8-1.2 mg / mL, and the use amount is 4-6 μL.

4. The T-2 toxin ratio-type electrochemical aptamer sensor according to claim 2, wherein, In step (4) of the preparation method, the use amount ratio of Fe3O4@COF@Au, methylene blue solution and aptamer is as follows: Fe3O4@COF@Au 8-12 mg; the concentration of methylene blue solution is 0.5-1.5 mM, and the use amount is 5 mL; the concentration of aptamer is 8-12 μM, and the use amount is 25-35 μL.

5. The T-2 toxin ratio-type electrochemical aptamer sensor according to claim 2, wherein, In step (4) of the preparation method, the treatment is oscillation at room temperature for 22-26 h, then washing 2-4 times, and finally re-dissolving with TMbuff solution.

6. The T-2 toxin ratio-type electrochemical aptamer sensor according to any one of claims 2-5 for use in qualitative or quantitative detection of T-2 toxin.

7. A method for detecting T-2 toxin based on the T-2 toxin ratio-type electrochemical aptamer sensor according to any one of claims 2 to 5, characterized in that, First, the solution to be tested is added to component 1 of the T-2 toxin ratio-type electrochemical aptamer sensor, and then component 2 is added.

8. The detection method according to claim 7, characterized in that, After adding 5-10 μL of the solution to be tested to component 1 of the T-2 toxin ratio-type electrochemical aptamer sensor, incubation is required at 35-39°C for 1.5-2.5 h, after which washing is performed, and then component 2 is added for incubation at 35-39°C for 1.5-2.5 h.

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