CN111398391B - Preparation method of electrochemical sensor for detecting T-2 toxin residue - Google Patents
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Abstract
The invention relates to a preparation method of an electrochemical aptamer sensor for detecting T-2 toxin residue, which comprises the following steps: preparing a functionalized carbon nano material by a microwave synthesis method, preparing a sulfur-doped metal nano sheet/carbon nano material composite material by a hydrothermal synthesis method, taking a proper amount of sulfur-doped metal nano sheet/functionalized carbon nano composite material/aptamer, and combining the proper amount of sulfur-doped metal nano sheet/functionalized carbon nano composite material/aptamer on a gold electrode in a covalent bonding mode to prepare a sulfur-doped metal nano sheet/functionalized carbon nano composite material/aptamer/gold electrode, taking a platinum wire electrode as a counter electrode and saturated silver chloride as a reference electrode, and obtaining the electrochemical aptamer sensor for detecting the residual T-2 toxin through the change of a signal molecule response signal. Compared with the traditional T-2 toxin detection method, the method has the advantages of high response speed, low detection limit, high sensitivity, good repeatability and high accuracy.
Description
Technical Field
The invention relates to a preparation method of an electrochemical aptamer sensor for detecting T-2 toxin residue, in particular to a preparation method of an electrochemical aptamer sensor for signal amplification based on a sulfur-doped metal nanosheet/functionalized carbon nanocomposite.
Background
T-2 toxin is trichothecene with thermal stability produced by fusarium, which is widely found in nature and is one of the main toxins polluting field crops and stored grains, and a report in China shows that the incidence rate of T-2 toxin in 420 feed samples is as high as 79.5%. T-2 toxin can be enriched and transmitted through food chains and organisms, pollutes animal-derived foods and further poses potential threats to human health. T-2 toxins have been shown to cause hematopoietic, gastrointestinal and reproductive dysfunction in humans and animals. There is also evidence that T-2 toxin can interact with aflatoxin to produce synergistic toxicity. Meanwhile, the food and agriculture organization (FAQ) and the World Health Organization (WHO) of the United nations use the T-2 toxin as well as aflatoxin as the most dangerous source of food contamination that exists naturally. Therefore, it is important to establish a T-2 toxin detection method. To date, various methods for detecting T-2 toxin have been developed, such as fluorimetry, gas chromatography with electron capture detection (GC-ECD), high performance liquid chromatography-mass spectrometry (HPLC-MS), ultra-high performance liquid chromatography (UPLC), etc., which have high specificity and accuracy, but generally require complicated pretreatment steps, expensive analytical instruments, high labor costs, and long signal response times, and thus are difficult to popularize in basic units and cannot meet the requirements of high-throughput and field testing in the food industry. Therefore, it is highly desirable to develop a novel, sensitive, low cost and easy to use method for detecting trace amounts of T-2 toxin in food and feed products. The electrochemical method has the advantages of simple operation, quick response and low detection cost, and is more and more widely applied to the field of food detection. The preparation of electrochemical sensors with high sensitivity, excellent performance and capability of on-site detection is the hot spot of current research. The identification strategy is important for enhancing the specificity of the sensor, the aptamer is an oligonucleotide fragment which is screened by an in vitro screening technology and can specifically bind to proteins or other small molecular substances, the oligonucleotide fragment is similar to the interaction of an antigen-antibody, the aptamer is bound with each target molecule of the aptamer through high specificity and affinity, and the aptamer has the characteristics of wide target molecule range and convenient and quick preparation and modification, the electrochemical aptamer sensor has the advantages of simple electrochemical analysis method and equipment, low cost and quick detection speed, and also has the advantages of high aptamer selectivity and strong specificity, but in practical application, the electrochemical sensor based on a single identification element aptamer still has the problem that the indexes such as sensitivity, stability and linear response are not ideal, and in order to further improve the response sensitivity of the sensor, the nano material is often widely applied to the construction of the biosensor as a signal amplification strategy, however, the single nano material has the defects of low conductivity and low catalytic performance, so that the design and synthesis of the functionalized nano composite material becomes the key for constructing the efficient T-2 toxin electrochemical sensor, the sulfur-doped metal nanosheet/functionalized carbon nano composite material not only can provide more ways and gaps for ion transfer and volume change, but also can serve as a good conductive agent to increase the conductivity, the technical method for preparing the sulfur-doped metal nanosheet/functionalized carbon nano composite material is rarely reported at present, and an aptamer sensor constructed by using the sulfur-doped metal nanosheet/functionalized carbon nano composite material as a load substrate composite nano material and an aptamer is not used for electrochemical analysis of T-2 toxin residues.
Disclosure of Invention
The invention relates to a preparation method of an electrochemical sensor for detecting T-2 toxin residue.
A preparation method of an electrochemical aptamer sensor for detecting residual T-2 toxin comprises the following steps:
the sulfur-doped metal nano sheet/functionalized carbon nano composite material is prepared by preparing a functionalized carbon nano material by adopting a microwave synthesis method, weighing 3-5 g of urea and 2-4 g of citric acid, adding the urea and the citric acid into 8-12 mL of deionized water, heating the solution in a microwave oven under the power of 500-1000W for 5-10 min, centrifuging the water solution of the functionalized carbon nano material for 10-20 min at the rotating speed of 2000-4000 rpm, freeze-drying to obtain functionalized carbon nano material powder, preparing the sulfur-doped metal nano sheet/carbon nano material composite material by adopting a hydrothermal synthesis method, dissolving 0.5-1 g of metal salt in 40-60 mL of ethylene glycol solution, carrying out ultrasonic treatment for 30-50 min at room temperature, then adding 1-1.2 g of thiourea, adding 0.05-0.1 g of the functionalized carbon nano material, carrying out ultrasonic treatment for 40-60 min, transferring the mixed solution into a stainless steel autoclave, carrying out thermal reaction for 10-15 h at 150-200 ℃, and drying for 10-14 h at 60-80 ℃ to obtain a sulfur-doped metal nanosheet/functionalized carbon nanocomposite material;
the signal tag affixes: 100 mu L of 3-5 mu M signal label DNA-S1Strand added 100. mu.L of signal tag DNA-S at the same concentration2Then incubating the mixed solution at 37-40 ℃ for 2-4 h; t-2Toxin aptamers were conjugated with helper DNA and: adding 100 mu L of 3-5 mu M T-2 toxin aptamer into 100 mu L of auxiliary DNA with the same concentration, and then incubating the mixed solution at 37-40 ℃ for 2-4 h. Adding 20-40 mu L of target objects with different concentrations into a mixed solution of 20-40 mu L T-2 toxin aptamer and auxiliary DNA, and then placing the mixed solution at 37-40 ℃ for incubation for 2-4 h to obtain conjugated T-2 toxin aptamer and auxiliary DNA;
constructing a sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode: placing a gold electrode in a piranha solution, soaking for 20-30 min, washing with ultrapure water, polishing with alumina powder, sequentially placing the polished gold electrode in ethanol and ultrapure water for ultrasonic treatment for 5-10 min, performing electrochemical activation with 0.5-1M of acid solution, dropwise adding 3-5 mu L of 2-4 mg/mL sulfur-doped metal nanosheet/functionalized carbon nanocomposite to the surface of the electrode, drying for 10-20 min under an infrared lamp, electrodepositing gold for 10-20 s with chloroauric acid solution, and transferring 3-5 mu L of 2-4 mu M DNA-H1Dropwise adding the mixed solution to the surface of an electrode, incubating for 2-4H at 37-40 ℃, washing and drying the mixed solution by buffer solution, dropwise adding 5-8 mu L of 0.1-0.4 mM MCH sealant, washing and drying the buffer solution, and dropwise adding 3-5 mu L of mixed solution containing a target and 3-5 mu L of DNA-H2And incubating for 2-4 h at 37-40 ℃, dropwise adding 3-5 mu L of conjugated signal label, incubating for 2-4 h at 37-40 ℃, washing with distilled water and drying to obtain the sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode.
The electrochemical sensor is an electrochemical aptamer sensor for detecting T-2 toxin residues, which is obtained by using a sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode and responding to the change of signals through signal molecules.
The metal salt is one or more of sodium ferrite, sodium molybdate, sodium hexanitrocobaltate and copper chloride.
The carbon nano material is one or more of graphene, carbon quantum dots, graphene quantum dots and single-walled carbon nanotubes.
The signal label is one or more of methylene blue, ferrocene and thionine.
The buffer solution is one or more of phosphate buffer solution, tris buffer solution and 3-morpholine propanesulfonic acid buffer solution.
The acid solution is one or more of sulfuric acid, nitric acid and phosphoric acid.
In the sensor, the functionalized carbon quantum dot/molybdenum disulfide composite material is used as an electrode surface substrate material, the aptamer is used as an identification element, and compared with other T-2 toxin residue detection sensors, the prepared novel electrochemical sensor has the advantages of high response speed, low detection limit, high sensitivity, good repeatability and high accuracy.
Detailed Description
The invention is described below with reference to specific examples:
example 1
The method comprises the following specific steps:
(1) preparing a functionalized graphene quantum dot by adopting a microwave synthesis method, weighing 3 g of urea and 2 g of citric acid, adding into 8 mL of deionized water, heating the solution in a microwave oven at 500W power for 5 min, centrifuging the aqueous solution of the functionalized graphene quantum dot at 2000 rpm for 10 min, and freeze-drying to obtain functionalized graphene quantum dot powder; preparing a sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material by adopting a hydrothermal synthesis method, dissolving 0.5 g of sodium molybdate in 40 mL of ethylene glycol solution, carrying out ultrasonic treatment at room temperature for 30 min, then adding 1 g of thiourea, adding 0.05 g of functionalized graphene quantum dot, carrying out ultrasonic treatment for 40 min, transferring the mixed solution into a stainless steel autoclave, carrying out thermal reaction at 150 ℃ for 10 h, and drying at 60 ℃ for 10 h to obtain the sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material;
(2) signal tag affix sum: 100. mu.L of 3. mu.M signal tag DNA-S1Strand added 100. mu.L of signal tag DNA-S at the same concentration2Then incubating the mixed solution at 37 ℃ for 2 h; t-2 toxin aptamer conjugated with helper DNA and: 100 μ LAdding 3 mu M of T-2 toxin aptamer into 100 mu L of auxiliary DNA with the same concentration, incubating the mixed solution at 37 ℃ for 2-4 h, adding 20 mu L of target objects with different concentrations into the mixed solution of 20 mu L T-2 toxin aptamer and the auxiliary DNA, and incubating at 37 ℃ for 2 h to obtain the conjugated T-2 toxin aptamer and the auxiliary DNA;
(3) constructing a sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material/aptamer/gold electrode: soaking a gold electrode in a piranha solution for 20 min, washing with ultrapure water, polishing with alumina powder, sequentially placing the polished gold electrode in ethanol and ultrapure water for ultrasonic treatment for 5 min, performing electrochemical activation with 0.5M acid solution, dropwise adding 3 mu L of 2 mg/mL sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material onto the surface of the electrode, drying for 10 min under an infrared lamp, electrodepositing gold for 10 s with chloroauric acid solution, and transferring 3 mu L of 2 mu M DNA-H1Dropwise adding to the surface of the electrode, incubating at 37 deg.C for 2H, washing with buffer solution, air drying, dropwise adding 5 μ L of 0.1 mM MCH sealant, washing with buffer solution, air drying, and dropwise adding 3 μ L of mixed solution containing target and 3 μ L of DNA-H2Incubating at 37 ℃ for 2 h, dripping 3 mu L of conjugated signal label, incubating at 37 ℃ for 2 h, washing with distilled water and drying to obtain the sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material/aptamer/gold electrode;
(4) the electrochemical sensor is an electrochemical aptamer sensor for detecting T-2 toxin residues, which is obtained by using a sulfur-doped molybdenum nanosheet/functionalized graphene quantum dot composite material/aptamer/gold electrode as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode and responding to the change of signals through signal molecules.
Example 2
The method comprises the following specific steps:
(1) preparing a sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material by adopting a microwave synthesis method, preparing functionalized carbon quantum dots, weighing 5 g of urea and 4 g of citric acid, adding the urea and the citric acid into 12 mL of deionized water, heating the solution in a microwave oven under 1000W of power for 10 min, centrifuging the aqueous solution of the functionalized carbon quantum dots for 20 min at the rotating speed of 4000 rpm, freeze-drying to obtain functionalized carbon quantum dot powder, preparing the sulfur-doped cobalt nanosheet/carbon quantum dot composite material by adopting a hydrothermal synthesis method, dissolving 1 g of sodium hexanitrocobaltate in 60 mL of ethylene glycol solution, carrying out ultrasonic treatment for 50 min at room temperature, then adding 1.2 g of thiourea, adding 0.1 g of functionalized carbon quantum dots, carrying out ultrasonic treatment for 60 min, transferring the mixed solution into a stainless steel autoclave, carrying out thermal reaction for 15 h at the temperature of 150-200 ℃, drying for 14 h at 80 ℃ to obtain the sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material;
(2) signal tag affix sum: 100. mu.L of 5. mu.M signal tag DNA-S1Strand added 100. mu.L of signal tag DNA-S at the same concentration2Then incubating the mixed solution at 40 ℃ for 4 h; t-2 toxin aptamer conjugated with helper DNA and: mu.L of 5. mu.M T-2 toxin aptamer was added to 100. mu.L of the same concentration of helper DNA, and the mixed solution was incubated at 40 ℃ for 4 h. Adding 40 mu L of target objects with different concentrations into a mixed solution of 40 mu L T-2 toxin aptamer and auxiliary DNA, and then placing the mixed solution at 40 ℃ for incubation for 4 h to obtain conjugated T-2 toxin aptamer and auxiliary DNA;
(3) constructing a sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material/aptamer/gold electrode: soaking a gold electrode in a piranha solution for 30 min, washing with ultrapure water, polishing with alumina powder, sequentially placing the polished gold electrode in ethanol and ultrapure water for ultrasonic treatment for 10 min, performing electrochemical activation with 1M acid solution, dropwise adding 5 mu L of 4 mg/mL sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material onto the surface of the electrode, drying for 20 min under an infrared lamp, electrodepositing gold for 20 s by using a chloroauric acid solution, and transferring 5 mu L of 4 mu M DNA-H1Dropping the mixture to the surface of an electrode, incubating at 40 ℃ for 4H, washing and drying the mixture in the buffer solution, dropping 8 mu L of 0.4 mM MCH sealant, washing and drying the mixture in the buffer solution, and dropping 5 mu L of the mixed solution containing the target substance and 5 mu L of DNA-H2Incubating at 40 ℃ for 4 h, dripping 5 mu L of conjugated signal label, incubating at 40 ℃ for 4 h, washing with distilled water and drying to obtain the sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material/aptamer/gold electrode;
(4) the electrochemical sensor is an electrochemical aptamer sensor for detecting T-2 toxin residues, which is obtained by using a sulfur-doped cobalt nanosheet/functionalized carbon quantum dot composite material/aptamer/gold electrode as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode and responding to the change of signals through signal molecules.
The prepared electrochemical sensor has the characteristics of high accuracy, wide linear range (1 fg/mL-100 ng/mL) and low detection lower limit (0.2 fg/mL) on the detection of the T-2 toxin. Meanwhile, the detection result of the actual sample (such as T-2 toxin in beer and feed) shows that the prepared sensor has very good practical application value.
The above examples are intended to illustrate the invention, but not to limit it. Many modifications and variations of the present invention are possible in light of the above teachings. Within the scope of the appended claims, the invention may be practiced other than as specifically described, and it is within the scope of the claims to select other reagent materials, adjust dispersion times, and the like.
Claims (3)
1. A preparation method of an electrochemical sensor for detecting residual T-2 toxin is characterized by comprising the following steps:
(1) preparing a sulfur-doped metal nanosheet/functionalized carbon nanocomposite material: preparing a functionalized carbon nano material by adopting a microwave synthesis method, weighing 3-5 g of urea and 2-4 g of citric acid, adding into 8-12 mL of deionized water, placing the solution in a microwave oven, heating for 5-10 min under the power of 500-1000W, centrifuging the aqueous solution of the functionalized carbon nano material for 10-20 min at the rotating speed of 2000-4000 rpm, and freeze-drying to obtain functionalized carbon nano material powder; preparing a sulfur-doped metal nanosheet/carbon nanomaterial composite material by adopting a hydrothermal synthesis method, dissolving 0.5-1 g of metal salt in 40-60 mL of ethylene glycol solution, carrying out ultrasonic treatment at room temperature for 30-50 min, then adding 1-1.2 g of thiourea, adding 0.05-0.1 g of functionalized carbon nanomaterial, carrying out ultrasonic treatment for 40-60 min, transferring the mixed solution into a stainless steel autoclave, carrying out thermal reaction at 150-200 ℃ for 10-15 h, and drying at 60-80 ℃ for 10-14 h to obtain the sulfur-doped metal nanosheet/functionalized carbon nanomaterial composite material; the metal salt is one or more of sodium molybdate and sodium hexanitrocobaltate;
(2) signal tag affix sum: 100 mu L of 3-5 mu M signal label DNA-S1The strand was added with 100. mu.L of signal tag DNA-S at the same concentration2Then incubating the mixed solution at 37-40 ℃ for 2-4 h; t-2 toxin aptamer conjugated with helper DNA and: adding 100 mu L of 3-5 mu M T-2 toxin aptamer into 100 mu L of auxiliary DNA with the same concentration, incubating the mixed solution at 37-40 ℃ for 2-4 h, adding 20-40 mu L of target objects with different concentrations into the mixed solution of 20-40 mu L T-2 toxin aptamer and auxiliary DNA, and incubating at 37-40 ℃ for 2-4 h to obtain the conjugated T-2 toxin aptamer and the auxiliary DNA; the signal label is one or more of methylene blue, ferrocene and thionine;
(3) constructing a sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode: placing a gold electrode in a piranha solution, soaking for 20-30 min, washing with ultrapure water, polishing with alumina powder, sequentially placing the polished gold electrode in ethanol and ultrapure water for ultrasonic treatment for 5-10 min, performing electrochemical activation with 0.5-1M of acid solution, dropwise adding 3-5 mu L of 2-4 mg/mL sulfur-doped metal nanosheet/functionalized carbon nanocomposite to the surface of the electrode, drying for 10-20 min under an infrared lamp, electrodepositing gold for 10-20 s with chloroauric acid solution, and transferring 3-5 mu L of 2-4 mu M DNA-H1Dropwise adding the mixed solution to the surface of an electrode, incubating for 2-4H at 37-40 ℃, washing and drying the mixed solution by buffer solution, dropwise adding 5-8 mu L of 0.1-0.4 mM MCH sealant, washing and drying the buffer solution, and dropwise adding 3-5 mu L of mixed solution containing a target and 3-5 mu L of DNA-H2Incubating at 37-40 ℃ for 2-4 h, dripping 3-5 mu L of conjugated signal label, incubating at 37-40 ℃ for 2-4 h, washing with distilled water and drying to obtain a sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode;
(4) the electrochemical sensor uses a sulfur-doped metal nanosheet/functionalized carbon nanocomposite/aptamer/gold electrode as a working electrode, a platinum wire electrode as a counter electrode and a saturated calomel electrode as a reference electrode, and an electrochemical aptamer sensor for detecting T-2 toxin residue is obtained through the change of a signal molecule response signal.
2. The method for preparing an electrochemical sensor for detecting T-2 toxin residue as claimed in claim 1, wherein in step (1), the carbon nanomaterial is one or more of graphene, carbon quantum dots, graphene quantum dots, and single-walled carbon nanotubes.
3. The method of claim 1, wherein the buffer solution in step (3) is one or more of phosphate buffer, tris buffer, and 3-morpholinopropanesulfonic acid buffer, and the acidic solution is one or more of sulfuric acid, nitric acid, and phosphoric acid.
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