CN110736829A - Rapid detection of T-2 toxin in food based on nucleic acid hydrogel - Google Patents

Abstract

The invention belongs to the technical field of food detection, and discloses methods for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel, which comprises the following steps of constructing the nucleic acid hydrogel taking T-2 toxin aptamer as linker cross-linking agent, uniformly embedding horseradish peroxidase in the nucleic acid hydrogel, and adding a sample to be detected into the nucleic acid hydrogel, wherein the concentration of T-2 toxin in the sample to be detected is 0.01ng mL^‑1‑10000ng mL^‑1The T-2 toxin aptamer is combined with the T-2 toxin, the gel is broken, horseradish peroxidase is released, hydrogen peroxide and potassium iodide are catalyzed by the horseradish peroxidase within time to react to generate iodine simple substance etched gold nanorods, and the content of the T-2 toxin is calculated according to an absorption peak of the gold nanorods under an ultraviolet spectrophotometer.

Description

Rapid detection of T-2 toxin in food based on nucleic acid hydrogel

technical field

The invention relates to the technical field of food detection, in particular to a method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel.

Background technique

The description of the background technology in the present invention belongs to the related art related to the present invention, and is only used to illustrate and facilitate the understanding of the invention content of the present invention, and should not be construed as the applicant's explicit belief or presumption that the applicant believes that the present invention is the first application of the present invention. the prior art of the filing date.

In recent years, food safety issues related to mycotoxins have emerged in an endless stream. The common ones include trichothecenes, aflatoxins, zearalenone, etc. Among them, T-2 toxin is the most important type of trichothecenes A toxin. , is also the most toxic one. T-2 toxin mainly acts on tissues and organs with vigorous cell division. It can inhibit the synthesis of DNA, RNA and protein. It has strong genotoxicity, immunotoxicity, blood toxicity, carcinogenicity, teratogenicity, and lethal effect. It is also toxic and induces apoptosis. The pure product of T-2 toxin is a white needle-like crystal, the molecular formula is C ₂₄ H ₃₄ O ₉ , the molecular weight is 466.51, the property is stable, and it has strong heat resistance and ultraviolet resistance. It is quite stable at room temperature. 6 to 7 years or heated to 100 to 120 ℃ for 1 hour, the toxicity is not reduced, so it is not easy to be inactivated by autoclaving during food production and processing. Studies have shown that T-2 toxin may be closely related to human food-induced toxic leukocytosis, Kashin-Beck disease and Keshan disease.

At present, many analytical techniques have been used for the detection of T-2 toxin, including Thin-layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Gas Chromatography (Gas Chromatography) -Mass Spectrometry, GC-MS) and enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA), these high sensitivity and high selectivity methods are the most commonly used methods for quantitative analysis of T-2 toxin. In addition, the immunological analysis method based on the specific recognition of antigen and antibody also provided technical support for the establishment of a quantitative and rapid detection method for T-2 toxin. However, these methods have expensive equipment and high detection costs, require samples to undergo strict pretreatment, and have a long detection time.

SUMMARY OF THE INVENTION

The purpose of the embodiments of the present invention is to provide a method for rapid detection of T-2 toxin in food based on nucleic acid hydrogel. The method of the present invention The method of the present invention is simple, fast, fast, has good selectivity and sensitivity, and can be used for food safety. Analysis of actual samples in testing.

The embodiment of the present invention provides a method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel, comprising the following steps:

Constructing a nucleic acid hydrogel using T-2 toxin aptamer as a linker cross-linking agent, wherein horseradish peroxidase is evenly embedded in the nucleic acid hydrogel;

A sample to be tested is added to the nucleic acid hydrogel, the concentration of T-2 toxin in the sample to be tested is 0.01ng mL ^-1 -10000ng mL ^-1 , the T-2 toxin aptamer and T-2 toxin bind, the gel breaks, releasing horseradish peroxidase;

In a certain period of time, horseradish peroxidase catalyzes the reaction of hydrogen peroxide and potassium iodide to generate iodine to etch gold nanorods, and the content of T-2 toxin is calculated according to the absorption peak of gold nanorods under UV spectrophotometer.

Further, the described construction of nucleic acid hydrogel using T-2 toxin aptamer as a cross-linking agent comprises the following steps:

100 μM of the complementary chain A and 100 μM of the complementary chain B modified with acrylamide were respectively added with a final concentration of 4wt% acrylamide, and then the air was evacuated at 35-40 ° C for 10-15 min to remove oxygen;

Add freshly prepared ammonium persulfate with a final concentration of 1.4% and freshly prepared tetramethylethylenediamine with a final concentration of 2.8%; extract the air at 35-40°C, and react for 10-15min to obtain high molecular complementary chains A and complementary strand B;

Mix the high molecular complementary strand A and the complementary strand B, incubate at 60-65 °C for 5-10 min, and repeat the heating twice to ensure that the reagents are completely mixed. Then add different concentrations of cross-linking agent (30, 35, 40, 45 μM) and horseradish peroxidase, incubate at 60-65 °C for 5-10 min, repeat heating 3 times to ensure that the reagents are mixed, and then cool to room temperature to form Nucleic acid hydrogels.

Wash the surface of the gel with phosphate buffered solution to remove excess horseradish peroxidase on the surface of the gel.

Further, the gold nanorods are prepared by the following method:

To prepare gold seeds: add 0.5-1.0mL of 0.2M cetyltrimethylammonium bromide solution to 0.5-1.0mL of 0.5mM tetrachloroauric acid solution; after shaking and mixing, immediately add 0.1-0.2mL of 6mM freshly prepared The sodium borohydride solution; Vigorously shake for 2-5min, the seed solution changes from yellow to brown, and then stand at room temperature for 30-45min to obtain gold seeds, store at room temperature for later use;

Preparation of growth solution: Weigh 0.9-1.1g cetyltrimethylammonium bromide and 0.11-0.13g 5-bromosalicylic acid in a 250mL round-bottom flask, dissolve with 25-30mL warm water (50-70℃) ; 1.2-1.5 mL of 4.0 mM silver nitrate solution was then added. The obtained solution was placed in a water bath at 30-37°C and allowed to stand for 15-20min, then 25mL of 1.0mM tetrachloroauric acid solution was added and stirred for 15-20min; finally, 0.2mL of 0.064-0.072M ascorbic acid was added and mixed for 30- 60s until the solution becomes colorless, the growth solution can be obtained;

Growth of gold nanorods: Add 80-85 μL of the obtained seed solution to the growth solution obtained above, mix well, place in a water bath at 30-37°C, and let stand for 12-14 hours to obtain the desired gold nanorods. Finally, the obtained gold nanorods were centrifuged and resuspended three times with 0.05M cetyltrimethylammonium bromide to remove the growth solution.

Further, when the substance ratio of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 110:100:45, the response time of the nucleic acid hydrogel is 15 minutes.

Further, the concentration of the potassium iodide is 0.10M.

Further, the concentration of the hydrogen peroxide is 0.075M.

Further, calculating the content of T-2 toxin according to the absorption peak presented by the gold nanorods under the UV spectrophotometer includes the following steps: establishing a standard curve: adding 10 μL of T-2 toxin standard samples of different concentrations to the above hydrogel, at room temperature After standing at room temperature for 15 min, the supernatant was removed and placed in 2 mL of 0.05 mg/mL gold nanorods, 5 mL of 0.10 M potassium iodide, 20 mL of 0.075 M hydrogen peroxide, and after standing at room temperature for 3 min, ultraviolet spectrum scanning was performed.

The embodiment of the present invention has the following beneficial effects:

The method of the invention is a rapid detection method of T-2 toxin in food based on gold nanorods and nucleic acid hydrogel. A nucleic acid hydrogel with T-2 toxin aptamer as a linker was constructed, and Horseradish Peroxidase (HRP) was evenly embedded in the hydrogel. The toxin aptamer is combined with the toxin, the gel is broken, and horseradish peroxidase is released. Within a certain period of time, horseradish peroxidase catalyzes the reaction of hydrogen peroxide and potassium iodide to generate iodine and etch the gold nanorods, so that the gold nanorods are etched. The rods appear in different colors and show absorption peaks at different positions under the UV spectrophotometer. The inventive method has a linear range of 0.01 ng/mL to 10000 ng/mL for detecting T-2 toxin, and the lowest detection limit is 0.87 pg mL ^-1 . Compared with the prior art, the method of the present invention is simple, fast and rapid, has good selectivity and sensitivity, and can be used for the analysis of actual samples in food safety detection.

Description of drawings

Fig. 1 is the schematic flow chart of the present invention based on nucleic acid hydrogel rapid detection of T-2 toxin in food;

Figure 2 is a TEM image of gold nanorods before etching;

Figure 3 is a TEM image of gold nanorods after etching;

Fig. 4 is the ultraviolet absorption spectrogram of gold nanorods before and after etching;

Figure 5 is a non-etchability verification diagram;

Fig. 6 is the optimization of hydrogel gelation conditions;

Fig. 7 is the optimization of hydrogel hydrolysis time;

Fig. 8 is the optimization of potassium iodide concentration;

Fig. 9 is the optimization of hydrogen peroxide concentration;

Figure 10 is a TEM image of the undisintegrated hydrogel;

Figure 11 is a TEM image of a completely disintegrated hydrogel;

Fig. 12 is the ultraviolet spectrum of rapid detection of T-2 toxin in food based on nucleic acid hydrogel;

Figure 13 is a standard curve diagram of rapid detection of T-2 toxin in food based on nucleic acid hydrogel;

Fig. 14 is the stability diagram of different placement time hydrogel;

Figure 15 is a specificity map with other mycotoxins;

Figure 16 is a graph of spike recovery.

Detailed ways

The present application will be further introduced below with reference to the embodiments.

In order to describe the embodiments of the present invention or the technical solutions in the prior art more clearly, in the following description, different "an embodiment" or "embodiments" do not necessarily refer to the same embodiment. Different embodiments can be replaced or combined, and for those skilled in the art, other implementations can also be obtained according to these embodiments without creative efforts.

With reference to Figure 1, a method for rapid detection of T-2 toxin in food based on nucleic acid hydrogel includes the following steps:

Constructing a nucleic acid hydrogel using T-2 toxin aptamer as a linker cross-linking agent, wherein horseradish peroxidase is evenly embedded in the nucleic acid hydrogel;

A sample to be tested is added to the nucleic acid hydrogel, the concentration of T-2 toxin in the sample to be tested is 0.01ng mL ^-1 -10000ng mL ^-1 , the T-2 toxin aptamer and T-2 toxin bind, the gel breaks, releasing horseradish peroxidase;

In a certain period of time, horseradish peroxidase catalyzes the reaction of hydrogen peroxide and potassium iodide to generate iodine to etch gold nanorods, and the content of T-2 toxin is calculated according to the absorption peak of gold nanorods under UV spectrophotometer.

In some embodiments of the present invention, the described construction of the nucleic acid hydrogel using T-2 toxin aptamer as a cross-linking agent comprises the following steps:

100 μM of the complementary chain A and 100 μM of the complementary chain B modified with acrylamide were respectively added with a final concentration of 4wt% acrylamide, and then the air was evacuated at 35-40 ° C for 10-15 min to remove oxygen;

Add freshly prepared ammonium persulfate with a final concentration of 1.4% and freshly prepared tetramethylethylenediamine with a final concentration of 2.8%; extract the air at 35-40°C, and react for 10-15min to obtain high molecular complementary chains A and complementary strand B;

Mix the high molecular complementary strand A and the complementary strand B, incubate at 60-65 °C for 5-10 min, and repeat the heating twice to ensure that the reagents are completely mixed. Then add different concentrations of cross-linking agent (30, 35, 40, 45 μM) and horseradish peroxidase, incubate at 60-65 °C for 5-10 min, repeat heating 3 times to ensure that the reagents are mixed, and then cool to room temperature to form Nucleic acid hydrogels.

Wash the surface of the gel with phosphate buffered solution to remove excess horseradish peroxidase on the surface of the gel.

In some embodiments of the present invention, the gold nanorods are prepared by the following methods:

To prepare gold seeds: add 0.5-1.0mL of 0.2M cetyltrimethylammonium bromide solution to 0.5-1.0mL of 0.5mM tetrachloroauric acid solution; after shaking and mixing, immediately add 0.1-0.2mL of 6mM freshly prepared The sodium borohydride solution; Vigorously shake for 2-5min, the seed solution changes from yellow to brown, and then stand at room temperature for 30-45min to obtain gold seeds, store at room temperature for later use;

Preparation of growth solution: Weigh 0.9-1.1g cetyltrimethylammonium bromide and 0.11-0.13g 5-bromosalicylic acid in a 250mL round-bottom flask, dissolve with 25-30mL warm water (50-70℃) ; 1.2-1.5 mL of 4.0 mM silver nitrate solution was then added. The obtained solution was placed in a water bath at 30-37°C and allowed to stand for 15-20min, then 25mL of 1.0mM tetrachloroauric acid solution was added and stirred for 15-20min; finally, 0.2mL of 0.064-0.072M ascorbic acid was added and mixed for 30- 60s until the solution becomes colorless, the growth solution can be obtained;

Growth of gold nanorods: Add 80-85 μL of the obtained seed solution to the growth solution obtained above, mix well, place in a water bath at 30-37°C, and let stand for 12-14 hours to obtain the desired gold nanorods. Finally, the obtained gold nanorods were centrifuged and resuspended three times with 0.05M cetyltrimethylammonium bromide to remove the growth solution.

In some embodiments of the present invention, when the amount ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 110:100:45, the response time of the nucleic acid hydrogel is 15min.

In some embodiments of the present invention, the concentration of the potassium iodide is 0.10M.

In some embodiments of the present invention, the concentration of the hydrogen peroxide is 0.075M.

In some embodiments of the present invention, calculating the content of T-2 toxin according to the absorption peak presented by gold nanorods under an ultraviolet spectrophotometer includes the steps of: establishing a standard curve: adding 10 μL of T-2 toxin in different concentrations to the above hydrogel 2. Toxin standard sample, after standing for 15min at room temperature, remove the supernatant and put it in 2mL 0.05mg/mL gold nanorods, 5mL 0.10M potassium iodide 20mL 0.075M hydrogen peroxide, after standing at room temperature for 3min, carry out UV spectrum scanning.

Preparation of gold nanorods

Prepare golden seeds. Add 0.5-1.0 mL of 0.2 M cetyltrimethylammonium bromide solution to 0.5-1.0 mL of 0.5 mM tetrachloroauric acid solution. Immediately after mixing by shaking, add 0.1-0.2 mL of 6 mM freshly prepared sodium borohydride solution. Vigorously shake for 2-5min, the seed solution changes from yellow to brown, and after standing at room temperature for 30-45min, gold seeds can be obtained, which can be stored at room temperature for later use.

Prepare growth fluid. Weigh 0.9-1.1 g of cetyltrimethylammonium bromide and 0.11-0.13 g of 5-bromosalicylic acid in a 250-mL round-bottom flask, and dissolve with 25-30 mL of warm water (50-70° C.). 1.2-1.5 mL of 4.0 mM silver nitrate solution was then added. The obtained solution was placed in a water bath at 30-37°C and allowed to stand for 15-20min, then 25mL of 1.0mM tetrachloroauric acid solution was added and stirred for 15-20min. Finally, 0.2mL of 0.064-0.072M ascorbic acid was added, and the solution was mixed for 30-60s until the solution became colorless, and the growth solution was obtained.

Growth of gold nanorods. Add 80-85 μL of the prepared seed solution to the above-obtained growth solution, mix well, place in a 30-37° C. water bath, and let stand for 12-14 hours to obtain the desired gold nanorods. Finally, the obtained gold nanorods were centrifuged and resuspended 3 times with 0.05M cetyltrimethylammonium bromide to remove the growth solution and used for subsequent experiments.

The TEM of the gold nanorods before etching is shown in Figure 2, and the TEM after etching is shown in Figure 3. The corresponding UV absorption spectra are shown in Figure 4. As shown in Figure 5, the non-etchability verification is carried out. In the absence of horseradish peroxidase, hydrogen peroxide and potassium iodide will not react for a certain period of time, and without iodine, the gold nanorods will not be etched to produce peak shifts. .

Preparation of Nucleic Acid Hydrogels

Acrylamide-modified complementary chain A and complementary chain B (100 μM each) were respectively added with a final concentration of 4 wt% acrylamide, and then air was evacuated at 35-40° C. for 10-15 min to remove oxygen.

Freshly prepared ammonium persulfate at a final concentration of 1.4% and freshly prepared tetramethylethylenediamine at a final concentration of 2.8% were added. At 35-40 ℃, the air was exhausted, and the reaction was carried out for 10-15 minutes to obtain the complementary chain A and the complementary chain B of the high molecular weight.

Mix the high molecular complementary strand A and the complementary strand B, incubate at 60-65 °C for 5-10 min, and repeat the heating twice to ensure that the reagents are completely mixed. Then add different concentrations of cross-linking agent (30, 35, 40, 45 μM) and horseradish peroxidase, incubate at 60-65 °C for 5-10 min, repeat heating 3 times to ensure that the reagents are mixed, and then cool to room temperature to form Nucleic acid hydrogels.

Wash the surface of the gel with phosphate buffered solution to remove excess horseradish catalase on the surface of the gel.

Optimization of hydrogel forming conditions

The ratio of the amount of the high molecular complementary chain A, the high molecular complementary chain B and the cross-linking agent was optimized, and the ratio ranged from 100:100:40-100:100:55. As shown in Figure 6, as shown in Figure 6a, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B and the cross-linking agent is 100:100:40, the supernatant of the experimental group after 20 minutes The absorbance value at 310nm was larger, indicating that the hydrogel was fully disintegrated; however, the absorbance value at 310nm of the supernatant in the control group increased significantly with the increase of time after standing for 20min, indicating that when the high molecular complementary chain A, high molecular complementary chain B. When the ratio of the three substances of the crosslinking agent is 100:100:40, the hydrogel is unstable, and even in the absence of the target, it will disintegrate itself. As shown in Figure 6b, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 100:100:45, with the increase of time, the supernatant in the experimental group is hot and spicy. The absorption value of root peroxidase gradually increased, and the absorption value remained almost unchanged after 20 min, indicating that the hydrogel was basically disintegrated after 20 min; at the same time, the 310 nm absorption value of the supernatant of the control group remained basically unchanged within 20 min, It shows that the hydrogel is very stable under this condition. As shown in Figures 6c and 6d, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 100:100:50/55, although the two groups of control groups were in the control group within 40min The absorption value of the supernatant at 310 nm remained almost unchanged, and the hydrogel was very stable. However, after standing for 40 minutes, the absorption value of the supernatant at 310 nm in the corresponding experimental group was smaller, indicating that the hydrogel was too stable under this condition. The toxin can only weakly disintegrate the hydrogel and respond poorly. To sum up, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B and the cross-linking agent is 100:100:45, the nucleic acid hydrogel has the best response to the toxin.

Optimization of hydrogel hydrolysis time

The ratio of the amount of the high molecular complementary chain A, the high molecular complementary chain B and the cross-linking agent was optimized, and the ratio ranged from 90:100:45-120:100:45. As shown in Figure 7, as shown in Figure 7a, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 90:100:45, the supernatant of the control group after 10 minutes The absorption value at 310 nm of the liquid remained almost unchanged, and the hydrogel was very stable. However, after standing for 10 min, the absorption value at 310 nm of the supernatant in the corresponding experimental group was smaller, indicating that under this condition, the hydrogel was too stable, and 1 μM toxin only The hydrogel can be weakly disintegrated, and the response effect is not good. As shown in Figure 7b, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 100:100:45, with the increase of time, the supernatant in the experimental group is hot and spicy. The absorption value of root peroxidase gradually increased, and the absorption value remained almost unchanged after 20 min, indicating that the hydrogel was basically disintegrated after 20 min; at the same time, the 310 nm absorption value of the supernatant of the control group remained basically unchanged within 20 min, It shows that the hydrogel is very stable under this condition. But compared to Figure 7c, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 110:100:45, the phenomenon is basically the same as Figure 7b, but at 15 minutes the experimental group The absorption value of horseradish peroxidase in the supernatant remained almost unchanged, indicating that the hydrogel had basically collapsed completely after 15 minutes. less time. As shown in Figure 7d, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 120:100:45, the absorbance at 310 nm of the supernatant in the experimental group is larger after 15 minutes. , indicating that the hydrogel was fully disintegrated; however, the absorbance at 310 nm of the supernatant in the control group increased significantly with time after standing for 15 min, indicating that when the linker concentration was 40 μM, the hydrogel was unstable, even in the absence of target When it exists, it also disintegrates itself. To sum up, when the ratio of the substances of the high molecular complementary chain A, the high molecular complementary chain B, and the cross-linking agent is 110:100:45, the nucleic acid hydrogel has the best response to the toxin, and the response time is the best. for 15min.

Optimization of potassium iodide concentration

Five concentrations were optimized, 0.08M, 0.09M, 0.10M, 0.11M, and 0.12M, respectively. As shown in Figure 8, when the potassium iodide concentration was between 0.08M and 0.10M, the peak shift gradually increased. At 0.10M, its peak shift reaches the maximum value, so the optimal concentration is 0.10M.

Optimization of hydrogen peroxide concentration

Five concentrations were optimized, which were 0.065M, 0.070M, 0.075M, 0.080M, and 0.085M, respectively. As shown in Figure 9, when the concentration of hydrogen peroxide was between 0.065M and 0.075M, its peak shift gradually increased. When the concentration of hydrogen oxide is 0.075M, its peak shift reaches the maximum value, so the optimal concentration is 0.075M.

Establishment of standard curve

Add 10 μL of T-2 toxin standard samples of different concentrations to the above hydrogel, and after standing at room temperature for 15 min, remove the supernatant and add 2 mL of 0.05 mg/mL gold nanorods, 5 mL of 0.10 M potassium iodide, and 20 mL of 0.075 M hydrogen peroxide After standing at room temperature for 3 min, the UV spectrum was scanned. 10 is the TEM image of the undisintegrated hydrogel, and FIG. 11 is the TEM image of the completely disintegrated hydrogel.

With the above experimental conditions, a standard curve was developed using a series of dilution concentrations of T-2 toxin standard samples. As shown in Figure 12, the peak shift increases with increasing T-2 toxin concentration, and this trend can be explained as follows: When different concentrations of T-2 toxin were added to the hydrogel, the cross-linking in the hydrogel The agent will specifically bind to the toxin, break the hydrogel, turn it into a solution, and release the horseradish peroxidase embedded in the hydrogel. The amount of horseradish peroxidase released is proportional to the toxin content. The liquid in the hydrogel system is moved into the gold nanorods, and horseradish peroxidase catalyzes the reaction of hydrogen peroxide and potassium iodide to generate iodine to etch the gold nanorods, so that the gold nanorods show different colors, and can be detected in ultraviolet spectrophotometry. There are absorption peaks at different positions under the meter, and the peak shift of gold nanorods is proportional to the concentration of T-2 toxin. As shown in Figure 13, taking the T-2 toxin concentration as the abscissa and the peak shift of the gold nanorods as the ordinate, the obtained standard curve is Y=32.3129lgX+54.0285, R ² =0.9991, and the T-2 toxin concentration is 0.01 ng/mL～10000ng/mL showed a good linear relationship, and the minimum detection limit was 0.87pgmL ^-1 .

Stability of the detection method

In order to evaluate the stability of the method of the present invention, hydrogels prepared for 30 days, 20 days, 10 days and 1 day were respectively taken, and standard samples of T-2 toxin with different concentrations of high, medium and low were added. As shown in Figure 14, the peak shifts obtained when the hydrogels were tested with different storage times were not significantly different. It shows that the hydrogel prepared by this method has good stability.

specificity of the detection method

In order to evaluate the specificity of the method of the present invention, the interfering substances ochratoxin (OTA), fumonisin (FB1), aflatoxin B1 (AFB1), vomitoxin (DON), which are both mycotoxins and T-2 toxins, were selected. ), zearalenone toxin (ZEN), theoretically, the specificity of this method mainly depends on the specific binding of T-2 toxin and T-2 toxin aptamer, if T-2 toxin aptamer and other Binding of mycotoxins produces a false positive signal. As shown in Figure 15, the peak shift of T-2 toxin was the most obvious after adding 10 ng/mL of different toxins. It shows that the hydrogel prepared by this method has good specificity.

Detection of actual samples

A spiked recovery experiment was used. Select corn, soybean, and coffee as actual samples. Weigh 1-1.5g corn flour, soybean flour and coffee powder into a 5mL centrifuge tube, then add 2-3mL T-2 toxin solution with a concentration of 100ng/mL (diluted with methanol:PBS=7:3), vortex Centrifuge at 10000rpm/min for 15-20min after vortexing for 5-7min, take the supernatant, filter with 0.45μm filter membrane, and then dilute to 50ng/mL, 1ng/mL, 0.1ng/mL (with methanol:PBS=7:3 dilution) for spike recovery experiments. Different concentrations (50ng/mL, 1ng/mL, 0.1ng/mL) were set in the detection range, and each concentration was measured in parallel for 3 times, and the recovery rate of standard addition was calculated. As shown in Figure 16, the standard addition recovery rate of the present invention was The rate was between 93.72% and 103.07%, and the relative standard deviation (RSD) was between 3.87% and 7.07%. It shows that the method of the present invention is accurate and reliable.

It should be noted that the above embodiments can be freely combined as required. The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1, A method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel, which is characterized by comprising the following steps:

constructing nucleic acid hydrogel taking T-2 toxin aptamer as linker cross-linking agent, wherein horseradish peroxidase is uniformly embedded in the nucleic acid hydrogel;

adding a sample to be detected into the nucleic acid hydrogel, wherein the concentration of the T-2 toxin in the sample to be detected is 0.01ng mL^-1-10000ng mL^-1The T-2 toxin aptamer is combined with the T-2 toxin, the gel is broken, and horseradish peroxidase is released;

in time, horseradish peroxidase catalyzes hydrogen peroxide and potassium iodide to react to generate iodine simple substance etching gold nanorods, and the content of T-2 toxin is calculated according to the absorption peak of the gold nanorods under an ultraviolet spectrophotometer.

2. The method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel of claim 1, wherein the step of constructing the nucleic acid hydrogel with T-2 toxin aptamer as a cross-linking agent comprises the following steps:

adding acrylamide with final concentration of 4 wt% into the complementary strand A100. mu.M and the complementary strand B100. mu.M modified with acrylamide, respectively, and then exhausting air at 35-40 deg.C for 10-15min to remove oxygen;

adding freshly prepared ammonium persulfate with a final concentration of 1.4% and freshly prepared tetramethylethylenediamine with a final concentration of 2.8%; pumping air at 35-40 deg.C, and reacting for 10-15min to obtain high-molecule complementary strand A and complementary strand B;

mixing the high-molecular complementary strand A and the complementary strand B, incubating at 60-65 ℃ for 5-10min, and repeatedly heating twice to ensure that the reagents are completely mixed; adding cross-linking agent (30, 35, 40, 45 μ M) and horse radish peroxidase with different concentrations, incubating at 60-65 deg.C for 5-10min, repeatedly heating for 3 times to ensure mixing of reagents, and cooling to room temperature to form nucleic acid hydrogel;

the glue plane was washed with phosphate buffer solution to remove excess horseradish peroxidase from the glue surface.

3. The method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel of claim 1, wherein the gold nanorods are prepared by the following method:

preparing gold seeds: adding 0.5-1.0mL of 0.2M hexadecyl trimethyl ammonium bromide solution into 0.5-1.0mL of 0.5mM tetrachloroauric acid solution; after shaking and mixing evenly, 0.1-0.2mL of 6mM freshly prepared sodium borohydride solution is added immediately; oscillating vigorously for 2-5min to turn the seed solution from yellow to brown, standing at room temperature for 30-45min to obtain gold seed, and storing at room temperature;

preparing a growth solution: weighing 0.9-1.1g of hexadecyl trimethyl ammonium bromide and 0.11-0.13g of 5-bromosalicylic acid into a 250mL round-bottom flask, and dissolving with 25-30mL warm water (50-70 ℃); then 1.2-1.5mL of 4.0mM silver nitrate solution is added; placing the obtained solution in a water bath kettle at 30-37 deg.C, standing for 15-20min, adding 25mL of 1.0mM tetrachloroauric acid solution, and stirring for 15-20 min; finally, 0.2mL of 0.064-0.072M ascorbic acid is added, and the mixture is uniformly mixed for 30-60s until the solution becomes colorless, so that a growth solution can be obtained;

and (3) growing the gold nanorods: adding 80-85 μ L of the prepared seed solution into the obtained growth solution, mixing uniformly, placing in a 30-37 deg.C water bath, and standing for 12-14h to obtain the desired gold nanorods; finally, the resulting gold nanorods were centrifuged and resuspended 3 times with 0.05M cetyltrimethylammonium bromide to remove the growth medium.

4. The method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel of claim 2, wherein the ratio of the amounts of the high-molecular complementary chain A, the high-molecular complementary chain B and the crosslinking agent is 110: 100: at 45 deg.C, the response time of the nucleic acid hydrogel is 15 min.

5. The method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel of claim 1, wherein the concentration of potassium iodide is 0.10M.

6. The method for rapid detection of T-2 toxin in food based on nucleic acid hydrogel of claim 1, wherein the concentration of hydrogen peroxide is 0.075M.

7. The method for rapidly detecting T-2 toxin in food based on nucleic acid hydrogel of claim 1, wherein the step of calculating the content of T-2 toxin according to the absorption peak of gold nanorods under an ultraviolet spectrophotometer comprises the steps of: establishing a standard curve: adding 10 mu L of T-2 toxin standard samples with different concentrations into the hydrogel, standing for 15min at room temperature, removing the supernatant into 2mL of 0.05mg/mL gold nanorods, 5mL of 0.10M potassium iodide and 20mL of 0.075M hydrogen peroxide, standing for 3min at room temperature, and then carrying out ultraviolet spectrum scanning.

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