Synthesis method of Cu2O and CDs probe and application of Cu2O and CDs probe in detection of ZEN and / or AFB1 toxin

Abstract

The invention discloses a synthesis method of a Cu2O and CDs probe and application of the Cu2O and CDs probe to detection of ZEN and / or AFB1 toxin, and belongs to the technical field of detection, Cu2O and CDs bionic nano-enzyme is formed by self-assembly through the synergistic effect between cuprous oxide and carbon dots, and meanwhile, a colorimetric fluorescent aptamer sensor capable of synchronously detecting ZEN and / or AFB1 is constructed based on the nano-enzyme. The whole detection time of the sensor can be controlled within 60 min, batch detection of at most 96 samples can be carried out, the sensor is short in detection time, high in detection sensitivity, simple to operate and high in detection efficiency, has excellent specificity, repeatability and storage stability, provides a new idea for rapid detection of mycotoxin, and has good application prospects. The method has potential application value in the aspect of food safety detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to an aptamer sensor, and in particular to a synthesis method of a Cu2O@CDs probe and application thereof in detection of ZEN and / or AFB1 toxins. BACKGROUND

[0002] Contamination of cereal crops and animal feed by various mycotoxins is a global problem. Among them, zearalenone (ZEN) has the widest contamination range in food, and aflatoxin B1 (AFB1) is the most toxic among many mycotoxins. Because of the high thermal stability of these two toxins, conventional food processing methods are difficult to remove. In addition, these two toxins often coexist in contaminated food and their products, and the synergistic and additive effects increase the toxicity, thereby increasing the threat to human and animal health and causing more serious food safety problems. It is necessary to develop a method for simultaneous detection of ZEN and AFB1 to accurately assess their contamination of food.

[0003] At present, the detection of mycotoxins mainly relies on instrumental analysis methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). These methods have high sensitivity and accuracy, but also have significant limitations such as high cost of instrument purchase and maintenance, long overall detection process (especially involving complex sample pretreatment), and the need for professional technical personnel. These factors make it difficult for existing technology to be widely applied in primary detection institutions, small and medium-sized food processing enterprises, and on-site rapid screening. More notably, the current mainstream detection system is mostly designed and optimized for a single mycotoxin. When multiple toxins (such as zearalenone (ZEN) and aflatoxin B1 (AFB1)) coexist in actual samples, two independent and complete detection procedures usually need to be performed. This "single target, multiple detection" mode not only significantly increases the detection cost per sample (including reagent consumption, labor, and time), but also may introduce uncontrollable cross-contamination risk due to multiple repeated sample preparation, transfer, and on-machine operation processes, thereby posing a potential threat to the reliability of the final detection results. Therefore, breaking through the bottleneck of existing detection technology and developing a new sensing technology that can simultaneously, quickly, and accurately identify multiple mycotoxins (i.e., with multiple detection capability), has a simple operation process (such as reducing or simplifying the pretreatment steps, easy to automate or portable), and has low overall cost, has become a crucial and urgent research direction in the field of food safety detection. The breakthrough of such technology will greatly improve the efficiency and coverage of mycotoxin risk monitoring, and better protect food safety and public health. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a synthesis method of Cu2O@CDs probe and its application in detecting ZEN and / or AFB1 toxins. The present application mediates the synthesis of Cu2O nanocrystals by changing the amount of reagents, and at the same time, different excitation wavelengths of CDs are prepared by different synthesis methods. Then, the Cu2O nanocrystals are assembled with CDs to construct a detection sensor system for fungal toxins ZEN and AFB1 in the field of food. In the construction process, no complex steps are needed. The aptamer is adsorbed on the surface of Cu2O@CDs to form a nano probe through the action of a crosslinking agent. The probe has excellent stability and specificity, and exhibits excellent performance in the rapid synchronous detection of ZEN and AFB1 toxins, and can be used as an efficient and rapid detection means.

[0005] To solve the deficiencies of the prior art, the technical scheme adopted by the present application is:

[0006] The synthesis method of the Cu2O@CDs probe comprises the following steps:

[0007] Step 1, Cu2O nanocrystals are added into an ethanol / water mixture, and then APTES is added. After ultrasonic dispersion for 20 min, reaction is carried out at 75 DEG C for 1 h. Then, the excess APTES is removed by washing with anhydrous ethanol, and the amino Cu2O solution is obtained by re-dispersing the product in 10 mL of an ethanol / water mixture;

[0008] Step 2, the amino Cu2O solution is uniformly dispersed in a PBS buffer, and CDs are added and ultrasonically treated for 20 min. The Cu2O@CDs crude product is collected by centrifugation;

[0009] Step 3, the Cu2O@CDs crude product is washed with deionized water for 3 times, and then dissolved in deionized water to obtain Cu2O@CDs.

[0010] As an improvement, the mass of Cu2O in step 1 is 0.1 g, the volume of the ethanol / water solution is 10 ml, and the volume of APTES is 600 μL.

[0011] As an improvement, when the particle size of the Cu2O nanocrystals is 30-100 nm, 0.5 mL, 0.6-1.2 mol·L -1 of deionized water and 0.5 mL, 1.2-2.4 mol·L -1 of NaOH solution are measured and added dropwise. -1 As an improvement, when the particle size of the Cu2O nanocrystals is 30-100 nm, 0.5 mL, 0.6-1.2 mol·L -1Ascorbic acid, then after stirring for 30 min, orange-red precipitate was obtained; after standing, centrifugal washing was performed with deionized water and anhydrous ethanol for 3 times, respectively, and finally vacuum drying at 60℃ for 6h, Cu2O nanoscale enzyme was obtained.

[0012] Further improvement is that when the concentration of trisodium citrate is 0.6-1.2M and the concentration of ascorbic acid is 0.6M, the particle size of the obtained nanoscale enzyme is 30nm-100nm cubic.

[0013] As an improvement, the synthesis method of Cu2O nanoscale enzyme in step 1 is as follows:

[0014] When the particle size of the Cu2O nanoscale enzyme is 500-900nm, the synthesis method of Cu2O nanoscale enzyme is as follows:

[0015] At 55℃, 10mL of 1-2M NaOH aqueous solution was added dropwise into 100mL of 0.01M CuCl2·2H2O aqueous solution under stirring by water bath at 1600rpm, and after stirring for 0.5h, 10mL of 0.6M ascorbic acid solution was added, and stirring was continued for 3h, then the precipitate was collected by centrifugation, washed with distilled water and ethanol, and freeze-dried, Cu2O nanoscale enzyme was obtained.

[0016] As an improvement, the hydrothermal reaction temperature in step 2 is 30℃, and the amount of CDs is 15μL.

[0017] As an improvement, the synthesis method of the CDs includes the following steps:

[0018] Step 1, dissolve citric acid and urea in 10mL of ultrapure water, and stir until a transparent and uniform solution is obtained;

[0019] Step 2, irradiate the mixture with a microwave oven, and after the reaction is completed, cool to room temperature;

[0020] Step 3, centrifugal washing of the brown-black solid with water and ethanol aqueous solution for 2-4 times;

[0021] Step 4, filter the CDs solution through a 0.22μm microporous membrane, and dry in a vacuum oven, CDs are obtained.

[0022] As an improvement, in step 2, the power of the microwave oven is 750W, and the irradiation time is 150s; in step 3, the rotation speed is 10000rpm, and the time is 10min.

[0023] The application of the above-mentioned biosensor based on Cu2O@CDs probe in detecting ZEN and / or AFB1 toxin in food.

[0024] As preferred, the specific steps of the application are as follows:

[0025] First step, construction of colorimetric fluorescence aptamer sensor:

[0026] Take 50 μL, 1 μM H1 and 50 μL, 1 μM H2 into 200 μL centrifuge tube, mix evenly, and then incubate at 37℃ in constant temperature oscillator for 60 min, and the TF-DNA double-stranded (H1 / H2) structure is prepared; add 100 μL of streptavidin solution with a concentration of 10 μg / mL to each well of the 96-well plate, and incubate at 37℃ for 3 h, then remove the liquid in the plate, and wash each well with 0.01M, 200 μL PBS solution for 3 times; then add 100 μL of 0.1% BSA solution to each well to block the non-specific binding sites, and immerse the 96-well plate at 25-30℃ overnight, then wash with PBS solution and dry, and drop 50 μL of TF-DNA double-stranded (H1 / H2) solution on the surface of the streptavidin-coated well plate, and incubate at 37℃ for 20 min, then wash with PBS solution for 3 times, and obtain the blank sensor based on Cu2O@CDs nanometer enzyme, and store at 4℃;

[0027] Second step, construction of probe:

[0028] Take 2 mL Cu2O@CDs probe, add 100 μL of streptavidin solution with a concentration of 100 μg / mL, stir at 25-30℃ for 30 min, and then stand at 37℃ for 2 h, then add the aptamer corresponding to CDs to the solution, stir at room temperature for 3 h, then add 200 μL of 0.01% BSA solution, and continue to stir at room temperature for 3 h, then centrifuge the mixed solution at 10000 rpm for 5 min, resuspend the solid collected after centrifugation with PBS buffer solution, and store at 4℃;

[0029] Third step, detection of mycotoxin:

[0030] Take a centrifuge tube, add 10 μL of ZEN solution with different concentrations (0-5 ng / mL) and 10 μL of AFB1 solution with different concentrations (0-5 ng / mL) in sequence, and incubate at 37℃ for 1 h, and obtain 40 μL of toxin and aptamer mixed solution. Add the mixed solution to the blank sensor based on Cu2O@CDs nanometer enzyme, mix evenly after oscillation, and then incubate the well plate at 37℃ for 1 h; then, transfer the toxin-aptamer mixed solution to the 96-well plate, stand for 20 min, then remove the solution, add Tris-HCl buffer solution with pH 7.4 to a total volume of 200 μL, and finally use a fluorescence spectrophotometer to record the fluorescence spectrum of the solution at the excitation wavelength;

[0031] Fourth step, application in food:

[0032] The corn flour or wheat flour solution containing 10 muL of ZEN and 10 muL of AFB1 was added into the same centrifuge tube, and incubated at 37 DEG C for 1h, 40 muL of toxin and aptamer mixed solution was obtained, and was added into the blank sensor based on Cu2O@CDs nanometer enzyme, after uniform shaking, the hole plate was placed in 37 DEG C, and incubated for 1h, then the toxin and aptamer mixed solution was added into the 96 hole plate, after 20 min, the solution was drawn out, and the Tris-HCl buffer solution with pH = 7.4 was supplemented to 200 muL, and the fluorescence spectrophotometer was used to record the fluorescence spectrum of the solution under the excitation wavelength.

[0033] Advantages:

[0034] Compared with the prior art, the synthesis method of the Cu2O@CDs probe and the application thereof in detecting ZEN and / or AFB1 toxin are provided.The different particle sizes of Cu2O are synthesized by changing the amount of trisodium citrate, the novel cubic Cu2O nanomaterial has a signal of rapid response to TMB and a fluorescence signal of carbon dots adsorbed on the cubic Cu2O, and the sensor constructed by the cubic Cu2O can be applied to the synchronous detection of ZEN and AFB1 toxin.Compared with the traditional detection method, the detection time is greatly shortened, and the sensor has good applicability, stability, good feasibility and specificity, provides a new idea for rapid detection of mycotoxins, and has potential application value in food safety detection.The specific advantages are as follows:

[0035] 1.The different particle sizes of Cu2O are synthesized by adjusting the amount of trisodium citrate, TMB is used as a chromogenic substrate to determine that the Cu2O has peroxidase characteristics, and the cubic Cu2O with a large specific surface area has the highest enzyme activity; different fluorescent colors of carbon dots are synthesized by adjusting the amount of different urea, and the carbon dots are adsorbed on the surface of the Cu2O, so that the Cu2O is directly endowed with the fluorescence ability that the Cu2O does not have.

[0036] 2.In the preparation of the Cu2O@CDs probe, the process is simple, the Cu2O@CDs-Apt probe can be directly constructed by cross-linking streptavidin and aptamer, and in the detection process, as the concentration of ZEN and AFB1 increases, the fluorescence signal is higher, the colorimetric signal is lower, the color is lighter, the detection signal has a certain linear relationship with the target concentration, the naked eye can directly distinguish, and the rapid quantitative detection of ZEN and AFB1 can also be realized.

[0037] 3.The sensor prepared by the Cu2O@CDs nanosenze probe has good environmental applicability and biocompatibility, has good anti-interference ability in the detection process, and can realize the detection of multiple samples at the same time by taking 96-well plates as carriers, greatly shortening the detection time. In addition, the problem of inactivation of natural enzymes in harsh environments does not need to be considered in the detection process, which has good applicability and can realize convenient detection, and can greatly reduce the detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a scanning electron microscope image and a transmission electron microscope image of Cu2O with different particle sizes in the application, wherein A: 30nm, B: 80nm, C: 200nm, D: 500nm, and E: 900nm;

[0039] Figure 2 It is a transmission electron microscope image of CDs with different excitation wavelengths (A: CDs-1; B: CDs-2);

[0040] Figure 3 It is an XRD diagram of Cu2O, CDs and Cu2O@CDs with different particle sizes in the application;

[0041] Figure 4 It is a response of Cu2O with different particle sizes to the substrate TMB at pH 6 when the concentration of H2O2 remains unchanged (A), a response of Cu2O with different shell thicknesses to the concentration of the substrate H2O2 at pH 6 when the concentration of TMB remains unchanged (B), and double-reciprocal diagrams (C) and (D) corresponding to (A) and (B);

[0042] Figure 5 It is a condition optimization of the colorimetric and fluorescent aptamer sensor based on the construction of Cu2O@CDs in the application (A: reaction time; B: TMB concentration; C, D: aptamer incubation time);

[0043] Figure 6 It is a single-target and double-mode detection of ZEN (concentration: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5ng / mL) by the colorimetric and fluorescent aptamer sensor based on Cu2O@CDs in the application (A: fluorescence spectrum; B: fluorescence standard curve; C: ultraviolet-visible absorption spectrum; D: ultraviolet standard curve);

[0044] Figure 7The AFB1 (concentration: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 ng / mL) single-target dual-mode detection (A: fluorescence spectrum; B: fluorescence standard curve; C: ultraviolet-visible absorption spectrum; D: ultraviolet standard curve) of the colorimetric fluorescence aptamer sensor based on Cu2O@CDs of the application;

[0045] Figure 8 The ZEN and AFB1 double-target synchronous rapid detection (A: fluorescence spectrum; B: fluorescence standard curve) of the colorimetric aptamer sensor based on Cu2O@CDs of the application;

[0046] Figure 9 (A) is the stability of the colorimetric aptamer sensor based on Cu2O@CDs of the application;

[0047] Figure 9 (B) is the specificity of the colorimetric aptamer sensor based on Cu2O@CDs of the application;

[0048] Figure 9 (C) is the recovery rate of the actual sample (wheat flour and corn flour) of the colorimetric aptamer sensor based on Cu2O@CDs of the application (A: ZEN; B: AFB1)

[0049] Figure 10 It is a schematic diagram of the synthesis of Cu2O@CDs material and the detection principle of the colorimetric aptamer sensor based on Cu2O@CDs of the application. DETAILED DESCRIPTION

[0050] Example 1

[0051] (1) 30 nm cuprous oxide nanoscale enzyme:

[0052] Take 200 mL of deionized water and 0.5 mL, 0.6 mol·L -1 Sodium citrate in a 250 mL beaker, stirring at 1200 rpm in a 30°C water bath for 20 min; quickly inject 0.5 mL, 1.2 mol·L -1 CuSO4 solution; after 5 min of reaction, add 0.5 mL, 4.8 mol·L -1 NaOH solution; continue to react for 5 min, add 0.5 mL, 1.2 mol·L -1 Ascorbic acid, then after 30 min of reaction, orange-red precipitate is obtained; after standing, centrifugal washing with deionized water and anhydrous ethanol for 3 times respectively, finally drying in a 60°C vacuum drying oven for 6 h, ready for use.

[0053] (2) 80 nm cuprous oxide nanoscale enzyme:

[0054] The concentration of trisodium citrate was changed to 1.2 mol·L -1 The rest was the same as (1).

[0055] (3) 500 nm cuprous oxide nanoszyme:

[0056] At 55 °C, 10 mL of 1 M NaOH aqueous solution was added dropwise into the transparent light green CuCl2·2H2O aqueous solution (100 mL, 0.01 M) with vigorous stirring. After stirring for 0.5 h, 10 mL of 0.6 M AA solution was added into the above dark brown turbid solution, and stirred for another 3 h. Finally, the precipitate was collected by centrifugation, and then washed thoroughly with distilled water and ethanol before freeze-drying.

[0057] (4) 900 nm cuprous oxide nanoszyme:

[0058] The concentration of NaOH aqueous solution was changed to 2 M, and the rest was the same as (4).

[0059] (5) Different color fluorescent carbon dots were synthesized by a simple, fast, one-step microwave pyrolysis method using citric acid and urea as precursors.

[0060] (5-1) Blue carbon dots:

[0061] 3 g of citric acid and 3 g of urea were dissolved in 10 mL of ultrapure water to form a transparent and homogeneous solution. After irradiating the mixture with a microwave oven (750 W) for 150 s, it was cooled to room temperature. Subsequently, the brown-black solid was collected by centrifugation, and then washed with water and ethanol aqueous solution for 4 times, respectively, at a centrifugal speed of 10000 rpm for 10 min. Finally, the CDs solution was filtered through a 0.22 μm microporous membrane to further purify and separate unreacted molecules and remove excess solvent. The CDs powder was dried in a vacuum oven at 60 °C for 1 hour. The CDs powder (denoted as CDs-1) was obtained by evaporation, re-dispersed in deionized water, and stored at 4 °C for further use.

[0062] (5-2) Orange carbon dots:

[0063] Dissolve 50 mg of citric acid and 50 mg of urea in 10 mL of ultrapure water, stir until uniform, then transfer to the reaction vessel of the microwave reactor, seal and keep at 200°C, heat for 20 min to obtain a red solid solution. Then, centrifuge the red solid, and then use water and ethanol solution to centrifuge the red solid 4 times, the centrifugal speed is 10000 rpm, the centrifugal time is 10 min, then filter the CDs solution through a 0.22 μm microporous membrane. In order to further purify and separate the unreacted molecules and remove excess solvent, the CDs powder is dried in a vacuum oven at 60°C for 1 h. Obtain CDs powder (marked as CDs-2) by evaporation, re-disperse in deionized water, and store at 4°C for further use.

[0064] (6) Cu2O@CDs synthesis method

[0065] Step 1, add 0.1 g of Cu2O to 10 mL of ethanol / water mixture, then add 600 μL of APTES, ultrasonic dispersion for 20 min, then react at 75°C for 1 h, then wash with anhydrous ethanol to remove excess APTES, then re-disperse in 10 mL of ethanol / water mixture to obtain an amino Cu2O solution;

[0066] Step 2, evenly disperse 5 mL of the amino Cu2O solution in 5 mL of PBS buffer, add CDs respectively, ultrasonic treatment for 20 min, centrifugal collection of Cu2O@CDs crude product;

[0067] Step 3, wash the Cu2O@CDs crude product with deionized water 3 times, then dissolve in 10 mL of deionized water to obtain Cu2O@CDs, wherein when CDs is CDs-1, the product is marked as Cu2O@CDs-1, and when CDs is CDs-2, the product is marked as Cu2O@CDs-2.

[0068] Figure 1 A~ Figure 1 E is the scanning electron microscope image and transmission electron microscope image of Cu2O with different particle sizes prepared in Example 1. As can be seen from the figure, different particle sizes of cubic cuprous oxide (A: 30 nm; B: 80 nm; C: 200 nm; D: 500 nm; E: 900 nm) are prepared by changing the copper source, temperature, amount of oxidizing agent and reducing agent during the synthesis stage. Cuprous oxide has a uniform three-dimensional morphology and presents a cubic shape.

[0069] Figure 2 A and Figure 2 B is the transmission electron microscope image of CDs with different excitation wavelengths. It can be seen that the particle size of CDs-1 is about 7 nm, and the particle size of CDs-2 is about 3.5 nm.

[0070] Figure 3The crystal properties of Cu2O, CDs and Cu2O@CDs with different particle sizes were studied by X-ray diffraction (XRD). Among them, Figure 3 In A, diffraction peaks appeared at 2θ of 36.4°, 42.3°, 61.3°, 73.5° and 77.3°, corresponding to (111), (200), (220), (311) and (222) crystal faces (JCPDS 05-0667), respectively. The results showed that the Cu2O nanoparticles exhibited cubic phase, and no other diffraction peaks were found, proving that the Cu2O cubes were successfully synthesized. Figure 3 In B, standard characteristic peaks of carbon dots appeared at about 20-30°, proving that the CDs were successfully synthesized. Figure 3 In C, the characteristic peaks of carbon dots and Cu2O cubes were obviously seen, proving that the carbon dots were successfully adsorbed on the surface of Cu2O cubes.

[0071] The steady-state kinetic experiments were performed on the above Cu2O with different particle sizes to verify their catalytic activities, from which Figure 4 As can be seen from A, B, C and D in the above table, the catalytic reaction of Cu2O on TMB and H2O2 followed the typical Michaelis equation. According to the Lineweaver-Burk double-reciprocal plot method, the Vmax and K max of the five different particle sizes of Cu2O were calculated. m Among them, the K m values of TMB as substrate were 0.207 mM; 0.310 mM; 0.224 mM; 0.36 mM; 0.403 mM, lower than that of horseradish peroxidase (HRP, K m = 0.43 mM), and the K m value of Cu2O with a particle size of 30 nm was lower, indicating that the Cu2O material had high affinity for TMB and showed stronger catalytic activity.

[0072] Example 2 Preparation of Cu2O@CDs-Apt Nanoprobes

[0073] 0.1 g of Cu2O with a particle size of 30 nm was added to 10 mL of an ethanol / water mixture (V1 / V2, 19:1), followed by the addition of 600 μL of APTES. After ultrasonic dispersion for 20 min, the reaction was carried out at 75°C for 1 h, followed by washing with anhydrous ethanol to remove excess APTES. Then it was re-dispersed in 10 mL of an ethanol / water mixture (V1 / V2, 19:1).

[0074] 5 mL of the amino-functionalized Cu2O prepared in Example 1 was uniformly dispersed into 5 mL of PBS buffer (pH = 7.4, 0.01 M) respectively. Then 15 μL of CDs-1 and CDs-2 (10 mM) mixture was added respectively, and the mixture was ultrasonically treated for 20 min, and Cu2O@CDs was collected by centrifugation (10000 rpm, 10 min). Finally, the collected Cu2O@CDs was washed with deionized water for 3 times, and then dissolved in 10 mL of deionized water.

[0075] 2 mL of the above Cu2O@CDs solution was taken, 100 μL of streptavidin solution (100 μg / mL) was added, and the mixture was stirred at room temperature for 30 min and stood at 37 °C for 2 h. Then aptamer S1 and S2 (10 μM, 20 μL) was added dropwise, and the mixture was stirred at room temperature for 3 h. Then 200 μL of 0.01% BSA solution was added, and the mixture was stirred at room temperature for another 3 h. Then the mixture was centrifuged at 10000 rpm for 5 min, and the obtained product was resuspended with PBS buffer and stored at 4 °C.

[0076] It should be noted that:

[0077] S1 (ZEN aptamer): Bio-AGC AGC ACA GAG GTC AGA TGC TCA TCT ATC TAT GGT S2 (AFB1 aptamer): TGCACG TGT TGT CTC TCT GTG TCT CGT GC-Bio

[0078] Example 3

[0079] 1. Construction of colorimetric fluorescent aptamer sensor

[0080] 50 μL of 1 μM H1 and 50 μL of 1 μM H2 (H1 and H2 were purchased from Shanghai Bioengineering Co., Ltd.) were added into a 200 μL centrifuge tube, and the mixture was uniformly mixed and then incubated at 37 °C for 60 min in a constant temperature shaker to prepare the TF-DNA double-stranded (H1 / H2) structure.

[0081] Add 100 μL of streptavidin solution (10 μg / mL) to each well of a 96-well plate and incubate at 37°C for 3 h. Wash away the liquid from the plate and wash each well with PBS solution (0.01 M, 200 μL), repeating 3 times. Then add 100 μL of 0.1% BSA solution to each well to block non-specific binding sites. Soak the 96-well plate overnight at room temperature, then wash with PBS solution and dry. Add 50 μL of TF-DNA double-stranded (H1 / H2) solution to the surface of the plate coated with streptavidin solution and incubate at 37°C for 20 min. After washing three times with PBS solution, a blank sensor based on Cu2O@CDs nanozyme is obtained and dried and stored at 4°C.

[0082] It should be noted that:

[0083] H1 (ZEN aptamer complementary chain): TGG AGT TGA GGT TGA GGG TAG TTT TTT TAC CAT AGATAG

[0084] H2 (AFB1 aptamer complementary chain): ACA ACA CGT GCA TTT TTT CTA CCC TCA ACC TCA ACTCCA TTT TTT-Bio

[0085] 2. Optimized testing conditions

[0086] The effects of incubation time, reaction time, and TMB dosage on the target analyte identification by a contrast-color fluorescent aptamer sensor were investigated. The catalytic activity of Cu2O@CDs and TMB were also examined. 2+ The absorption intensity of the colorimetric reaction system at 652 nm was correlated with that at the excitation wavelength of 365 nm, while the fluorescence effect of CDs was correlated with the absorption intensity of the fluorescence spectrum at 365 nm. Therefore, during the optimization of experimental conditions, the absorption peak intensity of the colorimetric reaction system at 652 nm was used as an indicator of the catalytic performance of Cu2O, and the fluorescence absorption peak intensity of the fluorescence reaction system at the excitation wavelength of 365 nm was used as a detection indicator. The detection results are as follows: Figure 5 As shown.

[0087] Figure 5 A shows that the UV absorption intensity of the reaction system gradually increases with the extension of reaction time, but the increase tends to level off at 8 min. Therefore, 8 min was selected as the reaction time for the reaction system.

[0088] Figure 5 B shows that the absorption intensity increases rapidly with the increase of TMB concentration. When the initial TMB concentration is greater than 10 mM, the absorption intensity no longer increases significantly. Therefore, a TMB concentration of not less than 10 mM should be selected in the reaction system.

[0089] Figure 5 C shows that the fluorescence absorption intensity increases with the increase of incubation time, and when the incubation time is greater than 60 min, the fluorescence absorption intensity no longer increases, so the incubation time in the reaction system is not less than 60 min.

[0090] 3、ZEN single-target dual-mode detection

[0091] 10 μL, 1 μM Cu2O@CDs-S1 and 10 μL of different concentrations of ZEN were added to the same centrifuge tube, and incubated at 37°C for 1 h to obtain a 20 μL mixed solution of toxins and aptamers. The mixed solution was added to the blank sensor based on Cu2O@CDs nanoscale enzyme, and after uniform shaking, the well plate was incubated at 37°C for 1 h. After incubation, the solution was added to a 96-well plate, and after 20 min, the solution was removed and supplemented with Tris-HCl buffer (pH = 7.4) to 200 μL. The fluorescence spectrum of the solution was recorded at an excitation wavelength of 365 nm using a fluorescence spectrophotometer. At the same time, 210 μL of 0.2M NaAc-HAc buffer (pH = 6), TMB (30 μL, 10 mM) and H2O2 (60 mL, 100 mM) were sequentially added to the well plate from which the solution was removed, and the reaction was carried out at room temperature for 8 min. Subsequently, the color change and ultraviolet-visible absorption spectrum of the reaction system were recorded.

[0092] The amount of Cu2O@CDs binding changes due to changes in ZEN concentration, so the ZEN concentration of the sample can be determined by identifying the color difference of the TMB produced by the nanoscale enzyme catalysis and the fluorescence signal intensity of the surface-adsorbed carbon dots. The results are shown in Figure 6 .

[0093] Figure 6 A is the fluorescence signal spectrum of the reaction system under different concentrations of ZEN. As the concentration of ZEN increases, the fluorescence absorption intensity of the reaction system at 365 nm gradually increases. As shown in Figure 6 B, the logarithm of the ZEN concentration in the range of 0.0005 ng / L-5 μg / L shows a good linear relationship with the gradually increasing fluorescence absorption intensity at 365 nm. Figure 6 C is the ultraviolet absorption-visible spectrum of the reaction system under different concentrations of ZEN. As the concentration of ZEN increases, the absorption intensity of the reaction system at 652 nm gradually increases. As shown in Figure 6 D, the logarithm of the ZEN concentration in the range of 0.0005 ng / L-5 μg / L shows a good linear relationship with the ultraviolet absorption intensity of the reaction system at 652 nm.

[0094] 4、AFB1 single-target dual-mode detection

[0095] 10 μL of 1 μM Cu2O@CDs-S2 and 10 μL of AFB1 at different concentrations were added to the same centrifuge tube and incubated at 37°C with shaking for 1 h to obtain a 20 μL toxin-aptamer mixed solution. This mixed solution was added to a blank sensor based on Cu2O@CDs nanozymes, shaken thoroughly, and then incubated at 37°C for 1 h. After incubation, the solution was added to a 96-well plate, and after 20 min, the solution was withdrawn, and Tris-HCl buffer (pH = 7.4) was added to bring the total volume to 200 μL. The fluorescence spectrum of the solution at an excitation wavelength of 365 nm was recorded using a fluorescence spectrophotometer. Simultaneously, 210 μL of 0.2 M NaAc-HAc buffer (pH = 6), 30 μL of TMB (10 mM), and 60 mL of H2O2 (100 mM) were added sequentially to the withdrawn solution in the wells, and the reaction was carried out at room temperature for 8 min. Subsequently, the color change and ultraviolet-visible absorption spectrum of the reaction system were recorded.

[0096] The binding amount of Cu2O@CDs changes due to variations in AFB1 concentration. Therefore, the AFB1 concentration in the sample can be determined by identifying the color differences produced by nanozyme catalysis of TMB and the intensity of the fluorescence signal generated by surface adsorbed carbon dots. The results are as follows: Figure 7 As shown.

[0097] Figure 7 A shows the fluorescence spectrum of the reaction system at different concentrations of AFB1. As the concentration of AFB1 increases, the fluorescence absorption intensity of the reaction system at 470 nm gradually increases.

[0098] Figure 7 B shows a good linear relationship between the logarithm of AFB1 concentration in the range of 0.0005 ng / L to 5 μg / L and the increasing fluorescence absorption intensity at 470 nm.

[0099] Figure 7 C represents the UV-Vis absorption spectrum of the reaction system at different concentrations of AFB1. As the concentration of AFB1 increases, the absorption intensity of the reaction system at 652 nm gradually decreases.

[0100] Figure 7 D represents a good linear relationship between the logarithm of AFB1 concentration in the range of 0.0005 ng / L to 5 μg / L and the UV absorption intensity of the reaction system at 652 nm.

[0101] 5. ZEN and AFB1 dual-target single-mode detection

[0102] Take 50 μL, 1 μM H1 and 50 μL, 1 μM H2 into 200 μL centrifuge tube, mix evenly, and incubate at 37℃ in a constant temperature oscillator for 60 min. The TF-DNA double-stranded (H1 / H2) structure is prepared.

[0103] Add 100 μL of streptavidin solution (10 μg / mL) to each well of the 96-well plate, and incubate at 37℃ for 3 h. Wash the liquid in the plate, and wash each well with PBS solution (0.01 M, 200 μL) for 3 times. Then add 100 μL of 0.1% BSA solution to each well to block the non-specific binding sites. Soak the 96-well plate at room temperature overnight, then wash with PBS solution and dry. And drop the TF-DNA double-stranded (H1 / H2) solution (50 μL) on the surface of the streptavidin-coated well plate, and incubate at 37℃ for 20 min. After washing with PBS solution for 3 times, the Cu2O@CDs nanoscale enzyme-based blank sensor is dried and stored at 4℃.

[0104] Add 10 μL, 1 μM Cu2O@CDs-S1 and 10 μL, 1 μM Cu2O@CDs-S2, 10 μL, 1 ng / mL ZEN and 10 μL, 1 ng / mL AFB1 into the same centrifuge tube, and incubate at 37℃ for 1 h. The 40 μL toxin and aptamer mixed solution is obtained. Add the above mixed solution to the prepared Cu2O@CDs nanoscale enzyme-based blank sensor, and then put the well plate into 37℃ for 1 h after shaking evenly. After incubation, add the solution to the 96-well plate, and after 20 min, remove the solution and supplement Tris-HCl buffer (pH=7.4) to 200 μL. Use a fluorescence spectrophotometer to record the fluorescence spectrum of the solution at 365 nm and 490 nm excitation wavelength, respectively.

[0105] With different concentrations of ZEN and AFB1 as analytes, the detection range and sensitivity of the designed aptamer colorimetric fluorescence sensor are determined. As shown in Figure 9 A, in the range of 0-5 ng / mL of ZEN concentration, the fluorescence intensity at 365 nm increases with the increase of ZEN concentration. In addition, after taking the logarithm of the ZEN concentration, the ΔF at 365 nm has a good linear correlation in the linear range of 0.001-10 ng / mL Figure 9 B). The linear equation is ΔF(OTA)=600.78755+195.57478Lg(C OTA ), the correlation coefficient R 2 =0.981, and the detection limit is 0.00085 ng / mL. In addition, as shown in Figure 9C shows that the fluorescence intensity at 470 nm increases with the increase of AFB1 concentration in the range of 0-10 ng / mL. After taking the logarithm of AFB1 concentration, the ΔF at 470 nm has a good linear correlation with the linear range of 0-10 ng / mL Figure 9 D). The linear equation is ΔF(AFB1) = 609.80582 + 197.91366Lg(C AFB1 ), the correlation coefficient R 2 = 0.970, and the LOD is 0.00091 ng / mL.

[0106] Example 4: Detection of ZEN and AFB1 mycotoxins in corn

[0107] 1. Detection of ZEN and AFB1 in actual samples

[0108] 1.1 Sensor specificity research

[0109] Figure 9 To judge the actual detection feasibility of the colorimetric fluorescence aptamer sensor by comparing its stability, reproducibility, and specificity.

[0110] Figure 9 In A, five groups of parallel ratio colorimetric fluorescence sensors were tested for UV absorption intensity and fluorescence intensity response in the presence of ZEN, and the reproducibility was calculated, indicating that the prepared different batches of colorimetric immunosensors showed good reproducibility for ZEN detection. AFB2, OTA, DON, T-2, and other structurally similar mycotoxins were used as interference substances to verify the specificity of the colorimetric fluorescence aptamer sensor for ZEN and AFB1 detection.

[0111] Figure 9 In B, the added interference substances were basically consistent with the blank control group, indicating that they had basically no effect on the detection signal of the colorimetric aptamer sensor. The designed sensor has high selectivity for ZEN, indicating that the prepared colorimetric fluorescence aptamer sensor has good specificity.

[0112] 1.2 Detection of spiked corn and wheat samples

[0113] In order to further study the feasibility and practicability of the colorimetric aptamer sensor, corn flour and wheat flour (Xinxiang City Xinliang Grain and Oil Processing Co., Ltd. in Henan Province) were used as actual samples for ZEN detection. The sample processing method was as follows: the corn sample was ground into powder, 0.50 g of corn flour was taken and then dried at 55°C, the dried sample was fully mixed with 5 mL of methanol-water (volume ratio 7:3) extraction solvent, placed in a shaker for 30 min, centrifuged at 10,000 r / min for 10 min, filtered with a 0.45 μm organic filter membrane to obtain the supernatant, the filtrate was diluted with deionized water, and then ZEN and AFB1 standard samples were added to prepare corn flour samples containing 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 ng / mL ZEN and AFB1, respectively. The same method was used to process the wheat flour, and then the same test scheme was used for detection, as shown in Figs. 1-3, 5-7 and 9-11, the recovery rates of the colorimetric fluorescence aptamer sensor for corn flour and wheat flour were 92.5-102.4% and 93.3-104.9%, respectively, indicating that the designed colorimetric fluorescence aptamer sensor can be used for actual sample detection. Figure 9 C、 Figure 9 D, the recovery rates of the colorimetric fluorescence aptamer sensor for corn flour and wheat flour were 92.5-102.4% and 93.3-104.9%, respectively, indicating that the designed colorimetric fluorescence aptamer sensor can be used for actual sample detection.

[0114] In summary, the colorimetric fluorescence nucleic acid aptamer sensor constructed in the application has the characteristics of high detection sensitivity, simple operation and high efficiency, and is suitable for rapid quantitative detection of ZEN and AFB1, with detection limits of 0.00085 ng / mL and 0.00091 ng / mL, respectively. In particular, the reliability of the sensor was evaluated by detecting the effectiveness of the food standard sample, and the test feedback recovery rates were 92.5-102.4% and 93.3-104.9%, respectively. It is expected to promote the development of the food hazard control monitoring industry towards convenience, automation and high throughput. The combination of the biosensor and automatic devices can replace manual operation and accurately detect the content of ZEN and AFB1 in food in batches.

[0115] The above describes the application and its embodiments in a schematic manner, which is not restrictive, and the embodiments shown in the drawings are only one of the embodiments of the application, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired thereby, without departing from the purpose of the application, similar structural modes and embodiments can be designed without creativity, which should all belong to the protection scope of the application.

Claims

1. A method for synthesizing Cu2O@CDs probes, characterized in that, Comprising the following steps: Step 1, Cu2O nanoszyme is added into ethanol / water mixture, then APTES is added, after ultrasonic dispersion for 20 min, reaction at 75℃ for 1h, then washed with anhydrous ethanol to remove excess APTES, then re-dispersed into 10mL ethanol / water mixture to obtain amino-Cu2O solution; Step 2, the amino-Cu2O solution is uniformly dispersed into PBS buffer, then CDs are added and ultrasonic treated for 20 min, then the Cu2O@CDs crude product is collected by centrifugation; Step 3, the Cu2O@CDs crude product is washed with deionized water for 3 times, then dissolved in deionized water to obtain Cu2O@CDs.

2. The synthesis method of Cu20@CDs probe according to claim 1, characterized in that, The mass of Cu2O in step 1 is 0.1g, the volume of ethanol / water solution is 10ml, and the volume of APTES is 600μL.

3. The synthesis method of Cu20@CDs probe according to claim 1, characterized in that, The synthesis method of the Cu2O nanoscale enzyme in step 1 is that when the particle size of the Cu2O nanoscale enzyme is 30-100 nm, 0.5 mL, 0.6-1.2 mol·L -1 Sodium citrate is stirred in a beaker in a water bath at a constant rate for 20 min, and 0.5 mL, 1.2-2.4 mol·L -1 CuSO4 solution, continue to stir for a period of time; Add 0.5 mL of 4.8–6 mol·L⁻¹ solution dropwise. -1 Add NaOH solution and stir for a while; then add 0.5 mL of 1.2–2.4 mol·L⁻¹ solution dropwise. -1 Ascorbic acid was added, and then stirred for 30 minutes to obtain an orange-red precipitate. After standing, the precipitate was washed three times each with deionized water and anhydrous ethanol, and finally dried under vacuum at 60°C for 6 hours to obtain Cu2O nanozyme.

4. The synthesis method of Cu20@CDs probe according to claim 3, characterized in that, When the concentration of trisodium citrate is 0.6-1.2M and the concentration of ascorbic acid is 0.6M, the particle size of the obtained nanoszyme is 30nm-100nm cubic.

5. The synthesis method of Cu20@CDs probe according to claim 1, characterized in that, The synthesis method of the Cu2O nanoszyme in step 1, when the particle size of the Cu2O nanoszyme is 500-900nm Cu2O nanoszyme synthesis method: 10mL of 1-2M NaOH aqueous solution is added dropwise into 100mL of 0.01M CuCl2·2H2O aqueous solution under stirring at 1600rpm by water bath at 55℃ for 0.5h, then 10mL of 0.6M ascorbic acid solution is added, and stirring is continued for 3h, then the precipitate is collected by centrifugation, washed with distilled water and ethanol, and freeze-dried to obtain Cu2O nanoszyme.

6. The synthesis method of Cu20@CDs probe according to claim 1, characterized in that, The hydrothermal reaction temperature in step 2 is 30℃, and the amount of CDs used is 15μL.

7. The synthesis method of Cu20@CDs probe according to claim 1, characterized in that, The synthesis method of the CDs in step 2, comprising the following steps: Step 1, citric acid and urea are dissolved in 10mL of ultrapure water, and stirred until a transparent and uniform solution is obtained; Step 2, the mixture is irradiated with a microwave oven, and after the reaction is completed, it is cooled to room temperature; Step 3, the brown-black solid is washed by centrifugation with water and ethanol aqueous solution for 2-4 times; Step 4, the CDs solution is filtered through a 0.22μm microporous membrane, and dried in a vacuum oven to obtain CDs.

8. The synthesis method of Cu20@CDs probe according to claim 7, characterized in that, The microwave oven power in step 2 is 750W, and the irradiation time is 150s; the rotation speed in step 3 is 10000rpm, and the time is 10min.

9. Application of the biosensor prepared by the Cu2O@CDs probe synthesized based on the synthesis method of claim 1 in the detection of ZEN and / or AFB1 toxins in food.

10. Use according to claim 9, characterized in that, The specific steps of the application are: First step, construction of colorimetric fluorescent aptamer sensor: Take 50 μL, 1 μM H1 and 50 μL, 1 μM H2 into 200 μL centrifuge tube, mix evenly, and incubate at 37°C in a constant temperature oscillator for 60 min, and the TF-DNA double-stranded (H1 / H2) structure is prepared; add 100 μL of streptavidin solution with a concentration of 10 μg / mL to each well of a 96-well plate, and incubate at 37°C for 3 h, then remove the liquid in the plate, and wash each well with 0.01M, 200 μL PBS solution for 3 times; then add 100 μL of 0.1% BSA solution to each well to block the non-specific binding sites, and immerse the 96-well plate at 25-30°C overnight, then wash with PBS solution and dry, and drop 50 μL of TF-DNA double-stranded (H1 / H2) solution on the surface of the streptavidin-coated well plate, and incubate at 37°C for 20 min, then wash with PBS solution for 3 times to obtain a blank sensor based on Cu2O@CDs nanoscale enzyme, and store at 4°C; Second step, construction of probe: Take 2 mL of Cu2O@CDs probe, add 100 μL of streptavidin solution with a concentration of 100 μg / mL, stir at 25-30°C for 30 min, and then add the corresponding aptamer of CDs to the solution, stir at room temperature for 3 h, then add 200 μL of 0.01% BSA solution, and continue to stir at room temperature for 3 h, then centrifuge the mixed solution at 10000 rpm for 5 min, resuspend the solid collected after centrifugation with PBS buffer solution, and store at 4°C; Third step, detection of mycotoxins: Take a centrifuge tube, add 10 μL of ZEN solution with different concentrations (0-5 ng / mL) and 10 μL of AFB1 solution with different concentrations (0-5 ng / mL) in sequence, and incubate at 37°C for 1 h to obtain 40 μL of toxin and aptamer mixed solution. Add the mixed solution to the blank sensor based on Cu2O@CDs nanoscale enzyme, mix evenly, then place the well plate in a 37°C incubator for 1 h; then, transfer the toxin-aptamer mixed solution to a 96-well plate, stand for 20 min, then remove the solution, add Tris-HCl buffer solution with pH 7.4 to a total volume of 200 μL, and finally use a fluorescence spectrophotometer to record the fluorescence spectrum of the solution at the excitation wavelength; Fourth step, application in food: Respectively add 10 μL of ZEN and 10 μL of AFB1 corn flour or wheat flour solution to the same centrifuge tube, and incubate at 37°C for 1 h to obtain 40 μL of toxin and aptamer mixed solution, and add it to the blank sensor based on Cu2O@CDs nanoscale enzyme, mix evenly, then place the well plate in a 37°C incubator for 1 h, then add the toxin and aptamer mixed solution to a 96-well plate, stand for 20 min, then remove the solution, and add Tris-HCl buffer solution with pH 7.4 to 200 μL, and use a fluorescence spectrophotometer to record the fluorescence spectrum of the solution at the excitation wavelength.

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