Preparation Method and Application of an Electrochemical Sensor Based on Nucleic Acid Aptamer

Abstract

The present invention discloses a preparation method of an aptamer electrochemical sensor, which comprises the following operation steps: (1) synthesizing a porous carbon material (SA-C); (2) taking the porous carbon material (SA-C) obtained in step (1) and refluxing it under concentrated acid to prepare a carboxyl-functionalized porous carbon material (SA-C-COOH); (3) constructing the aptamer electrochemical sensor. The current difference before and after the specific binding of the aptamer electrochemical sensor of the present invention and the logarithm of the patulin concentration show a good linear relationship in the range of 0.5-5000000 ng / L, with high detection sensitivity, good selectivity and accurate detection results.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical analysis and detection, and particularly relates to a preparation method and application of an aptamer electrochemical sensor for nucleic acids. Background Art

[0002] Patulin (PAT), a highly toxic fungal metabolite, is commonly present in rotten vegetables and fruits. It is one of the most harmful mycotoxins to humans, with toxicological effects such as affecting fertility, carcinogenicity, and teratogenicity. It can cause damage to the respiratory and urinary systems, etc. In severe cases, symptoms such as nerve paralysis, pulmonary edema, and renal failure may occur, posing a threat to human health. Therefore, establishing a simple, accurate, and sensitive analytical method for patulin is of great significance for the processing, transportation, and preservation of fruits and vegetables.

[0003] The main analytical methods for patulin include: high-performance liquid chromatography, high-performance liquid chromatography-tandem mass spectrometry, thin-layer chromatography, gas chromatography-mass spectrometry, and capillary electrophoresis, etc. However, these methods all have the disadvantages of cumbersome sample pretreatment, high toxicity of organic reagents used, high instrument operation cost, and long analysis time. Electrochemical methods have been widely used due to advantages such as fast response speed, simple operation, low cost, and easy miniaturization. However, the preparation process of traditional patulin molecularly imprinted electrochemical sensors is complex, with low sensitivity, small adsorption capacity, and difficult elution, making it difficult to meet the detection requirements of low-content patulin in complex-component samples. Therefore, it is urgent to establish a new type of patulin electrochemical sensor with simplicity, rapidity, good selectivity, and high sensitivity. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a preparation method of an aptamer electrochemical sensor for nucleic acids, aiming to obtain an aptamer electrochemical sensor for nucleic acids with high sensitivity, good selectivity, and accurate detection results for patulin.

[0005] To achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A preparation method of an aptamer electrochemical sensor for nucleic acids, comprising the following operating steps:

[0007] (1) Synthesize a porous carbon material (SA-C);

[0008] (2) Take the porous carbon material (SA-C) obtained in step (1) and reflux it under concentrated acid to prepare a carboxyl-functionalized porous carbon material (SA-C-COOH);

[0009] (3) Construction of the aptamer electrochemical sensor: Prepare the carboxyl-functionalized porous carbon material (SA-C-COOH) in step (2) into a carboxyl-functionalized porous carbon (SA-C-COOH) suspension, drop it on the surface of the pretreated glassy carbon electrode, dry it, and then immerse it in a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group. Rinse and dry to obtain the Act / SA-C-COOH / GCE modified electrode. Drop the aptamer solution and incubate to obtain the Aptamer / Act / SA-C-COOH / GCE modified electrode. Rinse and dry; Immerse the dried Aptamer / Act / SA-C-COOH / GCE modified electrode in a bovine serum albumin (BSA) solution for blocking, rinse and dry to obtain BSA / Aptamer / Act / SA-C-COOH / GCE, which is the aptamer electrochemical sensor;

[0010] Among them, the nucleotide sequence in the aptamer solution is: 5’-NH2-(CH2)6-CAGCTCAGAAGCTTGATCCT-GGCCCGCCAACCCGCATCA TCTACACTGATATTTTACCTT-GACTCGAAGTCGTGCATCTG-3’.

[0011] Furthermore, the synthesis of the porous carbon material (SA-C) in step (1) is to heat sodium alginate to 700-900 °C under inert gas protection for 1-2 h, and naturally cool it to room temperature. Wash the obtained black solid with HCl solution and water respectively, and filter to obtain the porous carbon material (SA-C).

[0012] Furthermore, 20 g of sodium alginate is under inert gas protection, and the heating program is 5 °C / min, heating to 800 °C and maintaining for 1.5 h. The molar concentration of the HCl solution is 5 mol / L; the inert gas is argon.

[0013] Furthermore, step (2) is to take the porous carbon material (SA-C) in step (1), add a mixed solution of concentrated H2SO4 and concentrated HNO3 and reflux for 1-3 h, cool to room temperature, wash the obtained solid with water, centrifuge at 8000 r / min for 5 min, and collect the product and dry it at 75 °C to obtain the carboxyl-functionalized porous carbon material (SA-C-COOH).

[0014] Furthermore, the volume ratio of concentrated H2SO4 to concentrated HNO3 in the mixed solution of concentrated H2SO4 and concentrated HNO3 is 3:1, the volume of the mixed solution of concentrated H2SO4 and concentrated HNO3 is 100 mL, the mass of the porous carbon material is 0.5 g, the reflux time is 2 h, and the reflux temperature is 60 °C.

[0015] Further, the rinsing in step (3) is all carried out by rinsing with Tris-HCl buffer solution. The molar concentration of the Tris-HCl buffer solution is 10 mmol / L and the pH value is 7.4. The drying in step (3) is all carried out by baking under an infrared lamp for 15 min.

[0016] Further, in step (3), the carboxyl-functionalized porous carbon material (SA-C-COOH) in step (2) is ultrasonically dispersed in water to prepare a uniformly stable carboxyl-functionalized porous carbon (SA-C-COOH) suspension with a mass concentration of 1.0 mg / mL, and the modification amount of the electrode is 6 μL.

[0017] Further, the pretreated glassy carbon electrode in step (3) is obtained by polishing the glassy carbon electrode (GCE) into a mirror surface with alumina, and then ultrasonically cleaning it successively with HNO3 solution, ethanol and ultrapure water, and naturally drying it.

[0018] Further, the pretreated glassy carbon electrode in step (3) is polished into a mirror surface with alumina with particle sizes of 0.3 μm and 0.05 μm respectively. The volume ratio of nitric acid (HNO3) to water in the HNO3 solution is 1:1. The ethanol is anhydrous ethanol.

[0019] Further, in step (3), the carboxyl group is activated by immersing it in a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 10 - 60 min. In the mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), the molar concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are both 0.5 mol / L.

[0020] Further, the incubation in step (3) is carried out at 30 - 40 °C for 1.5 - 3 h. The time for blocking in step (3) is 10 - 60 min.

[0021] Further, the molar concentration of the aptamer solution in step (3) is 6 μmol / L. The incubation is carried out at 37 °C for 2.5 h. The concentration of the bovine serum albumin (BSA) solution is 0.5%, and the time for blocking is 30 min.

[0022] The aptamer electrochemical sensor prepared by the method of the present invention is used for detecting patulin. The method for detecting patulin includes the following steps:

[0023] Step S1: Using the aptamer electrochemical sensor as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire as the counter electrode, a three-electrode system is formed.

[0024] Step S2: Prepare patulin standard solutions with different concentrations. Immerse the aptamer electrochemical sensor into the patulin standard solutions with different concentrations for specific binding respectively. Then insert the three-electrode system into the mixed solution containing [Fe(CN)6] 3- / 4- and KCl, perform differential pulse voltammetry scanning in the range of -0.10 - 0.50 V, record the current value at 0.160 ± 0.01 V, calculate the current difference ΔI before and after the specific binding of the aptamer electrochemical sensor with patulin through the DPV curve, and obtain the standard curve and linear equation according to the logarithm of the patulin concentration and the value of ΔI.

[0025] Step S3: When actually detecting patulin in the sample, immerse the aptamer electrochemical sensor into the sample to be tested for specific binding, and calculate the current difference ΔI according to the current values measured at 0.160 ± 0.01 V before and after the specific binding in the mixed solution containing [Fe(CN)6] 3- / 4- and KCl. Substitute the current difference ΔI into the linear equation to calculate the patulin concentration value in the sample to be tested.

[0026] Furthermore, the concentrations of the patulin standard solutions in Step S2 are respectively: 0.0 ng / L, 0.5 ng / L, 1 ng / L, 10 ng / L, 100 ng / L, 1000 ng / L, 10000 ng / L, 100000 ng / L, 500000 ng / L, 1000000 ng / L, 5000000 ng / L.

[0027] Furthermore, the linear equation in Step S2 is y = 4.8137x + 5.3212, and the correlation coefficient R 2 = 0.9972; where y is the current difference ΔI before and after the specific binding of patulin and the aptamer electrochemical sensor, and x is the logarithm of the patulin concentration (ng / L).

[0028] Furthermore, the specific binding time in Step S2 and Step S3 is 75 min.

[0029] Furthermore, in Step S2 and Step S3, the molar concentration of [Fe(CN)6] 3- / 4- in the mixed solution containing [Fe(CN)6] 3- / 4- and KCl is 5 mmol / L, and the molar concentration of KCl is 0.1 mol / L.

[0030] Compared with the prior art, the beneficial effects of the present invention:

[0031] (1) The method of the present invention uses a glassy carbon electrode as a substrate and for the first time applies the carboxyl-functionalized porous carbon material SA-C-COOH to the preparation of a patulin electrochemical aptasensor. This sensor is easy to fabricate, simple, highly sensitive, specific, stable, and low-cost.

[0032] (2) Based on the characteristics of the porous carbon material, such as large specific surface area, good conductivity, high biocompatibility, as well as the high biorecognition and strong affinity of nucleic acid aptamers, the present invention significantly improves the intensity and stability of the sensor current signal, and enhances the detection sensitivity and selectivity of the sensor.

[0033] (3) The current difference before and after the specific binding of the nucleic acid aptamer electrochemical sensor of the present invention to patulin shows a good linear relationship with the logarithm of the patulin concentration in the range of 0.5 - 5000000 ng / L. The detection sensitivity is high, the selectivity is good, and the detection result is accurate. Description of the Drawings

[0034] Figure 1 It is a scanning electron microscope (SEM) image of the carboxyl-functionalized porous carbon material (SA-C-COOH) prepared according to the present invention; the magnification is 2.00 μm.

[0035] Figure 2 It is an infrared (FTIR) image of the carboxyl-functionalized porous carbon material (SA-C-COOH) prepared according to the present invention.

[0036] Figure 3 It is a differential pulse voltammetry (DPV) curve graph of the nucleic acid aptamer electrochemical sensor constructed according to the present invention before (curve a) and after (curve b) specific binding to a 1000 ng / L patulin standard solution.

[0037] Figure 4 It is for the nucleic acid aptamer electrochemical sensor constructed according to the present invention in a mixed solution of 5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl, recording the DPV response graph after specific binding to different concentrations of patulin standard solutions for 75 min.

[0038] Figure 5 It is a standard curve graph of the current difference before and after the specific binding of the nucleic acid aptamer electrochemical sensor constructed according to the present invention to the patulin standard solution and the logarithm of the patulin standard solution concentration.

[0039] Figure 6 It is a specific binding experiment graph of the nucleic acid aptamer electrochemical sensor constructed according to the present invention for detecting patulin. Detailed Embodiments

[0040] The following is a detailed description of the specific implementation manners in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners.

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0042] The electrochemical workstation used in the following examples is of the PalmSens4C type, and the parameters of differential pulse voltammetry are set as follows: the potential increment is 4 mV, the amplitude is 50 mV, and the pulse width is 0.2 s.

[0043] The nucleic acid aptamer solution is a commercially available nucleic acid aptamer, which is configured into a solution with a molar concentration of 6 μmol / L using a 10 mmol / L Tris-HCl buffer solution with a pH value of 7.4 as the solvent and then used. The nucleotide sequence in the nucleic acid aptamer solution is as follows:

[0044] 5’-NH2-(CH2)6-CAGCTCAGAAGCTTGATCCT-GGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTT-GACTCGAAGTCGTGCATCTG-3’.

[0045] Example 1

[0046] A preparation method of a nucleic acid aptamer electrochemical sensor, the operation steps are as follows:

[0047] (1) Synthesize porous carbon material (SA-C): Put 20 g of sodium alginate into a porcelain boat, then place the porcelain boat in a tube furnace. Under argon protection, keep the heating program at 5 °C / min, heat up to 800 °C and keep it for 1.5 h, and then naturally cool to room temperature. Wash the obtained black solid with a 5 mol / L HCl solution and water three times respectively, filter by suction, collect the solid, that is, obtain the porous carbon material (SA-C), and dry it at 75 °C for 24 h for standby;

[0048] (2) Preparation of carboxyl-functionalized porous carbon material (SA-C-COOH): Take 0.5 g of the porous carbon material (SA-C) reserved in step (1) and put it into a 100 mL round-bottom flask. Then add 100 mL of a mixed solution of concentrated H2SO4 and concentrated HNO3 (the volume ratio of concentrated H2SO4 to concentrated HNO3 is 3:1). Reflux at 60 °C for 2 h. After cooling to room temperature, wash the obtained solid with water 3 times, centrifuge at 8000 r / min for 5 min, and collect the product and dry it at 75 °C for 24 h to obtain the carboxyl-functionalized porous carbon material (SA-C-COOH) powder; The morphology and chemical composition of the prepared carboxyl-functionalized porous carbon material (SA-C-COOH) were characterized. Among them, the scanning electron microscope (SEM) image of the carboxyl-functionalized porous carbon material (SA-C-COOH) is as Figure 1 shown. From Figure 1 it can be seen that the material has a porous structure with different sizes, indicating that the material is a porous carbon material; The infrared (FTIR) image of the carboxyl-functionalized porous carbon material (SA-C-COOH) is as Figure 2 shown. From Figure 2 it can be seen that characteristic peaks of carboxyl groups appear at 1130 cm -1 , 1727 cm -1 and 3424 cm -1 , indicating that the carboxyl-functionalized porous carbon (SA-C-COOH) was successfully synthesized;

[0049] (3) Construction of aptamer electrochemical sensor: Polish a glassy carbon electrode (GCE) with a diameter of 3 mm into a mirror surface using alumina with particle sizes of 0.3 μm and 0.05 μm respectively. Then ultrasonically clean it successively with HNO3 solution (the volume ratio of nitric acid (HNO3) to water is 1:1), absolute ethanol and ultrapure water, and let it dry naturally for later use;

[0050] Take 1.0 mg of the carboxyl-functionalized porous carbon material (SA-C-COOH) powder prepared in step (2) and ultrasonically disperse it in 1 mL of water to form a uniform and stable carboxyl-functionalized porous carbon (SA-C-COOH) suspension with a mass concentration of 1.0 mg / mL. Take 6 μL of the obtained suspension and drop it on the surface of the above-prepared glassy carbon electrode, and bake it under an infrared lamp for 15 min to obtain the SA-C-COOH / GCE modified electrode. Then immerse it in a mixed solution of 0.5 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 0.5 mol / L N-hydroxysuccinimide (NHS) for 30 min to activate the carboxyl groups on the surface of the SA-C-COOH / GCE modified electrode. Rinse it with 10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution with a pH value of 7.4 and bake it under an infrared lamp for 15 min to obtain the Act / SA-C-COOH / GCE modified electrode. Drop 5 μL of the nucleic acid aptamer solution (Aptamer) with a concentration of 6 μmol / L on the surface of the Act / SA-C-COOH / GCE modified electrode and incubate it in a constant temperature incubator at 37 °C for 2.5 h. The amino-modified nucleic acid aptamer (Aptamer) is assembled onto the surface of the Act / SA-C-COOH / GCE modified electrode through an amidation reaction. Subsequently, rinse it repeatedly with 10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution with a pH value of 7.4 to remove physical adsorption and bake it under an infrared lamp for 15 min to obtain the Aptamer / Act / SA-C-COOH / GCE modified electrode; Immerse the Aptamer / Act / SA-C-COOH / GCE modified electrode in a 0.5% bovine serum albumin (BSA) solution for 30 min to block non-specific adsorption sites, and finally rinse it repeatedly with 10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution and bake it under an infrared lamp for 15 min to obtain BSA / Aptamer / Act / SA-C-COOH / GCE, which is the nucleic acid aptamer electrochemical sensor; Store it at 4 °C for later use; Among them, the nucleotide sequence in the nucleic acid aptamer solution is:

[0051] 5’-NH2-(CH2)6-CAGCTCAGAAGCTTGATCCT-GGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTT-GACTCGAAGTCGTGCATCTG-3’.

[0052] Example 2

[0053] Use the nucleic acid aptamer electrochemical sensor reserved in Example 1 to detect patulin, and the method steps for detecting patulin are as follows:

[0054] (S1) Construct a three - electrode system: Use the aptamer - based electrochemical sensor reserved in Example 1 as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode to form a three - electrode system;

[0055] (S2) Preparation of standard solutions: Accurately weigh 0.0100 g of patulin standard, then dissolve it with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, and make the volume up to 10 mL in a volumetric flask to prepare a patulin stock solution with a concentration of 1000 mg / L; Pipette 0.10 mL of the patulin stock solution into a 100 - mL volumetric flask, and make the volume up to the mark with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, shake well to prepare a standard working solution of patulin with a concentration of 1000 μg / L; Pipette 0.10 mL of the standard working solution of patulin with a concentration of 1000 μg / L into a 100 - mL volumetric flask, and make the volume up to the mark with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, shake well to prepare a standard working solution of patulin with a concentration of 1 μg / L; Pipette 0.00 mL, 0.05 mL, 0.10 mL, 1.00 mL, 10.00 mL of the standard working solution of patulin with a concentration of 1 μg / L into 100 - mL volumetric flasks respectively, and make the volume up to the mark with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, shake well to obtain test standard solutions with concentrations of 0.0 ng / L, 0.5 ng / L, 1.0 ng / L, 10 ng / L, 100 ng / L respectively; Pipette 0.10 mL, 1.00 mL, 10.00 mL of the standard working solution of patulin with a concentration of 1000 μg / L into 100 - mL volumetric flasks respectively, and make the volume up to the mark with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, shake well to obtain test standard solutions with concentrations of 1000 ng / L, 10000 ng / L, 100000 ng / L respectively; Pipette 0.05 mL, 0.10 mL, 0.50 mL of the patulin stock solution with a concentration of 1000 mg / L into 100 - mL volumetric flasks respectively, and make the volume up to the mark with 10 mmol / L tris - (hydroxymethyl) aminomethane - hydrochloride (Tris - HCl) buffer solution with a pH value of 7.4, shake well to obtain test standard solutions with concentrations of 500000 ng / L, 1000000 ng / L, 5000000 ng / L respectively;

[0056] Standard curve plotting: Immerse the aptamer electrochemical sensor reserved in Example 1 into the above-mentioned patulin standard solutions with different concentrations for 75 min for specific binding, and then insert the three-electrode system into a mixed solution containing 5 mmol / L 3- / 4- and 0.1 mol / L KCl, and perform differential pulse voltammetry (DPV) scanning in the range of -0.10 - 0.50 V, record the current value at 0.160 ± 0.01 V, and plot the standard curve according to the logarithm of the patulin concentration and the current difference ΔI before and after the specific binding of the aptamer electrochemical sensor to patulin, then the optimal linear range and detection limit of patulin can be obtained; the current difference ΔI before and after the specific binding of the aptamer electrochemical sensor to patulin can be expressed by formula (Ⅰ):

[0057] ΔI = I0 – I1 (Ⅰ)

[0058] wherein, I0 is the peak current measured in a mixed solution of 5 mmol / L 3- / 4- and 0.1 mol / L KCl before the specific binding of the aptamer electrochemical sensor to the patulin standard solution, and I1 is the peak current measured in the same concentration of 3- / 4- mixed solution of and KCl after the specific binding of the aptamer electrochemical sensor to patulin standard solutions with different concentrations;

[0059] The logarithm of the current difference ΔI and the patulin concentration show a good linear relationship in the range of 0.5 - 5000000 ng / L, and the linear equation is: y = 4.8137x + 5.3212, and the correlation coefficient R 2 = 0.997; wherein y is the current difference ΔI before and after the specific binding of patulin to the aptamer electrochemical sensor, and x is the logarithm of the patulin concentration (ng / L); the detection limit of this method is: 0.25 ng / L;

[0060] (S3) Sample detection: Take 25 mL of commercially available apple juice and hawthorn juice in test tubes respectively, add 12.5 mL of ethyl acetate, extract by ultrasonic for 5 min, centrifuge at 6000 r / min for 5 min, collect the upper clear liquid, repeat the above steps once, collect the extraction liquid twice, and then rotary evaporate until the extraction liquid is nearly dry, dissolve it with 10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer solution with a pH value of 7.4 and make the volume up to 25 mL to obtain the sample to be tested; Take 5 mL of the above-mentioned sample to be tested and perform electrochemical testing according to the method and steps of (S2) "Standard curve plotting" in this example. According to the measured current difference ΔI, substitute it into the above linear equation to calculate the patulin concentration value corresponding to the sample to be tested;

[0061] Meanwhile, a certain amount of patulin was added to the samples respectively, so that the concentrations of patulin added to the samples were: 50 μg / L, 100 μg / L, 200 μg / L. The tests were carried out under the same conditions, and the spiked recoveries were calculated. The determination results of the samples and spiked recoveries are shown in Table 1:

[0062] Table 1 Contents of patulin in apple juice and hawthorn juice samples and determination results of spiked recoveries

[0063]

[0064] As can be seen from Table 1, the determination results of apple juice and hawthorn juice samples in the method for detecting patulin by the aptamer electrochemical sensor prepared by the method of the present invention are consistent with the determination results by high performance liquid chromatography. Moreover, the spiked recoveries of the method are 86.2% - 92.2% and 91.3% - 105.4% respectively, indicating that the method of the present invention can be used for the detection of patulin in actual samples. The present invention provides a detection scheme for the determination of patulin content in apple juice and hawthorn juice, which has the advantages of simple operation, high accuracy, high sensitivity, wide linear range and strong specificity.

[0065] In this embodiment, Figure 3 is the DPV curve graph of the aptamer electrochemical sensor constructed by the present invention before specific binding (curve a) and after specific binding (curve b) with a 1000 ng / L patulin standard solution. According to Figure 3 it can be known that after specific binding with patulin, the response current of the aptamer electrochemical sensor in a mixed solution of 5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl (curve b) is significantly smaller than that before specific binding (curve a), indicating that the aptamer has a significant specific binding effect on patulin and provides a basis for quantitative detection of patulin.

[0066] In this embodiment, Figure 4 is the DPV response graph recorded after the aptamer electrochemical sensor constructed by the present invention specifically binds with different concentrations of patulin standard solutions for 75 min in a mixed solution of 5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl; among them, curves a - k respectively represent the DPV curves of the electrode after specifically binding with patulin standard solutions with concentrations of 0.0 ng / L, 0.5 ng / L, 1.0 ng / L, 10 ng / L, 100 ng / L, 1000 ng / L, 10000 ng / L, 100000 ng / L, 500000 ng / L, 1000000 ng / L, 5000000 ng / L for 75 min; according to Figure 4It can be seen that the response current of the aptamer electrochemical sensor in a mixed solution of 5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl decreases with the increase of the patulin concentration, which can provide a basis for the drawing of the standard curve graph.

[0067] In this embodiment, Figure 5 is the standard curve graph of the logarithm of the current difference before and after the specific binding of the aptamer electrochemical sensor constructed in the present invention to the patulin standard solution and the concentration of the patulin standard solution; according to Figure 5 It can be seen that there is a good linear relationship between the logarithm of the current difference before and after the specific binding of the aptamer electrochemical sensor to patulin and the concentration of the patulin standard solution. The content information of patulin can be obtained through this standard curve.

[0068] In this embodiment, to verify the specificity of the aptamer electrochemical sensor constructed in the present invention, 1000 ng / L of patulin was respectively replaced with ochratoxin (OTB), zearalenone (ZEA), fumonisin (FB1), deoxynivalenol (DON), 3-nitropropionic acid (3-NPA), aflatoxin (AFT) of the same concentration and combined with the aptamer electrochemical sensor in the present invention, and electrochemical tests were carried out according to the methods and steps of "drawing of the standard curve" in (S2) of Example 2. The results are as Figure 6 shown. According to Figure 6 It can be seen that the current difference before and after the combination of the aptamer electrochemical sensor and ochratoxin (OTB), zearalenone (ZEA), fumonisin (FB1), deoxynivalenol (DON), 3-nitropropionic acid (3-NPA), aflatoxin (AFT) is tiny, indicating that the aptamer electrochemical sensor of the present invention will not specifically bind to the other 6 toxins except patulin, showing that the sensor has high specific recognition, good anti-interference effect and high selectivity.

[0069] Porous carbon material is a kind of porous material with a carbonaceous main framework. Due to its large specific surface area, many pore structures, good electrical conductivity and other advantages, it has become an excellent biological carrier to improve the electrochemical performance of the sensor. Carboxyl functionalization of porous carbon can introduce oxygen-containing functional groups such as hydroxyl and carboxyl on the surface of the porous carbon material, and provide a good binding interface for the amino-modified aptamer through amidation reaction; and then a highly sensitive and highly specific electrochemical determination method for patulin was established by using the specific recognition of aptamer and patulin.

[0070] The foregoing description of specific exemplary embodiments of the invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that, according to the above teaching, many modifications and variations are possible. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Application of a nucleic acid aptamer electrochemical sensor for detecting patulin, characterized in that, The preparation method of the nucleic acid aptamer electrochemical sensor comprises the following operation steps: (1) Synthesize porous carbon material. Under the protection of inert gas, heat sodium alginate to 700 - 900 °C and keep it for 1 - 2 h, then cool it. Wash the obtained black solid with HCl solution and water respectively, and filter to obtain porous carbon material (SA-C); (2) Take the porous carbon material (SA-C) in step (1) and reflux it under concentrated acid to prepare carboxyl-functionalized porous carbon material (SA-C-COOH); (3) Construction of the nucleic acid aptamer electrochemical sensor: Prepare a suspension of carboxyl-functionalized porous carbon (SA-C-COOH) in step (2), drop it on the surface of the pretreated glassy carbon electrode, dry it, and then immerse it in a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group. Rinse and dry to obtain the Act / SA-C-COOH / GCE modified electrode. Drop the nucleic acid aptamer solution and incubate to obtain the Aptamer / Act / SA-C-COOH / GCE modified electrode. Rinse and dry; Immerse the dried Aptamer / Act / SA-C-COOH / GCE modified electrode in bovine serum albumin solution for blocking, rinse and dry to obtain BSA / Aptamer / Act / SA-C-COOH / GCE, which is the nucleic acid aptamer electrochemical sensor; Among them, the nucleotide sequence in the nucleic acid aptamer solution is: 5’-NH2-(CH2)6-CAGCTCAGAAGCTTGATCCT-GGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTT-GACTCGAAGTCGTGCATCTG-3’.

2. Use of the aptamer electrochemical sensor according to claim 1 for detecting patulin, characterized in that: 20 g of sodium alginate, under the protection of inert gas, with a heating rate of 5 °C / min, heat it to 800 °C and keep it for 1.5 h. The molar concentration of the HCl solution is 5 mol / L; The inert gas is argon.

3. Use of the aptamer electrochemical sensor according to claim 1 for detecting patulin, characterized in that: Step (2) is to take the porous carbon material (SA-C) in step (1), add a mixed solution of concentrated H2SO4 and concentrated HNO3 and reflux for 1 - 3 h, cool it, wash the obtained solid with water, centrifuge it, collect the product and dry it to obtain carboxyl-functionalized porous carbon material (SA-C-COOH).

4. Use of the aptamer electrochemical sensor according to claim 3 for detecting patulin, characterized in that: The volume ratio of concentrated H2SO4 to concentrated HNO3 in the mixed solution of concentrated H2SO4 and concentrated HNO3 is 3:1, the volume of the mixed solution of concentrated H2SO4 and concentrated HNO3 is 100 mL, the mass of the porous carbon material is 0.5 g, the reflux time is 2 h, and the reflux temperature is 60 °C.

5. Use of the aptamer electrochemical sensor according to claim 1 for detecting patulin, characterized in that: The rinsing described in step (3) is all carried out by rinsing with tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution. The molar concentration of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution is 10 mmol / L and the pH value is 7.

4. The drying described in step (3) is all baking under an infrared lamp for 15 min. In step (3), the carboxyl-functionalized porous carbon material (SA-C-COOH) in step (2) is ultrasonically dispersed in water to prepare a uniformly stable carboxyl-functionalized porous carbon (SA-C-COOH) suspension with a mass concentration of 1.0 mg / mL, and the modification amount of the electrode is 6 μL. The pretreated glassy carbon electrode described in step (3) is obtained by polishing the glassy carbon electrode (GCE) into a mirror surface with alumina, ultrasonically cleaning it successively with HNO3 solution, ethanol and ultrapure water, and then naturally drying it. In step (3), it is immersed in a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 10 - 60 min to activate the carboxyl group. In the mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), the molar concentrations of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) are both 0.5 mol / L. The incubation described in step (3) is incubation at 30 - 40 °C for 1.5 - 3 h. The time for the blocking described in step (3) is 10 - 60 min.

6. Use of the aptamer electrochemical sensor according to claim 5 for detecting patulin, characterized in that: The pretreated glassy carbon electrode described in step (3) is obtained by polishing the glassy carbon electrode (GCE) into a mirror surface with alumina with particle sizes of 0.3 μm and 0.05 μm respectively. The volume ratio of nitric acid (HNO3) to water in the HNO3 solution is 1:

1. The ethanol is anhydrous ethanol.

7. Use of the aptamer electrochemical sensor according to claim 5 for detecting patulin, characterized in that: The molar concentration of the aptamer solution described in step (3) is 6 μmol / L. The incubation is incubation at 37 °C for 2.5 h. The concentration of the bovine serum albumin (BSA) solution is 0.5%, and the time for the blocking is 30 min.

8. Method for detecting patulin using the aptamer electrochemical sensor according to claim 1, characterized in that, It includes the following steps: Step S1, using the aptamer electrochemical sensor as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire as the counter electrode to form a three-electrode system; Step S2, prepare patulin standard solutions with different concentrations, immerse the aptamer electrochemical sensor into the patulin standard solutions with different concentrations respectively for specific binding, and then insert the three-electrode system into the mixed solution containing [Fe(CN)6] 3- / 4- and KCl, perform differential pulse voltammetry scanning in the range of -0.10 - 0.50 V, record the current value at 0.160 ± 0.01 V, calculate the current difference ∆I before and after the specific binding of the aptamer electrochemical sensor and patulin through the DPV curve, and obtain the standard curve and linear equation according to the logarithm of the ∆I value and the patulin concentration; Step S3: When actually detecting patulin in a sample, immerse the aptamer electrochemical sensor into the sample to be tested for specific binding, and calculate the current difference ∆I based on the current values of 0.160 ± 0.01 V measured in the mixed solution containing 3- / 4- [Fe(CN)6] and KCl before and after specific binding. Substitute the current difference ∆I into the linear equation to calculate the patulin concentration value in the sample to be tested.

9. The method for detecting patulin by the aptamer-based electrochemical sensor according to claim 8, characterized in that: The concentrations of the patulin standard solutions described in step S2 are respectively: 0.0 ng / L, 0.5 ng / L, 1 ng / L, 10 ng / L, 100 ng / L, 1000 ng / L, 10000 ng / L, 100000 ng / L, 500000 ng / L, 1000000 ng / L, 5000000 ng / L; the linear equation in step S2 is y = 4.8137x + 5.3212, and the correlation coefficient R 2 = 0.9972; where y is the current difference ∆I before and after the specific binding of patulin to the aptamer electrochemical sensor, and x is the logarithm of the patulin concentration.

10. The method for detecting patulin by the aptamer-based electrochemical sensor according to claim 8, characterized in that: The specific binding time of step S2 and step S3 is 75 min; [Fe(CN)6] is included in the mixed solution containing [Fe(CN)6] and KCl in step S2 and step S3 3- / 4- The molar concentration of [Fe(CN)6] in the mixed solution containing 3- / 4- [Fe(CN)6] and KCl is 5 mmol / L, and the molar concentration of KCl is 0.1 mol / L.

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