An electrochemical sensor for detecting malachite green with nucleic acid aptamer, its preparation method and application

Abstract

The present invention discloses a nucleic acid aptamer electrochemical sensor for detecting malachite green, its preparation method and application. In this preparation method, the thiolated complementary strand binds to the malachite green aptamer to form a double-stranded structure, which is connected to the gold nanoparticles through Au-S bonds to form a barrier for blocking the redox probe [Fe(CN)6] 3‑ / 4‑ The barrier in contact with the electrode causes the current to decrease; after adding malachite green, the aptamer specifically binds to malachite green, and the double-stranded structure is destroyed, and [Fe(CN)6] 3‑ / 4‑ can smoothly contact the electrode, resulting in an increase in current. This sensor is simple to prepare, the aptamer is inexpensive, has a wide linear range and a low detection limit. The current difference between different concentrations of malachite green can reach dozens of microamperes, with a significant difference, which can effectively improve the detection accuracy, reduce the detection cost and has good specificity. It can be applied to the accurate, efficient and sensitive detection of malachite green in water bodies.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a nucleic acid aptamer electrochemical sensor for detecting malachite green based on a nanocomposite material, and a preparation method and application thereof. Background Art

[0002] Malachite Green is an artificially synthesized triphenylmethane dye, which has the functions of killing bacteria and parasites, and is also a specific medicine for treating saprolegniasis. Therefore, malachite green is widely used in commercial aquaculture. In recent years, a large number of studies have shown that malachite green has cytotoxicity, mutagenicity and carcinogenicity, and even trace amounts of malachite green can damage the human immune system and reproductive system. In this regard, the European Union stipulates that the addition limit of malachite green in fish farming shall not exceed 2 μg / kg, and the Ministry of Agriculture of China completely prohibits the use of such compounds. However, in order to make more profits, some individual vendors still illegally use malachite green to improve the survival rate of fish during transportation. Therefore, it is necessary to develop a method for timely and rapid detection of malachite green.

[0003] At present, many methods for determining malachite green have been reported, including high performance liquid chromatography, high performance liquid mass spectrometry, high performance liquid chromatography - mass spectrometry, immunoassay, surface enhanced Raman scattering and molecularly imprinted polymers. These methods have good accuracy and sensitivity, but often require the use of expensive precision instruments and are operated by trained staff. In recent years, the biosensor technology with nucleic acid aptamers as recognition elements has become a research hotspot. Nucleic acid aptamers are artificial single-stranded DNA or RNA probes that can fold into different secondary structures to bind to target substances, and have the advantages of easy synthesis, high chemical stability and simple operation. Electrochemical aptamer sensors are an important type of aptamer sensors, which combine the high sensitivity of electrochemical detection and the specificity of aptamers, and have good application prospects.

[0004] Patent 200710070381.0 describes a method for rapidly detecting malachite green in aquatic products using aptamers, which employs an enzyme-linked colorimetric method for detection and uses single-stranded DNA with a specific sequence as the recognition element. However, this method requires elution and color development, and there may be false positives. Patent 201410008020.3 uses an electrochemical aptamer sensor to detect malachite green. The target is bound to the electrode surface through the interaction between the RNA aptamer and the target, and then the corresponding antibody to the target is combined to form a sandwich structure. This method avoids the problem of false positives to a certain extent, but the RNA aptamer has problems such as high cost, instability, and difficult labeling. Patent 201810625479.6 proposes a method for an aptamer electrochemical biosensor for detecting malachite green. The target is bound to the electrode surface through the specific binding between the aptamer and the target, and a sandwich structure is formed using the binding between avidin and biotin and horseradish peroxidase. However, this sensor has poor detection ability for low-concentration malachite green and can only detect malachite green with a concentration above 1 μg / L; moreover, the change in the sensing signal is small, and the current difference between malachite green with a concentration of 1 mg / L and the blank control sample is only 10 μA. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a nucleic acid aptamer electrochemical sensor for detecting malachite green. This sensor increases the current span, has more obvious sensing signal changes for malachite green at different concentrations, can effectively improve the detection accuracy, reduce the detection cost, and has good specificity.

[0006] Another object of the present invention is to provide a preparation method for the nucleic acid aptamer electrochemical sensor for detecting malachite green. In this preparation method, the thiolated complementary strand binds to the malachite green aptamer to form a double-stranded structure, which is connected to the gold nanoparticles through Au-S bonds to form a barrier against the redox probe [Fe(CN)6] 3- / 4- in contact with the electrode, causing the current to decrease; after adding malachite green, the aptamer specifically binds to malachite green, and the double-stranded structure is destroyed, and [Fe(CN)6] 3- / 4- can smoothly contact the electrode, resulting in an increase in current.

[0007] The third object of the present invention is to provide the application of the above nucleic acid aptamer electrochemical sensor for detecting malachite green.

[0008] The objects of the present invention are achieved by the following technical solutions:

[0009] An electrochemical sensor for detecting malachite green using nucleic acid aptamers, comprising a modification material, a recognition element, and a transducer device. The modification material includes a composite material of multi-walled carbon nanotubes and coupled titanium dioxide and gold nanoparticles; the recognition element consists of a malachite green aptamer, its complementary strand 1, and complementary strand 2. The preferred sequence of the malachite green aptamer is 5′-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3′ (China Patent Application No.: CN202210091617.3), which contains 36 bases. Two complementary strands (complementary strand 1 and complementary strand 2) designed according to the malachite green aptamer, the preferred sequence of complementary strand 1 is 5′-TCGCATGGTTTTT-3′, which contains 13 bases and is modified with -SH-(CH2)6- at the 3′ end; the preferred sequence of complementary strand 2 is 5′-TTTTTGGCTGATC-3′, which contains 13 bases and is modified with -SH-(CH2)6- at the 5′ end; the transducer device is a commonly used electrochemical workstation (preferably CHI660E electrochemical workstation), the working electrode is a gold electrode assembled with the modification material and the recognition element, the counter electrode is a platinum electrode, and the reference electrode is Ag / AgCl; the electrolyte is a potassium ferricyanide solution.

[0010] Its detection principle is as follows: The composite material and gold nanoparticles can significantly enhance the sensor current signal to amplify the current change brought by malachite green. Before adding malachite green, the aptamer DNA molecular layer forms a barrier on the surface of the modified electrode, preventing 3- / 4- [Fe(CN)6] from reaching the electrode surface, resulting in a decrease in the current signal. In the presence of malachite green, the aptamer separates from the complementary DNA and forms a complex with malachite green. The stripping of the aptamer causes the barrier blocking 3- / 4- [Fe(CN)6] to be broken, and 3- / 4- [Fe(CN)6] can freely contact the electrode surface, and the current intensity increases significantly. Moreover, the degree of increase is positively correlated with the concentration of malachite green. The detection of malachite green can be achieved through the change in the current signal.

[0011] A preparation method of an electrochemical sensor for detecting malachite green using nucleic acid aptamers includes the following steps:

[0012] (1) Preparation of the composite material of multi-walled carbon nanotubes and titanium dioxide: Dissolve multi-walled carbon nanotubes and titanium dioxide in absolute ethanol, mix well, and then perform ultrasonic treatment until a highly dispersed grayish-black solution is obtained to obtain the composite material of multi-walled carbon nanotubes and titanium dioxide;

[0013] (2) Polishing and activation treatment of the gold electrode: Immerse the gold electrode in the piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3), wash it with ultrapure water, and then polish the electrode surface to a mirror finish with alumina powder on a polishing cloth. Then, ultrasonically treat the gold electrode in ethanol and ultrapure water respectively, and dry it with a nitrogen stream for later use.

[0014] (3) Preparation of the recognition element: After thawing and diluting the malachite green aptamer, its complementary strand 1, and complementary strand 2 to the corresponding concentrations, take the same volume and concentration of complementary strand 1 and complementary strand 2, and twice the volume and half the concentration of the malachite green aptamer, vortex to mix them thoroughly, and incubate overnight at 2 - 8 °C (preferably 4 °C) to obtain a double-stranded DNA with thiol groups at both ends, which is the recognition element.

[0015] (4) Preparation of the modified electrode: Drop the multi-walled carbon nanotube and titanium dioxide composite prepared in step (1) onto the surface of the activated gold electrode in step (2). After natural drying, drop nano-gold particles onto the electrode surface and dry naturally. Then, drop the double-stranded DNA prepared in step (3) onto the electrode surface and dry naturally. Then, block the non-specific binding sites and dry naturally to complete the blocking. The modified electrode is prepared and then used for detection. When not in use, store it at 2 - 8 °C (preferably 4 °C).

[0016] In step (1),

[0017] The ratio of the multi-walled carbon nanotubes, titanium dioxide, and absolute ethanol is: 40 - 100 mg: 25 - 50 mg: 10 - 25 mL; preferably 100 mg: 50 mg: 25 mL.

[0018] Preferably, the mixing time is 3 - 5 minutes; further preferably 3 minutes.

[0019] The ultrasonication time is 40 - 60 minutes; preferably 60 minutes.

[0020] In step (2),

[0021] The soaking time is 5 - 10 min; preferably 10 min.

[0022] The ultrasonication time is 1 - 2 min; preferably 1 min.

[0023] In step (3), the vortex time is 3 - 5 minutes; preferably 3 minutes.

[0024] In step (4),

[0025] The blocking of non-specific binding sites is carried out with 0.5 mM to 1.5 mM 6-mercapto-1-hexanol; preferably, the non-specific binding sites are blocked with 1 mM 6-mercapto-1-hexanol;

[0026] The steps for the above sensor to establish a standard curve are as follows:

[0027] 1) Using the above modified electrode as the working electrode, a platinum electrode as the counter electrode, Ag / AgCl as the reference electrode, and a potassium ferricyanide solution as the electrolyte, differential pulse voltammetry is used for detection. The scanning potential range is -0.1 to 0.4 V, the amplitude is 0.05 V, the pulse time is 0.05 ms, and the differential pulse voltammogram is recorded; the peak current of the blank sample without malachite green is obtained.

[0028] 2) Take the detection bottom solution containing the malachite green standard and drop it on the working electrode. After incubating for 30 to 60 min, immerse the working electrode in the electrolyte and record the differential pulse voltammogram of the working electrode; in the concentration range of 10 1 ~10 6 ng / L, select a concentration every 10 times and repeat 3 times.

[0029] 3) Plot the logarithm of the concentration of the malachite green standard detection bottom solution lgC as the abscissa and the current value as the ordinate to establish a standard curve. According to the standard curve, the concentration of malachite green in different water samples can be detected.

[0030] The application of the above nucleic acid aptamer electrochemical sensor in the detection of malachite green.

[0031] The present invention has the following advantages and effects compared with the prior art:

[0032] (1) The linear range of the nucleic acid aptamer electrochemical sensor for detecting malachite green proposed by the present invention can span 5 orders of magnitude, and can detect malachite green at the pmol / L level, having good detection ability.

[0033] (2) The present invention solves the problems of the existing methods for detecting malachite green, such as high cost, long time consumption, complex detection scheme, and the need for professional personnel to operate. It provides a simple, fast, and efficient preparation method for the nucleic acid aptamer electrochemical sensor for malachite green. The sensor is simple to prepare, the aptamer is inexpensive, has a wide linear range and a low detection limit, and the current difference between malachite green at different concentrations can reach dozens of microamperes, with a significant difference. It can be applied to the accurate, efficient, and sensitive detection of malachite green in water bodies. Description of the Drawings

[0034] Figure 1Schematic diagram of the assembly process and detection principle of an electrochemical sensor based on a nucleic acid aptamer for detecting malachite green.

[0035] Figure 2 Graph showing the change in current response of an electrode modified with a composite of multi-walled carbon nanotubes and titanium dioxide and nano-gold.

[0036] Figure 3 Differential pulse voltammogram of an electrochemical sensor based on a nucleic acid aptamer for detecting malachite green at different concentrations of malachite green; among them, the concentrations of malachite green from a to g are 0, 10 1 ,10 2 ,10 3 ,10 4 ,10 5 and 10 6 ng / L.

[0037] Figure 4 Standard curve of an electrochemical sensor based on a nucleic acid aptamer for detecting malachite green. Detailed implementation mode

[0038] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.

[0039] For the test methods without specifying specific experimental conditions in the following embodiments, they are usually in accordance with conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0040] Example 1: Detection of malachite green at a concentration of 1 mg / L by the sensor

[0041] An electrochemical sensor based on a nucleic acid aptamer for detecting malachite green, including a modification material, a recognition element, and a transducer device. Among them, the schematic diagram of the assembly process and detection principle of the electrochemical sensor based on a nucleic acid aptamer for detecting malachite green is as Figure 1As shown. The modified materials include composite materials coupled with multi-walled carbon nanotubes and titanium dioxide and gold nanoparticles; the recognition element consists of three single-stranded DNAs, namely the malachite green aptamer, with a sequence of 5′-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3′, containing 36 bases. The two complementary chains of the malachite green aptamer, complementary chain 1 with a sequence of 5′-TCGCATGGTTTTT-3′ and complementary chain 2 with a sequence of 5′-TTTTTGGCTGATC-3′, each contain 13 bases, and are modified with -SH-(CH2)6- at the 3′ and 5′ ends respectively; the transducer is a commonly used CHI660E electrochemical workstation, the working electrode is a gold electrode assembled with the modified material and the recognition element, the counter electrode is a platinum electrode, and the reference electrode is Ag / AgCl; the electrolyte is a potassium ferrocyanide solution.

[0042] A method for preparing a nucleic acid aptamer electrochemical sensor for detecting malachite green, specifically comprising the following steps and parameters:

[0043] (1) Preparation of multi-walled carbon nanotubes and titanium dioxide composite materials: 100 mg of multi-walled carbon nanotubes and 50 mg of titanium dioxide were dissolved in 25 mL of anhydrous ethanol, mixed on a vortex mixer for 3 minutes, and then ultrasonically treated for 60 minutes until a highly dispersed gray-black solution was obtained, indicating that a multi-walled carbon nanotube and titanium dioxide composite material was obtained, which was recorded as MWCNTs@TiO2.

[0044] (2) Polishing and activation of gold electrodes: Soak the gold electrode in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3) for 10 minutes, then rinse with ultrapure water, dry with nitrogen flow, and place on top of a polishing cloth. Take 1.0μm alumina powder, place the electrode surface in the alumina powder and grind in circles for 2 minutes until the electrode surface is polished to a mirror surface. Then place the gold electrode in ethanol and ultrapure water for ultrasonic treatment for 1 minute, three times, and dry with nitrogen flow. Cyclic voltammetry was performed with a scan rate of 0.1V / s and a potential range of -0.4 to 0.8V for 20 cycles; rinse with ultrapure water, dry with nitrogen flow and set aside;

[0045] (3) Preparation of recognition element: Thaw the aptamer and its complementary chain, dilute them to the corresponding concentration with phosphate buffer, take 1 mL of 8 μM complementary chain 1, 1 mL of 8 μM complementary chain 2, and 2 mL of 4 μM malachite green aptamer, vortex for 3 minutes to mix them thoroughly, and incubate them at 4°C overnight to obtain a double-stranded DNA with thiol groups at both ends at a concentration of 2 μM, which is the recognition element.

[0046] (4) Preparation of the modified electrode: Take 2 μL of the composite material prepared in step (1) and drop-coat it on the surface of the gold electrode activated in step (2). After natural drying, take another 2 μL of gold nanoparticles (AuNPs) and drop-coat them on the electrode surface, then dry naturally. Subsequently, take 2 μL of the double-stranded DNA prepared in step (3) and drop-coat it on the electrode surface, and dry naturally. Then take 2 μL of 1 mM 6-mercapto-1-hexanol (MCH) to block the non-specific binding sites. After the natural drying of 6-mercapto-1-hexanol, the blocking is completed, and the preparation of the modified electrode is finished. Among them, the gold electrode is used as the blank control, and the gold electrode coated with MWCNTs@TiO2 composite material without coating gold nanoparticles is used as the experimental control. The electrode current response change diagram is as shown in Figure 2 shown. It can be seen from Figure 2 that when the gold electrode surface is modified with MWCNTs@TiO2 composite material, the peak current response increases from 58 μA to 172 μA; when the gold nanoparticles are further modified, the peak current response increases from 172 μA to 220 μA, indicating that the modified material is beneficial to enhancing the electrical signal conduction and thus amplifying the detection sensitivity.

[0047] (5) Using the modified electrode prepared in step (4) as the working electrode, the platinum electrode as the counter electrode, Ag / AgCl as the reference electrode, and the potassium ferricyanide solution as the electrolyte, differential pulse voltammetry is used for detection. The scanning potential range is -0.1 to 0.4 V, the amplitude is 0.05 V, the pulse time is 0.05 ms, and the differential pulse voltammogram is recorded (see Figure 3 ), and the peak current of the blank sample without malachite green is obtained, and the peak current is about 47 μA.

[0048] (6) Take 2 μL of the detection bottom solution containing the malachite green standard and drop-coat it on the working electrode. After incubating for 30 min, immerse the working electrode in the electrolyte, and record the differential pulse voltammogram of the working electrode. In the concentration range of 10 1 ~10 6 ng / L, select a concentration every 10 times and repeat 3 times. The differential pulse voltammogram is as shown in Figure 3 shown, indicating that within a certain range, the peak current response is proportional to the malachite green concentration.

[0049] (7) Plot the logarithm of the malachite green standard detection bottom solution concentration lgC as the abscissa and the current value as the ordinate to establish a standard curve, as shown in Figure 4 . In the concentration range of 10 1 ~10 6 ng / L, the current value shows a good linear relationship with the logarithm of the malachite green standard concentration (R 2= 0.99816), the linear equation is y = 51.0585 + 3.8892x, and the detection limit is 8.68 pg / mL. According to the standard curve, the concentration of malachite green in different water samples can be detected.

[0050] (8) Take 2 μL of the detection bottom solution with a malachite green concentration of 1 mg / L and drop it on the working electrode. After incubating for 30 min, immerse the working electrode in the electrolyte solution and record the differential pulse voltammogram of the working electrode. It can be seen that the peak current is about 74 μA, and the difference in peak current from the blank sample without malachite green is 27 μA.

[0051] Example 2: Detection of malachite green with a concentration of 10 ng / L by the sensor

[0052] Refer to Example 1 to prepare the sensor, with a malachite green concentration of 10 ng / L;

[0053] After the working electrode is prepared, take 2 μL of the detection bottom solution with a malachite green concentration of 10 ng / L and drop it on the working electrode. After incubating for 30 min, immerse the working electrode in the electrolyte solution and record the differential pulse voltammogram of the working electrode. It can be seen that the peak current is about 55 μA, and the difference in peak current from the blank sample without malachite green is 8 μA.

[0054] Example 3: Detection of sensor stability

[0055] Refer to Example 1 to prepare the sensor, and store the working electrode at 4 °C for 7 days.

[0056] After the working electrode is prepared, take 2 μL of the detection bottom solution with a malachite green concentration of 100 μg / L and drop it on the working electrode. After incubating for 30 min, immerse the working electrode in the electrolyte solution and record the differential pulse voltammogram of the working electrode. It can be seen that the peak current is about 63 μA, that is, the detection efficiency of the sensor stored at 4 °C for 7 days can still reach more than 90%.

[0057] Example 4: Specificity detection of the sensor

[0058] Refer to Example 1 to prepare the sensor, with a glyphosate concentration of 5 mg / L and a tricyclazole concentration of 5 mg / L;

[0059] After the working electrode was prepared, 2 μL of the detection base solution with a glyphosate concentration of 5 mg / L and 2 μL of the detection base solution with a tricyclazole concentration of 5 mg / L were respectively dropped onto different working electrodes. After incubation for 30 min, the working electrodes were immersed in the electrolyte solution, and the differential pulse voltammograms of the working electrodes were recorded. It can be seen that the peak current of the working electrode coated with glyphosate was about 55 μA, and the peak current of the working electrode coated with tricyclazole was about 58 μA. The peak current of the differential pulse voltammogram of the sensor for detecting malachite green at a concentration of 100 μg / L was about 70 μA. Even though the concentration difference was 50 times, the current difference still exceeded 12 μA, and the difference was obvious, indicating that the sensor had good specificity.

[0060] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of an electrochemical sensor for detecting malachite green with nucleic acid aptamer, characterized in that, It includes the following steps: (1) Preparation of multi-walled carbon nanotube and titanium dioxide composite: Dissolve multi-walled carbon nanotubes and titanium dioxide in absolute ethanol, mix well, and then perform ultrasonic treatment until a highly dispersed gray-black solution is obtained, thus obtaining the multi-walled carbon nanotube and titanium dioxide composite; (2) Polishing and activation treatment of gold electrode: Immerse the gold electrode in piranha solution and wash it with ultrapure water, then polish the electrode surface to a mirror surface with alumina powder on a polishing cloth; then place the gold electrode in ethanol and ultrapure water respectively for ultrasonic treatment, and dry it with nitrogen flow for later use; (3) Preparation of recognition element: After thawing and diluting malachite green aptamer and its complementary strand 1 and complementary strand 2 to the corresponding concentrations, take the same volume and concentration of complementary strand 1 and complementary strand 2 and twice the volume and half the concentration of malachite green aptamer, vortex to mix them well, and incubate overnight at 2 - 8 °C to obtain a double-stranded DNA with thiol groups at both ends, namely the recognition element; among them, the sequence of malachite green aptamer is 5′-CCATGCGACGGACAGCACGTGTCACCGCGATCAGCC-3′, the sequence of complementary strand 1 is 5′-TCGCATGGTTTTT-3′, and -SH-(CH2)6- is modified at the 3′ end; the sequence of complementary strand 2 is 5′-TTTTTGGCTGATC-3′, and -SH-(CH2)6- is modified at the 5′ end; (4) Preparation of modified electrode: Take the multi-walled carbon nanotube and titanium dioxide composite prepared in step (1) and drop-coat it on the surface of the activated gold electrode in step (2). After natural drying, then take gold nanoparticles and drop-coat them on the electrode surface, and dry it naturally. Subsequently, take the double-stranded DNA prepared in step (3) and drop-coat it on the electrode surface, and dry it naturally. Then block the non-specific binding sites, and after natural drying, the blocking is completed, and the preparation of the modified electrode is completed, and then it is used for detection.

2. The preparation method according to claim 1, characterized in that: When the modified electrode described in step (4) is not in use, it is stored at 2 - 8 °C.

3. The preparation method according to claim 1, characterized in that: In step (1), the ratio of the multi-walled carbon nanotubes, titanium dioxide, and absolute ethanol is: 40 - 100 mg : 25 - 50 mg : 10 - 25 mL.

4. The preparation method according to claim 3, characterized in that: In step (1), the ratio of the multi-walled carbon nanotubes, titanium dioxide, and absolute ethanol is: 100 mg : 50 mg : 25 mL.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (1), the mixing time is 3 - 5 minutes; In step (1), the ultrasonic treatment time is 40 - 60 minutes; In step (2), the soaking time is 5 - 10 min; In step (2), the ultrasonic treatment time is 1 - 2 min; In step (3), the vortex time is 3 - 5 minutes.

6. The preparation method according to any one of claims 1 to 4, characterized in that: In step (4), the blocking of non-specific binding sites is carried out with 0.5 mM - 1.5 mM 6-mercapto-1-hexanol.

7. The preparation method according to claim 6, characterized in that: In step (4), the blocking of non-specific binding sites is carried out with 1 mM 6-mercapto-1-hexanol.

8. The preparation method according to any one of claims 1 to 4, characterized in that: 1) Using the modified electrode prepared in step (4) as the working electrode, a platinum electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a potassium ferricyanide solution as the electrolyte, differential pulse voltammetry was used for detection. The scanning potential range was -0.1 to 0.4 V, the amplitude was 0.05 V, the pulse time was 0.05 ms, and the differential pulse voltammogram was recorded; the peak current of the blank sample without malachite green was obtained. 2) Drop the detection bottom solution containing malachite green standard on the working electrode. After incubating for 30 - 60 min, immerse the working electrode in the electrolyte solution and record the differential pulse voltammogram of the working electrode; within the concentration range of 10 1 ~10 6 ng / L, select a concentration every 10 times and repeat 3 times; 3) Using the logarithm of the concentration of the detection bottom solution lgC of the malachite green standard as the abscissa and the current value as the ordinate to plot a graph to establish a standard curve.

9. An electrochemical sensor for detecting malachite green with nucleic acid aptamer, characterized in that: Prepared according to the preparation method described in any one of claims 1 to 8.

10. Application of the electrochemical sensor for detecting malachite green with nucleic acid aptamer according to claim 9 in detecting malachite green.

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