Preparation method, product and application of electrochemical biosensor

Abstract

The invention relates to the field of electrochemical biosensors, in particular to a preparation method, a product and application of an electrochemical biosensor. The preparation method of the electrochemical biosensor comprises the following steps: dispersing phosphorus-doped carbon nitride in water, then adding cobalt salt, and uniformly mixing to obtain a mixed solution; a reducing agent is added into the mixed solution for a reaction, and Co-PCN is obtained; the Co-PCN and hexadecyl trimethyl ammonium bromide are dispersed in water, and CTABamp is obtained; a Co-PCN is used; performing electrochemical activation pretreatment on the electrode to obtain an activated electrode; the CTABamp is added to the surface of the activated electrode; and adding Co-PCN, and drying to obtain the electrochemical biosensor. The method is simple and easy for large-scale industrial preparation. The electrochemical biosensor prepared by the method has a wide detection range and a relatively low detection limit on dimethoate, which shows that the electrochemical biosensor has excellent dimethoate detection performance.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical biosensors, and particularly to a preparation method, product and application of an electrochemical biosensor. Background Art

[0002] As a highly effective and broad-spectrum organophosphorus insecticide, dimethoate can effectively control agricultural pests such as aphids and spider mites. However, it is neurotoxic to humans. Long-term exposure or excessive residue can lead to the inhibition of cholinesterase activity, causing neurological disorders and even endangering life. Therefore, the core significance of dimethoate detection lies in ensuring food safety and public health, especially for the monitoring of pesticide residues in agricultural products such as vegetables, fruits, tea and their processed products, which can effectively block the entry of toxic substances into the human body through the food chain.

[0003] From a technical perspective, the development of dimethoate detection methods reflects the support of scientific progress for public safety. Traditional instrumental analysis methods such as gas chromatography (GC) and liquid chromatography-mass spectrometry (LC-MS) have high precision, but they have limitations such as complex pretreatment and high cost. Therefore, a series of new dimethoate detection methods, such as immunoassay, colorimetry, fluorescence and surface-enhanced Raman scattering, have been established one after another. However, these methods also have some disadvantages. For example, antibodies are difficult to prepare and easily inactivate in immunoassay. Although colorimetry is fast, it has low sensitivity and is easily affected by the color of the sample itself. Fluorescence and surface-enhanced Raman scattering still rely on expensive professional equipment. Therefore, there is an urgent need to develop a new dimethoate detection method with simple operation, low cost and high sensitivity. Summary of the Invention

[0004] Based on the above, the present invention provides a preparation method, product and application of an electrochemical biosensor.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention, a preparation method of an electrochemical biosensor, includes the following steps:

[0007] Disperse phosphorus-doped carbon nitride in water, and then add a cobalt salt and mix well to obtain a mixed solution;

[0008] Add a reducing agent to the mixed solution and react to obtain Co-PCN;

[0009] Disperse the Co-PCN and cetyltrimethylammonium bromide in water to obtain CTAB&Co-PCN;

[0010] Perform electrochemical activation pretreatment on the electrode to obtain an activated electrode;

[0011] Add the CTAB&Co-PCN to the surface of the activated electrode and dry it to obtain the electrochemical biosensor.

[0012] The second technical solution of the present invention is an electrochemical biosensor prepared according to the above preparation method.

[0013] The third technical solution of the present invention is the application of the above electrochemical biosensor in detecting organophosphorus pesticides.

[0014] The present invention discloses the following technical effects:

[0015] The method of the present invention is simple and easy to prepare on a large scale industrially. The electrochemical biosensor (CTAB&Co-PCN / SPE) prepared by the method of the present invention has a wide detection range and a relatively low detection limit for dimethoate, indicating excellent dimethoate detection performance. Brief Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is a schematic diagram of the preparation process flow of the electrochemical biosensor in Example 1.

[0018] Figure 2 It is a TEM image of Co-PCN in Example 1.

[0019] Figure 3 In it, A is the CV curves of CTAB&Co-PCN / SPE in different test systems: among them, a is 1-NA, b is PbBE, and c is PbBE + 1-NA; B is the CV curves of CTAB&Co-PCN / SPE in the PbBE + 1-NA detection system when different concentrations of dimethoate exist: among them, a is without pesticide, b is 10 μg / L, and c is 100 μg / L.

[0020] Figure 4 It is a logarithmic linear relationship diagram of the inhibition rate and the dimethoate concentration (0.01 - 3000 μg / L).

[0021] Figure 5 It is the test result of the anti-interference ability of CTAB&Co-PCN / SPE: in the figure, a is dimethoate, b is KCl, c is glucose, d is oxalic acid, e is KNO 3 , f is MgCl 2 , g is MnCl 2, where h is ascorbic acid, i is citric acid, j is malic acid, and k is FeCl 3 , and l is CoCl 2 , and m is ZnCl 2 . Detailed implementation manners

[0022] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0023] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0026] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0027] The present invention first proposes a super-simple electrochemical biosensor based on cobalt nanoparticles-anchored phosphorus-doped carbon nitride (Co-PCN) nanomaterials and cetyltrimethylammonium bromide (CTAB) surfactant for trace detection of dimethoate. The sensor uses kidney bean esterase as a biorecognition element. This enzyme is derived from inexpensive kidney beans and has a simple extraction method, so the cost of sensing detection can be effectively reduced. To improve the detection sensitivity, Co-PCN is prepared by a simple method. Due to its high conductivity and redox catalytic activity, it can not only improve the electrocatalytic performance, but also promote the movement of electrons of the redox pair between the electrode interfaces, greatly enhancing the sensing signal. When dimethoate is present, the activity of kidney bean esterase is inhibited, resulting in a decrease in the amount of 1-naphthol, the hydrolysis product of 1-naphthyl acetate catalyzed by it, and a corresponding weakening of the electrochemical oxidation signal. By monitoring the fluctuation of the 1-naphthol oxidation electrical signal, simple, low-cost but highly sensitive detection of dimethoate is achieved. The electrochemical biosensor constructed by the present invention not only has the advantages of fast response speed, wide linear detection range, low detection limit, etc., but also provides a new detection platform for the qualitative and quantitative detection of dimethoate in food.

[0028] In the first aspect of the present invention, a preparation method of an electrochemical biosensor is provided, which is characterized by including the following steps:

[0029] Disperse phosphorus-doped carbon nitride in water, and then add a cobalt salt and mix well to obtain a mixed solution;

[0030] Add a reducing agent to the mixed solution and react to obtain Co-PCN;

[0031] Disperse the Co-PCN and cetyltrimethylammonium bromide in water to obtain CTAB&Co-PCN;

[0032] Perform electrochemical activation pretreatment on the electrode to obtain an activated electrode;

[0033] Add the CTAB&Co-PCN to the surface of the activated electrode and dry to obtain the electrochemical biosensor.

[0034] Cetyltrimethylammonium bromide plays a role in improving the electrochemical signal in the present invention. The present invention also experimented with the cases of replacing cetyltrimethylammonium bromide with dodecyltrimethylammonium bromide (DTAB) and stearyltrimethylammonium chloride (STAC) respectively. The results show that the improvement effects of dodecyltrimethylammonium bromide (DTAB) and stearyltrimethylammonium chloride (STAC) on the electrochemical signal are not as good as that of cetyltrimethylammonium bromide, and the specific reasons need to be further explored.

[0035] When preparing the Co-PCN, after the reaction, the steps of centrifugation, washing the centrifuged precipitate, and vacuum drying are also included.

[0036] In a preferred embodiment of the present invention, the mass ratio of the phosphorus-doped carbon nitride to the cobalt salt is 500:7; the cobalt salt is Co(NO 3 ) 2 ·6H 2 O; the reducing agent is NaBH 4 .

[0037] The present invention does not particularly limit the amount of the reducing agent, and the amount of the reducing agent can fully reduce the cobalt element in the cobalt salt to cobalt metal.

[0038] In a preferred embodiment of the present invention, the concentration of Co-PCN in the CTAB&Co-PCN is 0.6 mg / mL, and the concentration of cetyltrimethylammonium bromide is 0.8 mg / mL.

[0039] In a preferred embodiment of the present invention, the electrochemical activation pretreatment is specifically as follows: the electrode is placed in an acid solution and scanned 8 cycles in the range of -0.5 to 1.2 V at a scanning rate of 0.1 V / s, and then placed in a phosphate buffer solution with a pH of 7.0 for activation by chronopotentiometry, and then activated by cyclic voltammetry to obtain an activated electrode.

[0040] In a preferred embodiment of the present invention, the acid solution is a sulfuric acid solution with a concentration of 0.5 mol / L; the anodic current for activation by chronopotentiometry is 0.1 mV and the time is 40 s; the potential for activation by cyclic voltammetry is -0.7 to 1 V, the scanning rate is 0.1 V / S, and one cycle is scanned.

[0041] In a preferred embodiment of the present invention, the method for adding the CTAB&Co-PCN to the surface of the activated electrode is by dropping; the addition amount of the CTAB&Co-PCN on the surface of the activated electrode is 0.6 to 0.7 μL / mm 2 .

[0042] The present invention does not particularly limit the drying method, and natural drying or drying under an infrared lamp commonly used by those skilled in the art can be adopted.

[0043] In a preferred embodiment of the present invention, the preparation method of the phosphorus-doped carbon nitride includes the following steps:

[0044] Melamine and hydroxyethane diphosphonic acid are added to a mixed solvent in a mass ratio of 12:1 and mixed evenly, and then evaporated, vacuum dried, and calcined in sequence to obtain the phosphorus-doped carbon nitride;

[0045] The mixed solvent is a mixture of ethanol and water with a volume ratio of 1:15.

[0046] The calcination temperature is 550 °C and the time is 4 h. The mixing method is vigorous stirring for 30 min. The present invention does not particularly limit the stirring rate, and a stirring rate well-known to those skilled in the art can be used, such as 1200 rpm.

[0047] The second aspect of the present invention provides an electrochemically active biosensor prepared by the above preparation method.

[0048] The third aspect of the present invention provides the application of the above electrochemically active biosensor in detecting organophosphorus insecticides.

[0049] In a preferred embodiment of the present invention, the organophosphorus insecticide is dimethoate.

[0050] The excellent detection performance of dimethoate in the present invention benefits from the synergistic effect of the following three aspects: (1) the improvement of electrode performance by electrochemical activation pretreatment, (2) the high electrocatalytic activity of Co-PCN nanomaterials, and (3) the adsorption and enrichment effect of CTAB on 1-naphthol.

[0051] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are publicly available.

[0052] The Phaseolus vulgaris esterase (PbBE) used in the examples of the present invention was prepared by the following steps: Dry Phaseolus vulgaris was crushed, and the crushed material was added to distilled water at a weight ratio of 1:5 and stirred for 30 minutes, and then centrifuged at 8000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter membrane to obtain a crude enzyme solution. Further, the crude enzyme was purified by aqueous two-phase extraction. First, a two-phase aqueous system composed of 27.0% PEG1000 / 13.0% NaH 2 PO 4 (pH 5.0) was used for enzyme extraction, and at this time the enzyme was partitioned into the upper phase. After discarding the lower phase, (NH 4 ) 2 SO 4 was added to form a second two-phase aqueous system, and the enzyme was transferred to the lower phase. Subsequently, the lower phase was collected and dialyzed in 0.05 M phosphate buffer (pH 6.5) at 4 °C for 48 hours. Finally, the enzyme dialysate was freeze-dried to obtain powdered Phaseolus vulgaris esterase.

[0053] The screen-printed electrode (SPE) used in the examples of the present invention was purchased from Botan Technology (Weihai) Co., Ltd. The working electrode and the counter electrode of the SPE are both carbon electrodes, and the reference electrode is silver / silver chloride.

[0054] The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] Preparation of Co nanoparticles anchored PCN (Co-PCN): First, 4.8 g of melamine and 0.4 g of hydroxyethylidene diphosphonic acid were added to a mixed solution of 4 mL of ethanol and 60 mL of ultrapure water under vigorous stirring, and stirred vigorously for 30 min. Then, the mixed solution was evaporated to dryness at 80 °C and further dried in vacuo at 60 °C for 12 h. Finally, the dried mixture was heated to 550 °C at a heating rate of 2.5 °C / min and held for 4 h. The material obtained after calcination was phosphorus-doped carbon nitride (PCN).

[0057] Weigh 0.5 g of PCN and ultrasonically disperse it in 50 mL of ultrapure water. Under stirring conditions, 7 mg of cobalt nitrate hexahydrate (Co(NO 3 ) 2 ·6H 2 O) was added and stirring continued for 30 min. Subsequently, 10 mL of NaBH 4 (0.05 mol / L) was added dropwise and reacted for 20 min. After centrifugation at 6000 rmp / min for 10 min, the precipitate was washed with water and dried in vacuo (60 °C, 12 h) to obtain Co nanoparticles anchored PCN (Co-PCN). Its TEM image is shown in Figure 2 , and it can be clearly seen from Figure 2 the Co nanoparticles dispersed on the PCN support. In addition, the elemental contents were determined by ICP-OES, and the contents of Co and P in Co-PCN were 2.828 wt% and 2.118 wt% respectively (as shown in Table 1).

[0058] Table 1

[0059]

[0060] Preparation of CTAB&Co-PCN: Weigh 0.8 mg of cetyltrimethylammonium bromide (CTAB) and 0.6 mg of Co-PCN and ultrasonically disperse them in 1 mL of ultrapure water to obtain CTAB&Co-PCN.

[0061] Phaseolus bean esterase (PbBE) was prepared into an enzyme solution of 14.4 U / mL with ultrapure water.

[0062] SPE Electrochemical Activation and Modification: The screen-printed electrode (SPE) was scanned 8 cycles in a 0.5 mol / L sulfuric acid solution at a scanning rate of 0.1 V / s within the range of -0.5 to 1.2 V. Subsequently, it was placed in a phosphate buffer solution (PBS) with pH 7.0 and activated using chronopotentiometry (CP) (anodic current of 0.1 mV, time of 40 s), and then further activated using cyclic voltammetry (CV) (potential range of -0.7 to 1 V, scanning rate of 0.1 V / s, one cycle) to obtain the activated SPE. 12 μL of CTAB&Co-PCN was dropped onto the activated SPE and dried under an infrared lamp (power 175 W, distance 30 cm) until it was visually dry, obtaining CTAB&Co-PCN / SPE (i.e., the electrochemical biosensor). The schematic diagram of the preparation process flow of this electrochemical biosensor is shown in Figure 1 .

[0063] The CTAB&Co-PCN / SPE prepared in Example 1 was verified for its effectiveness as follows:

[0064] 1. Preparation of the test solution

[0065] Without pesticide: Ultra-pure water and PbBE (14.4 U / mL) were mixed at a volume ratio of 1:2.5. 40 μL was taken and added to 120 μL of PBS buffer solution with pH 6.5, and then 10 μL of 32 mmol / L 1-naphthyl acetate was added. After reacting for 7 min, reaction solution I was obtained.

[0066] With pesticide: Different concentrations of dimethoate standard solutions and PbBE (14.4 U / mL) were mixed at a volume ratio of 1:2.5 and incubated for 12 min. Subsequently, 40 μL was taken and added to 120 μL of PBS buffer solution with pH 6.5, and then 10 μL of 32 mmol / L 1-naphthyl acetate was added. After reacting for 7 min, reaction solution II was obtained.

[0067] 2. Preparation of the actual sample

[0068] Spinach was selected as the actual detection sample, and the specific operation was as follows. 1 g of crushed spinach leaf sample was placed in 5 mL of methanol and stirred for extraction for 15 min, and the supernatant was collected. After the solvent evaporated, 10 mL of PBS (pH 7.0) buffer solution was added for re-dissolution to obtain the sample solution.

[0069] 3. Electrochemical testing

[0070] The detection of dimethoate was carried out in 0.1 mol / L PBS using differential pulse voltammetry (DPV) within the range of -0.1 V to 0.6 V. The specific steps were as follows: First, 120 μL of reaction solution I was dropped onto the CTAB&Co-PCN / SPE for DPV testing, and the peak current value was recorded as I 0, then 120 μL of Reaction Solution II was added dropwise onto CTAB&Co-PCN / SPE, and the peak current value at this time was denoted as I 1 . The current inhibition rate was calculated according to the following formula.

[0071]

[0072] The results are as follows:

[0073] 1. Response of CTAB&Co-PCN / SPE to 1-naphthol and feasibility of dimethoate electrochemical detection

[0074] The hydrolysis of 1-naphthyl acetate (1-NA) by PbBE can generate the reducing product 1-naphthol. Figure 3 A in [reference] records the CV response of CTAB&Co-PCN / SPE in PBS solution containing 1-naphthyl acetate (1-NA) and PbBE. It can be seen that an obvious oxidation peak appeared at about 0.3 V, which was attributed to the electrochemical oxidation of 1-naphthol. In the PBS solution containing only 1-NA or PbBE, since no 1-naphthol was generated, the corresponding CV curves were all smooth and no oxidation peak appeared.

[0075] Subsequently, the feasibility of using CTAB&Co-PCN / SPE for the electrochemical detection of dimethoate was further investigated, and the results are shown in Figure 3 B in [reference]. It can be seen that after incubating dimethoate with PbBE, the oxidation peak of 1-naphthol weakened, and the greater the concentration of dimethoate, the more the Ip decreased. Therefore, the detection of pesticides can be achieved according to the change of the oxidation peak of 1-naphthol in the presence of dimethoate.

[0076] 2. Detection of dimethoate by an electrochemical sensor

[0077] Under the optimal detection conditions, the detection of dimethoate by CTAB&Co-PCN / SPE was carried out using differential pulse voltammetry (DPV). The experimental results showed that as the concentration of dimethoate increased, the oxidation peak of 1-naphthol became smaller, and its corresponding peak current Ip gradually decreased. The corresponding detection mechanism was based on the inhibition of the activity of PbBE by dimethoate. The more pesticides there were, the weaker the hydrolysis ability of PbBE to 1-NA, and the less 1-naphthol was generated, resulting in a decrease in the oxidation peak current. Taking the inhibition rate of the current signal (ΔI / I) as a quantitative index, in the range of 0.01 - 3000 μg / L, there was a quantitative relationship between the logarithm of the dimethoate concentration and the inhibition rate, and the results are shown in Figure 4 , and its linear equation was ΔI / I(%) = 10.89lgC + 30.08 (R 2 = 0.997), and the detection limit LOD was 3.3 ng / L (S / N = 3).

[0078] 3. Anti-interference ability

[0079] The influence on detection when dimethoate at 10 μg / L coexists with other interfering substances was investigated. The results are as Figure 5 shown. The interfering substances were 100 μg / L KCl, 100 μg / L glucose, 100 μg / L oxalic acid, 100 μg / L KNO 3 , 100 μg / L MgCl 2 , 100 μg / L MnCl 2 , 100 μg / L ascorbic acid, 100 μg / L citric acid, 100 μg / L malic acid, 100 μg / L FeCl 3 , 100 μg / L CoCl 2 , 100 μg / L ZnCl 2 which had no significant effect on the response current of the sensor, indicating good anti-interference ability.

[0080] 4. Detection of actual samples

[0081] Taking spinach as a sample, the feasibility of the fabricated electrochemical biosensor for detecting actual samples was investigated. After pretreatment of spinach, different concentrations of dimethoate were added for detection. Each experiment was measured in parallel 3 times, and the experimental results are shown in Table 2. It can be seen that the recoveries were in the range of 92.5% - 103.3%, and the relative standard deviation (RSD) was 3.2% - 4.8%. The above results indicate that the sensor has good accuracy for detecting actual samples and is expected to be applied to the detection of pesticides in actual samples.

[0082] Table 2

[0083]

[0084] This invention studied a novel electrochemical biosensor for highly sensitive detection of dimethoate in food. PbBE can catalyze the generation of electrochemically active 1-naphthol from 1-NA, while dimethoate can effectively inhibit the activity of PbBE, resulting in a decrease in the generated 1-naphthol. Dimethoate was indirectly detected based on the amount of 1-naphthol generated. Compared with the previously reported detection methods, the linear range of this method was 0.01 - 3000 μg / L, and the detection limit was extremely low, being 3.3 ng / L. In addition, the sensor had good stability (the response current decreased to 87.0% of that on the first day after 90 days of storage) and anti-interference ability, and could be applied to the qualitative and quantitative detection of dimethoate in food.

[0085] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing an electrochemical biosensor, characterized in that: The following steps are involved: Dispersing phosphorus-doped carbon nitride in water, then adding cobalt salt and mixing well to obtain a mixed solution; Adding a reducing agent to the mixed solution for reaction to obtain Co-PCN; Dispersing the Co-PCN and hexadecyltrimethylammonium bromide in water to obtain CTAB&Co-PCN; The electrode is subjected to electrochemical activation pretreatment to obtain an activated electrode; The CTAB & Co-PCN are added to the surface of the activated electrode and dried to obtain the electrochemical biosensor.

2. The method for preparing an electrochemical biosensor according to claim 1, characterized in that: The mass ratio of the phosphorus-doped carbon nitride to the cobalt salt is 500:(6-8); the cobalt salt is Co(NO3)2·6H2O or CoCl2·6H2O; and the reducing agent is NaBH4.

3. The method for preparing an electrochemical biosensor according to claim 1, characterized in that: The concentration of Co-PCN in the CTAB&Co-PCN is 0.6 mg / mL, and the concentration of hexadecyltrimethylammonium bromide is 0.8 mg / mL.

4. The method for preparing an electrochemical biosensor according to claim 1, characterized in that: The electrochemical activation pretreatment is specifically as follows: placing the electrode in an acid solution, scanning 8 cycles in the range of -0.5 to 1.2 V at a scanning speed of 0.1 V / s, then placing it in a phosphate buffer solution at pH 7.0 and activating it using a chronopotentiometry method, and then activating it using a cyclic voltammetry method to obtain an activated electrode.

5. The method for preparing an electrochemical biosensor according to claim 4, characterized in that: The acid solution is a sulfuric acid solution with a concentration of 0.5 mol / L; the anode current for activation using chronopotentiometry is 0.1 mV, and the time is 40 s; the potential for activation using cyclic voltammetry is -0.7 to 1 V, and the scan rate is 0.1 V / S for one circle.

6. The method for preparing an electrochemical biosensor according to claim 1, characterized in that: The method of adding the CTAB & Co-PCN to the surface of the activated electrode is dropwise addition; the amount of the CTAB & Co-PCN added to the surface of the activated electrode is 0.6 to 0.7 μL / mm 2 .

7. The method for preparing an electrochemical biosensor according to claim 1, characterized in that: The preparation method of phosphorus-doped carbon nitride comprises the following steps: Melamine and hydroxyethylidene diphosphonic acid are added to a mixed solvent in a mass ratio of 12:1 and mixed evenly, and then evaporated, vacuum dried, and calcined in sequence to obtain the phosphorus-doped carbon nitride; The mixed solvent is a mixture of ethanol and water in a volume ratio of 1:

15.

8. The electrochemical biosensor prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the electrochemical biosensor as claimed in claim 8 in detecting organophosphorus pesticides.

10. The use according to claim 9, characterized in that: The organophosphorus insecticide is dimethoate.

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