阅读说明：本技术 一种AChE和CHO双酶介导的磁弛豫开关传感器及其制备方法和应用 (AChE and CHO double-enzyme mediated magnetic relaxation switch sensor and preparation method and application thereof ) 是由 吴龙 周敏 陈翊平 吴正奇 陈小强 于 2020-06-30 设计创作，主要内容包括：本发明公开一种AChE和CHO双酶介导的磁弛豫开关传感器及其制备方法和应用,用于神经毒性农药残留的快速灵敏检测。氯化乙酰胆碱(ACh)可被乙酰胆碱酯酶(AChE)催化生成胆碱,再被胆碱氧化酶(CHO)水解成甜菜碱和过氧化氢(H<Sub>2</Sub>O<Sub>2</Sub>)；啶虫脒会使AChE失活,从而导致产生的H<Sub>2</Sub>O<Sub>2</Sub>减少；其中ACh、AChE和CHO为反应底物,引入顺磁离子Fe<Sup>2+</Sup>；H<Sub>2</Sub>O<Sub>2</Sub>会引起Fe<Sup>2+</Sup>/Fe<Sup>3+</Sup>的转化,从而影响周围水分子中氢质子的横向弛豫时间(T<Sub>2</Sub>),并作为啶虫脒检测的信号读出。检出限为2.66ng·mL<Sup>-1</Sup>(S/N＝3,n＝3)。该方法无需洗涤可一步检测啶虫脒,是复杂样品中农药残留检测的有效分析工具。(The invention discloses an AChE and CHO double-enzyme mediated magnetic relaxation switch sensor, a preparation method and application thereof, which are used for quickly and sensitively detecting neurotoxicity pesticide residues. Acetylcholine chloride (ACh) is catalyzed by acetylcholinesterase (AChE) to produce choline, which is then hydrolyzed by choline oxidase (CHO) to betaine and hydrogen peroxide (H) 2 O 2 ) (ii) a Acetamiprid inactivates AChE, resulting in the production of H 2 O 2 Reduction; wherein ACh, AChE and CHO are used as reaction substrates, and paramagnetic ion Fe is introduced 2+ ；H 2 O 2 Will cause Fe 2+ /Fe 3+ Thereby influencing the transverse relaxation time (T) of hydrogen protons in the surrounding water molecules 2 ) And read out as the signal for acetamiprid detection. The detection limit is 2.66 ng/mL ‑1 (S/N＝3，n3). The method can detect the acetamiprid in one step without washing, and is an effective analysis tool for detecting pesticide residues in complex samples.)

1. A preparation method of AChE and CHO double-enzyme mediated magnetic relaxation switch sensor is characterized in that,

preprocessing: AChE is dissolved and diluted by Tris-HCl buffer solution, CHO is dissolved and diluted by PBS buffer solution, ACh and neurotoxicity pesticide solution are prepared by water, and before testing, AChE and CHO are activated;

② Standard Curve is prepared by incubating a series of neurotoxic pesticide solutions of different concentrations with pretreated AChE for a period of time, adding excess pretreated ACh and CHO in sequence, incubating for a period of time, and mixing with Fe²⁺Mixing for a period of time, collecting T with a low-field NMR spectrometer₂Establishing a standard curve by taking the logarithm of the concentration of the sample as a horizontal coordinate and the transverse relaxation time as a vertical coordinate, and calculating to obtain a linear regression equation of the standard curve;

③ measurement of sample to be tested, incubating sample to be tested with appropriate amount of pretreated AChE for a certain time, sequentially adding excess pretreated ACh and CHO, incubating for a certain time, and further incubating with Fe²⁺Mixing for a period of time, collecting T with a low-field NMR spectrometer₂And substituting the value into a linear regression equation, and calculating to obtain the concentration of the neurotoxicity pesticide of the sample to be detected.

2. The method of claim 1, wherein the neurotoxic pesticide solution of step ② and step ③ is incubated with a suitable amount of pretreated AChE at 37 ℃ for 30-60min, then added with excess pretreated ACh and CHO in sequence, incubated for 20-40min, and then incubated with Fe²⁺Mixing for 0-20 min.

3. The method of claim 1, wherein the AChE and CHO two-enzyme-mediated magnetic relaxation switch sensor is prepared from Fe²⁺The optimum concentrations of ACh, AChE and CHO were 5mM, 500 U.L, respectively^-1And 2000 U.L^-1。

4. The method of claim 1, wherein the concentration of Tris-HCl buffer is 10mM, the pH is 7.2-8.5, the concentration of PBS buffer is 10mM, the pH is 7-8, and the solution of ACh and acetamiprid is prepared with ultra pure water.

5. The method of claim 1, wherein the AChE and CHO are activated at room temperature for 10min and used within 7 days before testing.

6. The method of claim 1, wherein the linear regression equation is as follows: Y63.49X +139.17, R²0.98, Lg [ acetamiprid concentration (μ g/mL) ]^-1)，Y＝ΔT₂，LOD＝2.66ng mL^-1，S/N＝3,n＝3,RSD＝3.8％。

7. The method of claim 1, wherein the Fe is Fe and CHO two-enzyme mediated magnetic relaxation switch sensor²⁺From FeSO₄。

8. An AChE and CHO two-enzyme mediated magnetic relaxation switch sensor prepared according to the method of any one of claims 1-7.

9. The AChE and CHO dual-enzyme mediated magnetic relaxation switch sensor of claim 8 for detecting residues of neurotoxic pesticides.

10. The AChE and CHO duzyme-mediated magnetic relaxation switch sensor of claim 9 for detecting imidacloprid, acetamiprid or imidaclothiz residue.

Technical Field

The invention belongs to the field of biosensors, and particularly relates to an AChE and CHO double-enzyme mediated magnetic relaxation switch sensor and a preparation method and application thereof.

Background

Since the invention of agricultural chemicals, agricultural chemicals such as organochlorine, organophosphorus, carbamate, pyrethroid, etc. have been rapidly applied to various fields. With the emergence of neonicotinoid compounds, the development of pesticides enters a new stage. Because of the unique action mechanism, the neonicotinoid pesticide has no cross resistance with the traditional pesticide, and is widely used. However, neonicotinoid pesticides have been found to pose a great threat to living organisms, with potential risks of mutagenicity, carcinogenicity, and teratogenicity. Therefore, specific and sensitive detection of these pesticides is required to initiate rapid measures. At present, there are three main types of analytical methods for detecting pesticide residues, including liquid chromatography, gas chromatography, mass spectrometry/liquid chromatography, immunoassay methods such as enzyme-linked immunosorbent assay (ELISA) and Lateral Flow Immunoassay (LFIA), and spectrophotometry. The existing pesticide residue detection method is either complex and time-consuming, or has low sensitivity, and cannot meet the requirements of quick application of low-level detection, especially the field analysis of pesticide residue. Therefore, the establishment of a rapid and sensitive pesticide residue detection method is of great significance.

The magnetic relaxation sensor has the advantages of one-step detection, no need of multiple washing, strong anti-interference capability and the like, so the magnetic relaxation sensor has wide application prospect in food sample analysis science. The signal readout of the magnetic sensor comes mainly from Magnetic Nanoparticles (MNPs) and paramagnetic ions. The former signal depends on the state change (dispersion/aggregation) of MNPs, and the latter signal comes from unpaired electrons. In consideration of the instability of MNPs, paramagnetic ions are stable and uniform in an aqueous solution, and can provide Fe³⁺And Fe²⁺Transverse relaxation time (T) of a plasma₂) As an alternative to developing magnetic assays free of MNPs. In electronic structure, Fe³⁺For semi-filling the d-orbit, the Electron Spin Relaxation (ESR) is about Fe²⁺30 times of the total weight of the powder. Results tableMing, Fe³⁺/Fe²⁺T in hydrogen protons in water₂Different values, at the same concentration, Fe²⁺T of₂Value greater than Fe³⁺T of₂Value, description of Fe³⁺Influence on the protons of the surrounding Water (Δ T)₂) Greater than Fe²⁺Influence on the protons of the surrounding water. Thus, use is made of Fe²⁺/Fe³⁺It is attractive to construct a magneto-sensitive sensor by mutual transformation.

In view of Fe²⁺And Fe³⁺Can be easily carried out in a redox reaction, and H₂O₂Related to all enzymes, it is a popular biochemical marker. On the other hand, neonicotinoid insecticides, such as acetamiprid, can damage the nervous system of insects by inhibiting the activity of acetylcholinesterase (AChE), which is also called enzyme inhibition. In an enzymatic reaction, H can be produced in the presence of acetylcholine chloride (ACh), acetylcholinesterase (AChE) and choline oxidase (CHO)₂O₂Can cause Fe²⁺To Fe³⁺By transformation of (1) to present T₂A change in the signal.

Disclosure of Invention

In order to overcome the problem of unstable signal read by Magnetic Nanoparticles (MNPs), the invention provides a preparation method of an AChE and CHO double-enzyme-mediated magnetic relaxation switch sensor, which uses paramagnetic ion Fe³⁺And Fe²⁺Transverse relaxation time (T)₂) The method is more stable and reliable as an alternative method for developing magnetic analysis without MNPs.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for preparing an AChE and CHO double-enzyme mediated magnetic relaxation switch sensor,

preprocessing: AChE is dissolved and diluted by Tris-HCl buffer solution, CHO is dissolved and diluted by PBS buffer solution, ACh and neurotoxicity pesticide solution are prepared by water, and before testing, AChE and CHO are activated;

② Standard Curve is prepared by incubating a series of neurotoxic pesticide solutions of different concentrations with appropriate amounts of pretreated AChE for a period of time, and sequentially adding an excess of pretreated AChEFor a period of time, and then with Fe²⁺Mixing for a period of time, collecting T with a low-field NMR spectrometer₂Establishing a standard curve by taking the logarithm of the concentration of the sample as a horizontal coordinate and the transverse relaxation time as a vertical coordinate, and calculating to obtain a linear regression equation of the standard curve;

③ measurement of sample to be tested, incubating sample to be tested with appropriate amount of pretreated AChE for a certain time, sequentially adding excess pretreated ACh and CHO, incubating for a certain time, and further incubating with Fe²⁺Mixing for a period of time, collecting T with a low-field NMR spectrometer₂And substituting the value into a linear regression equation, and calculating to obtain the concentration of the neurotoxicity pesticide of the sample to be detected.

In the method for preparing the AChE and CHO double-enzyme mediated magnetic relaxation switch sensor, the neurotoxic pesticide solution in the step ② and the step ③ is firstly incubated with a proper amount of pretreated AChE at 37 ℃ for 30-60min, then excessive pretreated ACh and CHO are added in sequence, the incubation is continued for 20-40min, and then the neurotoxic pesticide solution is incubated with Fe²⁺Mixing for 0-20 min.

The preparation method of the AChE and CHO double-enzyme mediated magnetic relaxation switch sensor comprises the step of preparing Fe²⁺The optimum concentrations of ACh, AChE and CHO were 5mM, 500 U.L, respectively^-1And 2000 U.L^-1。

In the method for preparing the AChE and CHO double-enzyme mediated magnetic relaxation switch sensor, the concentration of the Tris-HCl buffer solution is 10mM, the pH is 7.2-8.5, the concentration of the PBS buffer solution is 10mM, the pH is 7-8, and the ACh and acetamiprid solutions are prepared by using ultrapure water.

In the method for preparing the AChE and CHO two-enzyme mediated magnetic relaxation switch sensor, before testing, AChE and CHO should be activated at room temperature for 10 minutes and used within 7 days.

The preparation method of the AChE and CHO double-enzyme mediated magnetic relaxation switch sensor comprises the step of preparing Fe²⁺From FeSO₄。

The preparation method of the AChE and CHO two-enzyme mediated magnetic relaxation switch sensor comprises the following steps: y ═63.49X+139.17，R²0.98, Lg [ acetamiprid concentration (μ g/mL) ]^-1)，Y＝ΔT₂，LOD＝2.66ngmL^-1，S/N＝3,n＝3,RSD＝3.8％。

An AChE and CHO two-enzyme mediated magnetic relaxation switch sensor prepared by the method of any one of the above.

The AChE and CHO double-enzyme mediated magnetic relaxation switch sensor is used for detecting residues of neurotoxic pesticides.

The AChE and CHO double-enzyme mediated magnetic relaxation switch sensor is used for detecting the residue of imidacloprid, acetamiprid or imidaclothiz.

In the patent, a novel magnetic sensor is constructed by combining the enzyme inhibition property of a neurotoxic pesticide with an enzyme reaction system. Wherein, two enzymes of acetylcholinesterase (AChE) and choline oxidase (CHO) are adopted, ACh is taken as a substrate, and is simultaneously introduced into the solution containing Fe together with AChE and CHO²⁺In the sample (2). The acetylcholinesterase (AChE) catalyzes the substrate ACh to generate choline, which is then oxidized by CHO to generate H₂O₂Realization of Fe³⁺And Fe²⁺To be transformed in between. In the presence of neurotoxic pesticide, acetylcholinesterase (AChE) is deactivated to cut off ACh reaction, so reducing choline and H₂O₂Is generated. H₂O₂Fe can be oxidized and reduced in oxidation-reduction reaction²⁺Conversion to Fe³⁺Result in Δ T₂An increase, which is related to the concentration of the neurotoxic pesticide. The "one-step mixing method" (acetylcholinesterase (AChE), Cholinesterase (CHO) and acetylcholine chloride (ACh)) has been successfully applied to the detection of acetamiprid. The introduction of the dual-enzyme mediated cascade reaction is expected to improve the detection sensitivity, while Fe²⁺/Fe³⁺The switching system enables the magnetic sensor to remain stable.

The magnetic relaxation time sensor prepared by the invention has the advantages that:

1. the rapid detection method of pesticide residue, namely an enzyme inhibition method, is combined with a low-field nuclear magnetic resonance spectrometer, so that the defects that the traditional optical signal is easily interfered by a sample matrix, the sensitivity is insufficient and the like are overcome, and the analysis of the target substance in the low concentration range of the pesticide residue can be met.

2. The magnetic sensor has the advantages of low background signal, small equipment, low installation requirement, low detection cost, strong external interference resistance and easy realization of local instant detection.

Drawings

FIG. 1 is a schematic diagram of the present invention for detecting neurotoxic pesticides.

Fig. 2 is a representation of the present sensor.

FIG. 3 is an optimization plot of experimental conditions.

FIG. 4 is a fitting curve of quantitative detection of acetamiprid by the sensor prepared by the invention and compared with a traditional method.

FIG. 5 is a diagram showing the measurement of an actual sample of the present sensor.

Detailed Description

The technical scheme of the invention is further illustrated by the following specific examples, the neurotoxic pesticide can be imidacloprid, acetamiprid, imidaclothiz and the like, and the acetamiprid is mainly used at present, and the acetamiprid is taken as an example for illustration. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The reagent instrument equipment sources used:

1. 0.5T low field nmr spectrometer: shanghai Numag Inc.;

2. acetylcholine chloride, acetylcholinesterase: shanghai Michelin Biochemical technology, Inc.;

3. choline oxidase, acetamiprid: shanghai-sourced leaf Biotechnology, Inc.;

4. tomato and grape samples: a local supermarket.

5. Pretreatment: AChE was dissolved and diluted with Tris-HCl buffer at pH 8.0 and 10mM, and CHO was dissolved and diluted with PBS buffer at pH 7.4 and 10 mM. The ACh and acetamiprid solutions were prepared with ultra pure water. Prior to testing, AChE and CHO should be activated for 10 minutes at room temperature and used within 7 days. On storage, all solutions were stored at-4 ℃ for further use.