Self-powered electrochemical sensor for detecting acetamiprid and preparation method thereof

Abstract

The invention discloses a self-powered electrochemical sensor for detecting acetamiprid, the self-powered electrochemical sensor comprises an anode, a cathode and an electrolyte, the anode is an AuNPs / GDY / GOD biological anode, and the cathode is an AuNPs / GDY / DNA chain biological sensor. The self-energized electrochemical sensor integrates a self-energized sensing technology, a multi-cascade nucleic acid amplification technology, capacitor auxiliary amplification and auxiliary reading by using a smart phone or a computer and the like. The sensor is used for high-sensitivity detection of acetamiprid ACE residues in food. The ACE detection method provided by the invention has the advantages of simplicity, rapidness, ultrasensitivity, intelligence and the like, has remarkable and excellent performance, and shows huge application potential in accurate monitoring of ACE in food and environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sensors, in particular to a self-powered electrochemical sensor for detecting acetamiprid and a preparation method thereof. BACKGROUND

[0002] As a representative of the first generation of neonicotinoid insecticides, acetamiprid (ACE) has been widely used in agricultural production, environmental health pest control, and pet parasite prevention, etc. However, if it is used in excess, it may produce persistent residues in environmental media (such as water environment, food chain and soil system), which not only destroys the ecological balance and biodiversity, but also poses a potential threat to human health, and may induce adverse health effects such as teratogenicity, carcinogenicity and mutagenicity. Therefore, it is extremely critical to accurately and sensitively detect the content of ACE in food and environment.

[0003] Currently, the detection of acetamiprid mainly includes the following methods: 1. Liquid chromatography-tandem mass spectrometry, which separates acetamiprid from other components in the sample by liquid chromatography, and then performs qualitative and quantitative analysis by tandem mass spectrometry. 2. Gas chromatography / gas chromatography-mass spectrometry, which is an effective separation and detection tool for some pesticides. However, the polarity and thermal stability of acetamiprid may not be suitable for direct gas chromatography, and usually requires complex derivatization steps, so it is not as widely used as LC-MS / MS. 3. High performance liquid chromatography, which separates using liquid chromatography and detects using ultraviolet or diode array detector. 4. Rapid detection methods, including enzyme-linked immunosorbent assay: based on the specific reaction of antigen and antibody. There is a special acetamiprid ELISA detection kit. Immune chromatography test strip: similar to early pregnancy test paper, simple operation, results can be obtained within a few minutes. However, the existing detection methods have the problems of low sensitivity, poor anti-interference ability, and inconvenience to carry, etc.

[0004] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art of this application. SUMMARY

[0005] The purpose of the present application is to provide a self-powered electrochemical sensor for detecting acetamiprid, so as to overcome the shortcomings of poor sensitivity, poor anti-interference ability, inconvenience to carry, etc. in detecting acetamiprid.

[0006] Another purpose of the present application is to provide a preparation method of a self-powered electrochemical sensor for detecting acetamiprid.

[0007] To achieve the above object, the application provides a self-powered electrochemical sensor for detecting acetamiprid, comprising: the self-powered electrochemical sensor comprises an anode, a cathode and an electrolyte, the anode is an AuNPs / GDY / GOD biological anode, and the cathode is an AuNPs / GDY / DNA chain biosensor.

[0008] A preparation method of the self-powered electrochemical sensor for detecting acetamiprid, the biosensor is based on gold nanoparticles / graphite dyne AuNPs / GDY, carbon cloth, DNA chain and biological enzyme to construct the cathode and anode of the self-powered electrochemical sensor.

[0009] Preferably, in the technical scheme, the preparation of the anode of the self-powered electrochemical sensor comprises: taking the carbon cloth coated with gold nanoparticles / graphite dyne AuNPs / GDY as an electrode substrate, and then coating glucose oxidase GOD on the surface of the electrode substrate to obtain an AuNPs / GDY / GOD biological anode.

[0010] Preferably, in the technical scheme, the preparation of the cathode of the self-powered electrochemical sensor comprises:

[0011] (1) Preparation of double-stranded DNA: mixing acetamiprid ACE aptamer and target DNA stock solution, heat treatment, then cooling to obtain a partially complementary double-stranded DNA solution specific to ACE, that is, an A-T complex solution;

[0012] (2) Preparation of biological cathode: taking the carbon cloth coated with gold nanoparticles / graphite dyne AuNPs / GDY as an electrode substrate, adding H1 solution dropwise on the electrode substrate and reacting, washing, then adding A-T complex solution, H2 solution and acetamiprid solution with different concentrations dropwise and reacting, then adding H3 and H4 solutions, and then incubating to obtain the cathode of the self-powered electrochemical sensor.

[0013] Preferably, in the technical scheme, the preparation of the electrode substrate comprises: coating 1-5 mg / mL gold nanoparticles / graphite dyne AuNPs / GDY mixed solution on the surface of the carbon cloth electrode, drying at 35-40℃ for 1-4 h, then immersing in 0.5-3 mg / mL N-(3-dimethylaminopropyl)-N'-ethylcarboxy imidazole EDC and N-hydroxysuccinimide NHS solution, washing after reaction to obtain the electrode substrate.

[0014] Preferably, in the technical scheme, the step (1) of preparing double-stranded DNA comprises: mixing equal volumes of acetamiprid ACE and target DNA stock solution, heat treating at 90-95℃ for 1-10 min, then cooling the mixture to 37℃ at a speed of 0.1-0.5℃ / s and keeping at 37℃ for 0.5-2 h to obtain an A-T complex solution.

[0015] Preferably, in the above technical solution, step (2) is to drop 30-60 μL of 0.5-2 μmol / L H1 solution on the electrode substrate, and react at 3-6°C for 8-16 h, then wash, and then sequentially add 30-60 μL of A-T solution, 0.5-2 μmol / L H2 solution and different concentrations of acetamiprid solution on the electrode, and react at 3-6°C for 1-5 h, then add 30-60 μL of 0.5-2 μmol / L H3 and H4 solutions, and further incubate at 3-6°C for 30-120 min.

[0016] Preferably, in the above technical solution, the nucleotide sequence of the aptamer Apt of acetamiprid is as shown in SEQ ID NO: 1.

[0017] The nucleotide sequence of the target DNA is as shown in SEQ ID NO: 2.

[0018] The nucleotide sequence of H1 is as shown in SEQ ID NO: 3.

[0019] The nucleotide sequence of H2 is as shown in SEQ ID NO: 4.

[0020] The nucleotide sequence of H3 is as shown in SEQ ID NO: 5.

[0021] The nucleotide sequence of H4 is as shown in SEQ ID NO: 6.

[0022] An application of a self-powered electrochemical sensor for detecting acetamiprid.

[0023] A method for detecting acetamiprid by using a self-powered electrochemical sensor, the method comprising the following steps:

[0024] (1) The biological cathode and the biological anode after reacting with different concentrations of acetamiprid target are immersed in an electrolyte, a multimeter is used to measure the electrochemical signal of the sensor, the detected electrochemical signal is amplified by a capacitor, data is transmitted to a smartphone or a computer for reading, and data is obtained; wherein the electrochemical signal includes one or both of E OCV value and current;

[0025] (2) A standard curve between the electrochemical signal and the concentration of acetamiprid is drawn, and the determination of acetamiprid is completed;

[0026] The support electrolyte of the biosensor built above is a 0.01 M PBS buffer system containing 5 mM glucose and 5 mM [Ru(NH3)6] 3+ The pH value of the system is 7.4.

[0027] The detection principle of the self-powered electrochemical sensor for detecting acetamiprid according to the present application is as follows:

[0028] The present application uses graphdiyne loaded gold nanoparticles (AuNPs / GDY) as a biological electrode base material, and glucose oxidase (GOD) is modified on the surface of a carbon cloth (CP) electrode to construct a biological anode. In the preparation process of the biological cathode, the H1 probe is fixed on the surface of the CP electrode through an Au-S bond. When there is no acetamiprid in the detection system, the hairpin structure of the H1 probe is in a closed state, which cannot trigger the subsequent molecular hybridization reaction, so that the electron acceptor cannot be effectively combined to the electrode interface, and finally the open circuit voltage of the system is maintained at a low level (E OCV ). However, when acetamiprid exists, the aptamer in A-T binds to acetamiprid with high affinity, and releases tDNA. According to the principle of DNA base complementary pairing, H1 is opened by tDNA to form a partially complementary double-stranded structure (H1-tDNA), which exposes the binding site of H2. H2 then hybridizes with H1 to form a complex of H1, H2 and tDNA. Under the driving of strand displacement free energy, tDNA is gradually replaced by H2, and the displaced tDNA serves as an initiator to hybridize with H1 again, starting the catalytic hairpin self-assembly (CHA) cycle, and finally forming a large number of H1-H2 complexes on the electrode sensing interface. The reaction mechanism promotes the unfolding of H3 and H4 hairpin probe structures, thereby triggering the hybridization chain reaction (HCR) to form a large number of double helix DNA (dsDNA) long chains on the surface of the biological cathode. Trivalent ruthenium ammonia complex [Ru(NH3)6] 3+ is embedded in the DNA double helix structure through electrostatic interaction. When the electrons generated by the glucose oxidation of the biological anode are transferred to the biological cathode, [Ru(NH3)6] 3+ is reduced to divalent state [Ru(NH3)6] 2+ , producing a significant electrochemical oxidation current (E OCV ) response. To improve the detection sensitivity, the present application also introduces a matching capacitor as a signal amplification element, which significantly improves the performance of the biosensor. The detection signal is amplified by several times. When the sensor generates an electrical signal for the analyte, the smart phone APP receives these data in real time through Bluetooth.

[0029] Compared with the prior art, the self-powered electrochemical sensor for detecting acetamiprid and the preparation method thereof according to the present application have the following beneficial effects: the self-powered electrochemical sensor for detecting acetamiprid and the preparation method thereof according to the present application integrate self-powered sensing technology, multi-level nucleic acid amplification technology, capacitor auxiliary amplification, and use a smart phone or a computer for auxiliary reading. The sensor according to the present application is used for high-sensitivity detection of acetamiprid ACE residues in food. The linear range of the detection is 1 fg / L (10 -15 g / L) to 50 pg / L (10 -12The detection limit reaches 0.14 fg / L, the relative standard deviation (RSD) is 5.0 % in 5 parallel detections of 10 fg / L ACE solution, the recovery rate of the ACE in the tomato and cucumber samples is between 91.2-98.6 %, and the detection result is also in good agreement with the detection result of the national standard method, the ACE detection method has the advantages of simplicity, high sensitivity, intelligence and the like, has remarkable excellent performance, and has great application potential in the accurate monitoring of ACE in food and environment. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a detection principle diagram of a self-powered electrochemical sensor for detecting acetochlor according to the application;

[0031] Figure 2 is an open circuit voltage E of the self-powered electrochemical sensor for detecting acetochlor according to the application in detection of different concentrations of acetochlor OCV response diagram;

[0032] Figure 3 is a change diagram of the open circuit voltage E of the self-powered electrochemical sensor for detecting acetochlor according to the application in detection of different concentrations of acetochlor and the open circuit voltage E OCV ;

[0033] Figure 4 is a linear relationship diagram of different concentrations of acetochlor and the open circuit voltage E of the self-powered electrochemical sensor for detecting acetochlor according to the application OCV ;

[0034] Figure 5 is a relationship diagram between the current intensity received by the smart phone and different concentrations of acetochlor;

[0035] A is a change value of different concentrations of acetochlor and the current before capacitor amplification, and B is a linear relationship diagram of different concentrations of acetochlor and the current before capacitor amplification;

[0036] C is a real-time current value received by the smart phone before the self-powered electrochemical sensor for detecting acetochlor, i.e., the enzyme biological fuel cell EBFCs, is connected in series with the capacitor;

[0037] D is a change value of different concentrations of acetochlor and the current after capacitor energy storage and amplification, and E is a linear relationship diagram of different concentrations of acetochlor and the current after capacitor energy storage and amplification;

[0038] F is a real-time current value received by the smart phone after the self-powered electrochemical sensor for detecting acetochlor, i.e., the enzyme biological fuel cell EBFCs, is connected in series with the capacitor. DETAILED DESCRIPTION

[0039] The present application will be described in detail below with reference to the drawings, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments.

[0040] The raw materials and reagents used in the examples are commercially available unless otherwise specified. Examples

[0041] A self-powered electrochemical sensor for detecting acetamiprid

[0042] As shown in Figure 1 A self-powered electrochemical sensor for detecting acetamiprid, comprising: the self-powered electrochemical sensor comprises an anode, a cathode and an electrolyte, the anode is an AuNPs / GDY / GOD biological anode, and the cathode is an AuNPs / GDY / DNA chain biosensor.

[0043] Preparation of a self-powered electrochemical sensor for detecting acetamiprid

[0044] 1. Preparation of double-stranded DNA

[0045] An equal volume (30 μL each) of ACE aptamer and target DNA (tDNA) stock solution was mixed and heat-treated at 95°C for 5 min to eliminate secondary structures in the DNA chain and promote effective binding of ACE aptamer to tDNA. Subsequently, to avoid mismatching during the binding process, the mixture was slowly cooled to 37°C at a rate of 0.1°C / s and kept at this temperature for 1 h to form a partially complementary double-stranded DNA solution specific to ACE, i.e., an A-T complex. This preparation step ensures the structural stability of the double-stranded DNA and high sensitivity to the target analyte.

[0046] 2. Preparation of electrode substrate

[0047] A carbon cloth (CP) electrode with a size of 1 cm × 1 cm was coated with 50 μL of a 1 mg / mL gold nanoparticle / graphyne (AuNPs / GDY) mixed solution and dried in a vacuum environment at 37°C for 120 min. Subsequently, the electrode and its substrate material were immersed in a 1 mg / mL solution of N-(3-dimethylaminopropyl)-N'-ethylcarboxyimidazole (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl groups of AuNPs. After that, the electrode was thoroughly washed with ultrapure water to remove unreacted EDC and NHS.

[0048] 3. Preparation of biological anode

[0049] The above-processed electrode substrate was treated with 50 μL of a 5 mg / mL glucose oxidase (GOD) solution to realize stable combination of GOD on the electrode surface through a reaction at 4°C for 12 h, thereby constructing a bioanode structure.

[0050] 4. Preparation of a biocathode

[0051] 50 μL of a 1 μmol / L H1 solution was added dropwise to the prepared electrode substrate, and reacted at 4°C for 12 h, followed by washing with distilled water to remove excess H1. 50 μL of an A-T solution, a 1 μmol / L H2 solution and different concentrations of acetamiprid solution were added dropwise to the electrode in sequence, and reacted at 4°C for 2 h. The density of the acetamiprid solution was 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 pg / L, respectively.

[0052] Subsequently, 50 μL of 1 μmol / L H3 and H4 solutions were added, and further incubated at 4°C for 70 min. Finally, the prepared electrode was stored at 4°C to maintain its biological activity.

[0053] The nucleotide sequence of the aptamer Apt of acetamiprid is shown as SEQ ID NO: 1; the nucleotide sequence of the target DNA is shown as SEQ ID NO: 2; the nucleotide sequence of H1 is shown as SEQ ID NO: 3; the nucleotide sequence of H2 is shown as SEQ ID NO: 4; the nucleotide sequence of H3 is shown as SEQ ID NO: 5; and the nucleotide sequence of H4 is shown as SEQ ID NO: 6. Details are shown in Table 1.

[0054] Table 1. Oligonucleotide sequences used in the present application

[0055]

[0056] III. Use of the self-powered electrochemical sensor of the present application for detection of acetamiprid

[0057] 1. 4 mL of a solution containing 5 mM glucose and 5 mM [Ru(NH3)6]Cl3was added to the electrode, and the electrode was incubated at 4°C for 12 h. 3+A 0.01 M PBS buffer system (pH 7.4) was used as the supporting electrolyte to prepare acetamiprid solutions at concentrations of 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 pg / L. After reacting with different concentrations of acetamiprid, the biocathode and bioanode were immersed in the electrolyte for 10 min, and then the acetamiprid standard solution was measured according to the above detection procedure. A multimeter was used to measure the electrochemical signal of the sensor, including E... OCV The values ​​and current are measured and the data is obtained in real time via mobile phone software.

[0058] 2. Plot a standard curve between the electrochemical signal and the concentration of acetamiprid, i.e., use the concentration value (c) or lgc and the corresponding E. OCV Plot a standard curve using the current. Complete the determination of acetamiprid. Figures 2-5 As shown.

[0059] like Figures 2-4 As shown, the open-circuit potential (E) of the sensor without the addition of acetamiprid OCV The voltage reading stabilized at around 0.15 volts. As the acetamiprid concentration gradually increased, the E voltage of the detection device... OCV The values ​​show an increasing trend (from line a to line j). Within the acetamiprid concentration range of 1 fg / L to 50 pg / L, E OCV A good linear relationship was observed between the logarithm of the acetamiprid concentration and the concentration of acetamiprid. Figure 4 The linear equation is E. OCV = 0.04633 lg c + 0.90094, correlation coefficient R² = 0.997, and detection limit (LOD) of 1.26 fg / L (signal-to-noise ratio S / N = 3), indicating that the sensor has high sensitivity and low detection limit.

[0060] like Figure 5 As shown, this invention employs an optimized strategy of parallel connection of electrochemical energy storage devices and enzyme biofuel cells to improve detection sensitivity. Experimental data show that without parallel capacitors ( Figure 5 C), the instantaneous current signal generated by the EBFC system is weak, but its current intensity shows a significant positive correlation with the concentration of acetamiprid. Figure 5 B), the linear regression equation is Y = 1.8119 lgC + 31.86246 (R2=0.998), and the detection limit is 0.14 fg / L (S / N=3).

[0061] After optimization with parallel capacitors, the system first completes the charging process of the capacitors, and then releases a significantly enhanced instantaneous current signal. Figure 5 D), such asFigure 5 E, the linear relationship equation is Y = 17.38363 lgC + 300.95391 (R2=0.996), and the detection limit is 0.27 fg / L (S / N=3). The sensitivity of the optimized system is 9.59 times higher than that of the original system, and the detection performance is significantly improved.

[0062] 3. Determining acetamiprid in a sample to be measured using the self-powered electrochemical sensor of the present application

[0063] Sample extraction

[0064] Using cucumbers and tomatoes as samples, the content of ACE in cucumbers and tomatoes was determined. First, the samples were pretreated using a high-speed homogenizer. 10.0 g of homogenized sample was accurately weighed, mixed with 20.0 mL of acetonitrile in a 50 mL centrifuge tube, vortexed for 5 minutes, then centrifuged at 4000 r / min for 5 minutes, the supernatant was collected, diluted with acetonitrile to 100 mL, and used for detection according to the above determination procedure of acetamiprid. The electrochemical signal was read and the corresponding concentration was obtained according to the standard curve. The determination results are shown in Table 2.

[0065] Table 2 Determination results of ACE content in cucumber and tomato samples

[0066]

[0067] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms described. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application and to allow others skilled in the art to understand the application for various exemplary embodiments with various modifications being suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and their equivalents.

Claims

1. A self-powered electrochemical sensor for detecting acetamiprid, characterized in that, The application relates to a self-powered electrochemical sensor for detecting acetamiprid. The self-powered electrochemical sensor comprises an anode, a cathode and an electrolyte, the anode is an AuNPs / GDY / GOD biological anode, and the cathode is an AuNPs / GDY / DNA chain biological sensor.

2. A method for preparing a self-powered electrochemical sensor for detecting acetamiprid according to claim 1, characterized in that, The biological sensor is based on gold nanoparticles / graphite dyryl (AuNPs / GDY), carbon cloth, DNA chains and biological enzymes to construct the cathode and anode of the self-powered electrochemical sensor.

3. The preparation method according to claim 2, characterized in that, Preparation of the anode of the self-powered electrochemical sensor comprises the following steps: taking carbon cloth coated with gold nanoparticles / graphite dyryl (AuNPs / GDY) as an electrode substrate, and then coating the electrode substrate surface with a glucose oxidase (GOD) reaction to obtain an AuNPs / GDY / GOD biological anode.

4. The production method according to claim 2, characterized by, Preparation of the cathode of the self-powered electrochemical sensor comprises the following steps: (1) Preparation of double-stranded DNA: mixing acetamiprid ACE aptamer and target DNA stock solutions, heat treating, and then cooling to obtain a partially complementary double-stranded DNA solution specific to ACE, namely an A-T complex solution; (2) Preparation of a biological cathode: taking carbon cloth coated with gold nanoparticles / graphite dyryl (AuNPs / GDY) as an electrode substrate, dropping and reacting H1 solution on the electrode substrate, cleaning, and then sequentially dropping and reacting A-T complex solution, H2 solution and different concentrations of acetamiprid solution, and then adding H3 and H4 solutions and incubating to obtain the cathode of the self-powered electrochemical sensor.

5. The production method according to claim 3 or 4, characterized by, Preparation of the electrode substrate comprises the following steps: coating the surface of a carbon cloth electrode with 1-5 mg / mL gold nanoparticles / graphite dyryl (AuNPs / GDY) mixed solution, drying at 35-40 DEG C for 1-4 h, and then immersing in 0.5-3 mg / mL N-(3-dimethylaminopropyl)-N'-ethylcarboxyimidazole (EDC) and N-hydroxysuccinimide (NHS) solutions, cleaning after reaction to obtain the electrode substrate.

6. The preparation method according to claim 4, characterized in that, Step (1) Preparation of double-stranded DNA comprises the following steps: mixing equal volumes of acetamiprid ACE and target DNA stock solutions, heat treating at 90-95 DEG C for 1-10 min, and then cooling the mixture to 37 DEG C at a speed of 0.1-0.5 DEG C / s and keeping at 37 DEG C for 0.5-2 h to obtain an A-T complex solution.

7. The preparation method according to claim 4, characterized in that, Step (2) dropping 30-60 muL of 0.5-2 mu mol / L H1 solution on the electrode substrate and reacting at 3-6 DEG C for 8-16 h, cleaning, and then sequentially dropping A-T solution, 0.5-2 mu mol / L H2 solution and different concentrations of acetamiprid solution each 30-60 muL on the electrode and reacting at 3-6 DEG C for 1-5 h, and then adding 0.5-2 mu mol / L H3 and H4 solutions each 30-60 muL and further incubating at 3-6 DEG C for 30-120 min.

8. The preparation method according to claim 4, characterized in that, The nucleotide sequence of the acetamiprid aptamer (Apt) is shown in SEQ ID NO: 1; The nucleotide sequence of the target DNA is shown in SEQ ID NO: 2; The nucleotide sequence of H1 is shown in SEQ ID NO: 3; The nucleotide sequence of H2 is shown in SEQ ID NO: 4; The nucleotide sequence of the H3 is shown as SEQ ID NO: 5; The nucleotide sequence of the H4 is shown as SEQ ID NO:

6.

9. Use of a self-powered electrochemical sensor according to any one of claims 1 to 8, characterized in that, The self-powered electrochemical sensor is used for detecting acetamiprid.

10. A method for detecting acetamiprid using a self-powered electrochemical sensor as described in any one of claims 1-9, characterized in that, The method comprises the following steps: (1) the bio-cathode and bio-anode after the reaction of different concentrations of acetamiprid target are immersed in the electrolyte, the electrochemical signal of the sensor is measured, the detected electrochemical signal is amplified by the capacitor, the data is transmitted to the smart phone or computer for reading, and the data is obtained; wherein the electrochemical signal includes one or both of E OCV value and current; (2) drawing a standard curve between the electrochemical signal and the acetamiprid concentration, and completing the determination of acetamiprid; Wherein, the above-mentioned biological sensor support electrolyte is 0.01 MPBS buffer system containing 5 mM glucose, 5 mM [Ru(NH3)6] 3+ , and the pH value of the system is 7.4.

Images