CN113109406A - Method for constructing electrochemiluminescence aptamer sensor for omethoate detection - Google Patents
Method for constructing electrochemiluminescence aptamer sensor for omethoate detection Download PDFInfo
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 53
- PZXOQEXFMJCDPG-UHFFFAOYSA-N omethoate Chemical compound CNC(=O)CSP(=O)(OC)OC PZXOQEXFMJCDPG-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 32
- 229910016021 MoTe2 Inorganic materials 0.000 claims abstract description 61
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- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000002096 quantum dot Substances 0.000 claims abstract description 19
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- 239000000243 solution Substances 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 22
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 claims description 20
- 239000006185 dispersion Substances 0.000 claims description 20
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- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 description 6
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- YNXICDMQCQPQEW-UHFFFAOYSA-N 1-naphthyl dihydrogen phosphate Chemical compound C1=CC=C2C(OP(O)(=O)O)=CC=CC2=C1 YNXICDMQCQPQEW-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a method for constructing an electrochemiluminescence aptamer sensor for sensitively detecting omethoate, which comprises the following steps of: step 1, preparing molybdenum telluride (MoTe)2) Quantum dots; step 2, preparing molybdenum telluride/2-methylimidazole zinc salt (MoTe)2/ZIF-8) nanocomposites; and 3, constructing an electrochemiluminescence aptamer sensor for sensitively detecting omethoate. The electrode material of the sensor adopts ZIF-8 with defects caused by quantum dots, so that the excitation potential of the material is effectively reduced, and the sensitivity of the sensor is greatly improved. Based on MoTe2The excellent ECL performance of the ZIF-8 is combined with an OMT aptamer with specific recognition, and an ECL aptamer sensor is successfully prepared for OMT monitoring. Meanwhile, the electrochemical luminescence detection method of OMT provided by the invention has operationSimple and flexible, simple instrument and equipment, wide detection range, low detection limit, low detection cost and the like.
Description
Technical Field
The invention belongs to the field of electrochemical analysis and detection, and discloses an electrochemiluminescence aptamer sensor for detecting omethoate in a vegetable sample.
Background
Organophosphorus pesticides (OPPs) refer to phosphorus-containing organic compound pesticides that are widely used in agricultural production, primarily as insecticides, and rarely as herbicides, fungicides, and plant growth regulators. Plays an active role in controlling agricultural plant diseases and insect pests and improving the quality and the yield of agricultural products. Omethoate (OMT) is one of organophosphorus pesticides, and is widely applied by farmers in various countries in the world due to the advantages of broad spectrum, high efficiency, rapidness, low price and the like. The abuse of omethoate for a long time in large quantities makes the pesticide residue problem increasingly serious. The residues of the organophosphorus pesticide not only pollute the ecological environment, but also cause different degrees of harm to human health and life safety. Its toxicity is based on the inhibition of acetylcholinesterase in humans, which is essential for the functioning of the mammalian central nervous system. A series of symptoms of neurotoxicity appear when OPPs are ingested in a large amount, and respiratory paralysis and even death appear in severe cases. Organ damage, teratogenesis, carcinogenesis, mutagenesis, and the like can occur when OPPs are taken in low doses for a long time or through diet.
The existing methods for detecting OMT comprise research methods such as a gas chromatography-mass spectrometry combination method, an enzyme inhibition method, a high performance liquid chromatography-mass spectrometry combination method and the like, and although the methods can meet the requirements of sensitivity and specificity detection, the methods have certain limitations in the aspect of practical application. For example, chromatography can be used for qualitative and quantitative detection, and the detection result is relatively accurate, reliable, high in sensitivity and good in reproducibility, but the used instruments and equipment are expensive, the operation is complex, and professional technicians are required, so that the method is not suitable for processing and analyzing large-batch samples and performing rapid detection on the spot. Electrochemiluminescence (ECL) is a light emission phenomenon that is generated when an excited state of a reaction product is generated by efficient electron transfer by applying a voltage or current to an electrode surface, and the excited state returns to a ground state. As a novel analysis and detection means combining chemiluminescence phenomenon and electrochemical technology, ECL couples the advantages of both (such as high sensitivity, low background interference, low detection limit, good selectivity and reproducibility, etc.), and thus, is widely used in the fields of food quality and safety, clinical treatment, environmental monitoring, etc.
Disclosure of Invention
The invention provides a method for constructing an electrochemiluminescence aptamer sensor, which is used for preparing defect-rich molybdenum telluride/2-methylimidazole zinc salt (MoTe)2ZIF-8) electrochemiluminescence active material, which is used for establishing an ECL sensing platform and is applied to detection of omethoate OMT in agricultural products. The invention constructs an ECL aptamer sensor for quickly and sensitively detecting OMT based on the characteristic of aptamer specificity identification, establishes the corresponding relation between the OMT concentration and the ECL response value, and realizes the aim of simply, sensitively and quickly detecting OMT. Thus, the prepared sensor can be used to detect OMT content in vegetable samples.
The adopted scheme is summarized as follows:
adopting room temperature synthesis method to mix MoTe2The particles introduced ZIF-8, causing the ligand of ZIF-8 to be deleted, resulting in defects.
To prepared defect-rich MoTe2the/ZIF-8 is used as an electrochemiluminescence active material to create an ultra-sensitive electrochemiluminescence sensing platform. Utilizing defect rich MoTe2The ZIF-8 nano composite has the characteristics of catalytic performance, good conductivity and the like, plays a role in signal amplification on a detection system, and increases the detection sensitivity. When adding target OMT, MoTe into the solution2the/ZIF-8 nano compound is excited by voltage to generate efficient electron transfer, an excited state of a reaction product is generated, and light is generated when the excited state returns to a ground state. And establishing a relation between the ECL response value and the OMT concentration so as to achieve the aim of quickly, sensitively and selectively detecting the OMT content in the soybean and spinach samples.
The invention is realized by the following specific technical scheme:
a method of constructing an electrochemiluminescent aptamer sensor for omethoate detection, comprising the steps of:
mixing the block MoTe2Freezing with liquid nitrogen, immediately transferring into cosolvent, performing ultrasonic disruption under ice water-rich condition, and centrifuging to obtain MoTe2Quantum dots;
the prepared MoTe2Fully mixing the quantum dot dispersion liquid with a dimethyl imidazole methanol solution, adding a zinc nitrate methanol solution, standing at room temperature for reaction, fully washing after the reaction is finished, and performing vacuum centrifugal drying to obtain MoTe2/ZIF-8 solid;
mixing the compound MoTe2the/ZIF-8 is dispersed in Naphthol Phosphate Buffer Solution (PBS) to obtain Naphthol/MoTe2The ZIF-8 dispersion liquid is dropwise coated on a polished glassy carbon electrode GCE; obtaining the Naphthol/MoTe2ZIF-8 modified GCE electrodes, i.e. Naphthol/MoTe2/ZIF-8/GCE; after air drying, dropping OMT aptamer on the electrode, reacting for a period of time, and leaching with PBS to obtain aptamer modified material electrode, namely aptamer/Naphthol/MoTe2ZIF-8/GCE, namely an ECL aptamer sensor for OMT detection is obtained. In step 1, MoTe2The preparation method of the quantum dots comprises the following steps: MoTe is prepared by a typical freezing and crushing method2Quantum dot, 60mg bulk MoTe2Placing in a centrifuge tube containing liquid nitrogen, standing for freezing for several hours, adding 20mL of tert-butanol/water as cosolvent into the above solution to form mixed solution, immediately transferring into a cell pulverizer for ultrasonic treatment, and centrifuging the dispersion with a centrifuge 13000rmp for 15-30 min to obtain MoTe2And (3) dispersing the mixture.
Wherein, as the cosolvent, the volume ratio of the tert-butyl alcohol to the water is 1: 1.
in step 2, MoTe2The volume ratio of the quantum dot dispersion liquid to the dimethyl imidazole methanol solution to the zinc nitrate methanol solution is 1: 10: 10, wherein MoTe2The concentration of the quantum dot dispersion liquid is 1-10mg/L, the concentration of the dimethyl imidazole methanol solution is 10mM, and the concentration of the zinc nitrate methanol solution is 10 mM.
The mixing mode is ultrasonic mixing; standing at room temperature for 2-4 hr, washing with methanol, and vacuum drying at 60 deg.C overnight.
In step 3, the polishing and cleaning steps of the glassy carbon electrode GCE are as follows: firstly, a glassy carbon electrode GCE with phi of 3.0mm is respectively used with Al of 1.0,0.3 and 0.05 mu m2O3Polishing powder is polished on polishing cloth into a mirror surface, then ultrasonic cleaning is carried out in ultrapure water, ethanol and ultrapure water in sequence, and blow-drying is carried out by nitrogen gas under the condition of room temperature.
In step 4, MoTe2The dosage proportion of the/ZIF-8 to the Naphthol Naphthol phosphate buffer solution is 2 mg: 1 mL; wherein, the mass percentage concentration of the naphthol is 0.5 percent;
the dropping amount of the dispersion was 6.0. mu.L;
the dropping amount of OMT aptamer is 6 μ L, wherein the concentration of OMT aptamer solution is 3 × 10-6mol/L。
For comparison, Naphthol/MoTe was obtained using similar procedure, respectively2(ii)/GCE and Naphthol/ZIF-8/GCE.
ECL aptamer sensors prepared in accordance with the present invention were successfully constructed and applied for the analytical detection of OMT in soybean and spinach samples.
The invention has the beneficial effects that:
(1) the invention prepares MoTe2the/ZIF-8 nano-composite is used as an ECL active material to construct an ECL sensor, and the ECL signal of the nano-composite is greatly enhanced relative to that of a monomer. This is probably because of the passage of MoTe2The doping of the nano-particles causes more defects of ZIF-8, obviously improves the catalytic performance of the ZIF-8, improves the electron transmission and reduces the excitation voltage.
(2) The invention relies on MoTe2The ZIF-8 aptamer sensor has excellent ECL performance and specificity. The sensor exhibits a wider linear range (10)-10-10-5g/L) and lower detection limit (3.33X 10)- 11g/L)。
(3) In addition, ECL aptamer sensors were constructed to detect OMT in soybean and spinach samples with satisfactory results. This work not only demonstrated the enrichment of defect-rich MoTe2ZIF-8 is potentially attractive for ECL-related applications, but also for foodA novel ECL sensing platform is constructed in the fields of product detection, biomedicine, environmental monitoring and the like.
(4) Compared with the traditional detection method, the ECL detection method of the OMT provided by the invention has the characteristics of simpler and more flexible operation, simpler instruments and equipment, less reagent dosage, low detection cost and the like.
Drawings
FIG. 1 shows ZIF-8(A) and MoTe2SEM representation of/ZIF-8 (B);
FIG. 2 shows MoTe2A photoelectron energy spectrum and a high-resolution photoelectron energy spectrum of/ZIF-8;
FIG. 3 is a schematic diagram of the aptamer sensing process (A), ECL signal variation graph of the aptamer sensing process (B) and impedance signal variation graph of the aptamer sensing process (C)
FIG. 4 is a graph of (A) the photocurrent response of the ECL aptamer sensor constructed to detect OMT at different concentrations, (B) the corresponding OMT linearity curve, (C) the stability of the ECL sensor constructed to be an aptamer ECL sensor at 28 cycles, and (D) the OMT aptamer sensor selectivity
Detailed Description
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
Example 1:
(1)MoTe2preparation of quantum dots
60mg of the purchased MoTe was weighed in bulk2The mixture was placed in a 40mL centrifuge tube, and frozen by immersing the tube in liquid nitrogen for 2 hours. 10mL of t-butanol and 10mL of water were measured and mixed to prepare a cosolvent. The tube was removed and immediately added to the above-mentioned cosolvent to form a mixed solution. Immediately transferred to a cell disruptor for 2 hours. Finally, the dispersion liquid is centrifuged by a centrifuge for 15 minutes at the rotating speed of 13000rmp, and the supernatant is collected to obtain MoTe2And (3) dispersing the mixture.
(2)MoTe2Preparation of/ZIF-8
Preparing 10mM 2-methylimidazole (HMeIM) in methanol, adding 1mL 1-10mg/L MoTe2And ultrasonically mixing the dispersion liquid for 0.5 hour to obtain a solution A. Then 10mM zinc nitrate (Zn (NO) was prepared3)2) Methanol solutionB, 10mL of A and 10mL of B were mixed well and allowed to stand at room temperature for 2 hours. Washing with methanol 10000rmp by centrifugation for 2-3 times. Then dried overnight under vacuum at 60 ℃.
ZIF-8(A) and MoTe2SEM representation of/ZIF-8 (B) (FIG. 1) and MoTe2The photoelectron spectrum and high-resolution photoelectron spectrum of/ZIF-8 (FIG. 2) show MoTe2Successful preparation of ZIF-8.
(3) Preparation steps of modified electrode
First, a glassy carbon electrode (GCE, Φ 3.0mm) was polished with 1.0,0.3 and 0.05 μm of polishing powder (Al), respectively2O3) And polishing the glass to be a mirror surface on a polishing cloth. Then ultrasonic cleaning is carried out in ultrapure water, ethanol and ultrapure water in sequence, and nitrogen is used for blow-drying under the condition of room temperature.
2mg of MoTe was weighed2the/ZIF-8 nano functional material is ultrasonically dispersed in 1mL of 0.5 percent Nafion/water solution to obtain 2.0mg/mL of MoTe2Naphthol/MoTe dispersion liquid of/ZIF-8 nano functional material2/ZIF-8). Transferring 6.0 μ L of the dispersion, dropping on the surface of the pre-treated GCE electrode, and air drying at room temperature to obtain Naphthol/MoTe2ZIF-8 modified GCE electrode (Naphthol/MoTe)2/ZIF-8/GCE)。
For comparison, Naphthol/MoTe was obtained using similar procedure, respectively2(ii)/GCE and Naphthol/ZIF-8/GCE.
(4) Construction steps of ECL sensor
In Naphthol/MoTe2the/ZIF-8/GCE electrode was coated with 6. mu.L OMT aptamer solution (3X 10)-6mol/L). The adapter sequence number of the OMT is: 5'-AAGCT TTTTT GACTG ACTGC AGCGATTCTT GATCG CCACG GTCTG GAAAA AGAG-3' are provided. Prepared aptamer modified Naphthol/MoTe2the/ZIF-8/GCE was reacted in a refrigerator at 4 ℃ for 12 hours and washed with 0.1mol/L PBS to remove the excess unadsorbed aptamer, yielding aptamer/Naphthol/MoTe2/ZIF-8/GCE。
Thereafter, 20. mu.L of 10 concentration was added-10,10-9,10-8,10-7,10-6,10-5g/L OMT was added to aptamer/Naphthol/MoTe, respectively2On a/ZIF-8/GCE electrode and incubated in a 37 ℃ incubator for a period of time。
Fig. 3 is a schematic and characterization diagram of an aptamer sensing process, demonstrating the process and successful construction of ECL aptamer sensors. The ECL experiment is carried out on an MPI-A type electrochemical luminescence detector, the high voltage of a photomultiplier is set to be 600V, the voltage range is-2.0-0V, and the test system contains 50mM K2S2O8And finally ECL aptamer sensors were successfully constructed and applied for analytical detection of OMT in soybean and spinach samples.
FIG. 4 is a graph showing the photocurrent response of the ECL aptamer sensor constructed in (A) to detect OMT with different concentrations, and (B) the corresponding OMT detection linearity curve at 10-10～10-5In the concentration interval of g/L, the ECL signal value and the OMT concentration log value present good linear relation, and the detection limit can reach 3.33 multiplied by 10-11g/L. Graph (C) constructed stability of the aptamer ECL sensor over 28 cycle periods and (D) OMT aptamer sensor selectivity graph showed good selectivity.
Example 2:
(1)MoTe2preparation of quantum dots
40mg of the purchased MoTe was weighed in bulk2The mixture was placed in a 40mL centrifuge tube, and frozen by immersing the tube in liquid nitrogen for 2 hours. 10mL of t-butanol and 10mL of water were measured and mixed to prepare a cosolvent. The tube was removed and immediately added to the above-mentioned cosolvent to form a mixed solution. Immediately transferred to a cell disruptor for 2 hours. Finally, the dispersion liquid is centrifuged by a centrifuge for 15 minutes at the rotating speed of 13000rmp, and the supernatant is collected to obtain MoTe2And (3) dispersing the mixture.
(2)MoTe2Preparation of/ZIF-8
Preparing 10mM 2-methylimidazole (HMeIM) in methanol, adding 1mL 1-10mg/L MoTe2And ultrasonically mixing the dispersion liquid for 0.5 hour to obtain a solution A. Then 10mM zinc nitrate (Zn (NO) was prepared3)2) The methanol solution was thoroughly mixed with 10mL of A and 10mL of B, and then allowed to stand at room temperature for 2 hours. Washing with methanol 10000rmp by centrifugation for 2-3 times. Then dried overnight under vacuum at 60 ℃.
Steps (3) and (4) are the same as Steps (3) and (4) of example 1.
Example 3:
(1)MoTe2preparation of quantum dots
40mg of the purchased MoTe was weighed in bulk2The mixture was placed in a 40mL centrifuge tube, and frozen by immersing the tube in liquid nitrogen for 2 hours. 10mL of t-butanol and 10mL of water were measured and mixed to prepare a cosolvent. The tube was removed and immediately added to the above-mentioned cosolvent to form a mixed solution. Immediately transferred to a cell disruptor for 2 hours. Finally, the dispersion liquid is centrifuged by a centrifuge for 15 minutes at the rotating speed of 13000rmp, and the supernatant is collected to obtain MoTe2And (3) dispersing the mixture.
(2)MoTe2Preparation of/ZIF-8
Preparing 20mM 2-methylimidazole (HMeIM) in methanol, adding 1mL 1-10mg/L MoTe2And ultrasonically mixing the dispersion liquid for 0.5 hour to obtain a solution A. Then 10mM zinc nitrate (Zn (NO) was prepared3)2) The methanol solution was thoroughly mixed with 10mL of A and 10mL of B, and then allowed to stand at room temperature for 2 hours. Washing with methanol 10000rmp by centrifugation for 2-3 times. Then dried overnight under vacuum at 60 ℃.
Steps (3) and (4) are the same as Steps (3) and (4) of example 1.
Claims (10)
1. A method of constructing an electrochemiluminescent aptamer sensor for omethoate detection, comprising the steps of:
step 1, MoTe2Preparing quantum dots:
mixing the block MoTe2Freezing with liquid nitrogen, immediately transferring into cosolvent, performing ultrasonic disruption under ice water-rich condition, and centrifuging to obtain MoTe2Quantum dots;
step 2, MoTe2Preparation of ZIF-8:
the prepared MoTe2Fully mixing the quantum dot dispersion liquid with a dimethyl imidazole methanol solution, adding a zinc nitrate methanol solution, standing at room temperature for reaction, fully washing after the reaction is finished, and performing vacuum centrifugal drying to obtain MoTe2/ZIF-8 solid;
step 3, polishing and cleaning the glassy carbon electrode GCE;
and 4, constructing an ECL sensor for detecting omethoate electrochemiluminescence:
mixing the compound MoTe2the/ZIF-8 is dispersed in Naphthol Naphthol phosphate buffer solution PBS to obtain Naphthol/MoTe2The ZIF-8 dispersion liquid is dropwise coated on a polished glassy carbon electrode GCE; obtaining the Naphthol/MoTe2ZIF-8 modified GCE electrodes, i.e. Naphthol/MoTe2/ZIF-8/GCE; after air drying, dropping OMT aptamer on the electrode, reacting for a period of time, and leaching with PBS to obtain aptamer modified material electrode, namely aptamer/Naphthol/MoTe2the/ZIF-8/GCE is an ECL aptamer sensor for omethoate OMT detection.
2. The method of constructing an electrochemiluminescent aptamer sensor for omethoate detection according to claim 1, wherein MoTe is used in step 12The preparation method of the quantum dots comprises the following steps: MoTe is prepared by a typical freezing and crushing method2Quantum dot, 60mg bulk MoTe2Placing in a centrifuge tube containing liquid nitrogen, standing for freezing for several hours, adding 20mL of tert-butanol/water as cosolvent into the above solution to form mixed solution, immediately transferring into a cell pulverizer for ultrasonic treatment, and centrifuging the dispersion with a centrifuge 13000rmp for 15-30 min to obtain MoTe2And (3) dispersing the mixture.
3. The method for constructing an electrochemiluminescent aptamer sensor for omethoate detection as claimed in claim 2, wherein the volume ratio of tert-butanol to water in the cosolvent is 1: 1.
4. the method for constructing an electrochemiluminescent aptamer sensor for omethoate detection according to claim 1, wherein MoTe is used in step 22The volume ratio of the quantum dot dispersion liquid to the dimethyl imidazole methanol solution to the zinc nitrate methanol solution is 1: 10: 10, wherein MoTe2The concentration of the quantum dot dispersion liquid is 1-10mg/L, the concentration of the dimethyl imidazole methanol solution is 10mM, and the concentration of the zinc nitrate methanol solution is 10 mM.
5. The method for constructing the electrochemiluminescence aptamer sensor for omethoate detection according to claim 1, wherein in the step 2, the mixing mode is ultrasonic blending; standing at room temperature for 2-4 hr, washing with methanol, and vacuum drying at 60 deg.C overnight.
6. The method for constructing an electrochemiluminescent aptamer sensor for omethoate detection according to claim 1, wherein in the step 3, the polishing and cleaning steps of the glassy carbon electrode GCE are as follows: firstly, a glassy carbon electrode GCE with phi of 3.0mm is respectively used with Al of 1.0,0.3 and 0.05 mu m2O3Polishing powder is polished on polishing cloth into a mirror surface, then ultrasonic cleaning is carried out in ultrapure water, ethanol and ultrapure water in sequence, and blow-drying is carried out by nitrogen gas under the condition of room temperature.
7. The method for constructing an electrochemiluminescent aptamer sensor for omethoate detection according to claim 1, wherein MoTe is used in step 42The dosage proportion of the/ZIF-8 to the Naphthol Naphthol phosphate buffer solution is 2 mg: 1 mL; wherein the mass percentage concentration of the naphthol is 0.5 percent.
8. The method for constructing an electrochemiluminescent aptamer sensor for omethoate detection according to claim 1, wherein in step 4, the dispersion is dispensed in an amount of 6.0 μ L; the dropping amount of OMT aptamer is 6 μ L, wherein the concentration of OMT aptamer solution is 3 × 10-6mol/L。
9. Use of the electrochemiluminescence aptamer sensor prepared by the method of claims 1-8 for detecting omethoate.
10. The use according to claim 9, wherein the electrochemiluminescent aptamer sensor is used for detecting omethoate in soybean and spinach samples.
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