CN115060777A - Ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate, preparation method and application - Google Patents
Ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate, preparation method and application Download PDFInfo
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Abstract
The invention relates to the technical field of electrochemical sensing, in particular to a ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate and a preparation method and application thereof, which is obtained by sequentially assembling a CNHs/AQ/AuNPs composite material, hDNA, MCH, MB-Apt1 and Fc-Apt2 on a glassy carbon electrode. When MAL or OMT is present, its specific binding to the aptamer causes the stripping of MB-Apt1 or Fc-Apt2 from the electrode surface, resulting in a ratiometric signal I MB /I AQ Or I Fc /I AQ And the ratio of MAL and OMT is reduced and the simultaneous detection is realized. The linear detection range of the MAL is 3 pg/mL-3 ng/mL, and the detection limit is 1.3 pg/mL; the detection linear range of the OMT is 10 pg/mL-10 ng/mL, and the detection limit is 2.8 pg/mL. The sensor has the advantages of high sensitivity, good selectivity, high precision and the like.
Description
Technical Field
The invention relates to the technical field of electrochemical sensing, in particular to a ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate.
Background
Organophosphorus pesticides are a class of commonly used phosphorus-containing organic compound pesticides in agricultural production, and are mainly used for preventing, controlling or eliminating diseases, pests and weeds so as to improve the quality and yield of crops. Malathion (MAL) and Omethoate (OMT) are two common organophosphorus pesticides, and are widely used by farmers in various countries in the world due to the advantages of broad spectrum, high efficiency, low price and the like.
Organophosphorus pesticide has high neurotoxicity, and can inhibit the activity of acetylcholinesterase in nervous system even at low concentration to accumulate acetylcholine, so as to cause a series of neurotoxicity symptoms, and can be killed by pulmonary edema, cerebral edema and respiratory paralysis in severe cases. Due to the widespread use of organophosphorus pesticides, their residues in agricultural products have caused serious human health and environmental problems. Therefore, there is a need to develop effective detection techniques to achieve efficient detection and analysis of MAL and OMT in agricultural products.
Currently, technologies for simultaneously detecting MAL and OMT mainly include elisa, gas chromatography, hplc-mass spectrometry, etc., but these technologies have the disadvantages of expensive instrument, long time consumption, complex operation, etc., and it is necessary to develop a simple, fast and efficient detection means.
The electrochemical sensing technology is expected to become a promising method for simultaneously detecting various pesticides with high efficiency by the advantages of low cost, quick response, simple operation and the like. At present, electrochemical aptamer sensors for single pesticides have good sensitivity and selectivity, but the detection accuracy needs to be further improved.
Therefore, the development of a technical scheme capable of realizing the simultaneous detection of the MAL and the OMT with high sensitivity and high accuracy has important research significance.
Disclosure of Invention
The invention aims to provide a ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate, and a preparation method and application thereof.
The ratio electrochemical aptamer sensor based on the ratio strategy has double response signals aiming at a target object, and the concentration of the target object is quantified by measuring the ratio of the double signals, so that the accuracy of the electrochemical sensor can be effectively improved. The invention uses carbon nanohorn/anthraquinone-2-carboxylic acid/gold nanoparticle composite material (CNHs/AQ/AuNPs), hairpin DNA (hDNA), Mercaptohexanol (MCH), methylene blue marked malathion aptamer (MB-Apt1) and ferrocene marked omethoate aptamer (Fc-Apt2) to be sequentially assembled on a glassy carbon electrode to construct a ratio electrochemical aptamer sensor, thereby realizing the high-efficiency simultaneous detection of malathion and omethoate.
Specifically, the invention provides the following technical scheme:
a method of making a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate, comprising the steps of:
(1) sequentially polishing the glassy carbon electrode GCE by using alumina powder with different particle sizes, sequentially performing ultrasonic treatment in ethanol and pure water to remove surface residues, and airing at room temperature;
(2) dropwise adding the prepared carbon nanohorn/anthraquinone-2-carboxylic acid/gold nanoparticle composite material, namely CNHs/AQ/AuNPs, onto the electrode treated in the step (1), and airing at room temperature;
(3) modifying hairpin DNA, namely hDNA, on the electrode treated in the step (2), storing for a period of time at room temperature, and fixing the hDNA on the surface of the electrode by utilizing a gold-sulfur bond;
(4) carrying out Mercaptohexanol (MCH) modification on the electrode treated in the step (3), and incubating for a period of time at room temperature to block non-specific binding sites of gold;
(5) modifying the electrode treated in the step (4) with methylene blue labeled malathion aptamer as MB-Apt1, reacting for a period of time at a certain temperature, and assembling MB-Apt1 on the electrode by utilizing complementary hybridization of the malathion aptamer and hDNA;
(6) and (3) modifying the ferrocene-labeled omethoate aptamer as Fc-Apt2 on the electrode treated in the step (5), reacting for a period of time at a certain temperature, and further assembling Fc-Apt2 on the electrode by utilizing complementary hybridization to obtain a ratiometric electrochemical aptamer sensor as MB-Apt1& Fc-Apt 2/MCH/hDNA/CNHs/AQ/AuNPs/GCE.
Preferably, in the step (1), the diameter of the glassy carbon electrode is 3 mm; the grain sizes of the aluminum oxide powders used are 0.3 μm and 0.05 μm in this order.
Preferably, in the step (2), the CNHs/AQ/AuNPs composite material is prepared by the following steps: firstly, dispersing 1-10 mg of carbon nanohorns in 2-20 mL of water, and stirring for 0.5-2 hours at room temperature to obtain a solution A; adding 0.1-1 mg of anthraquinone-2-carboxylic Acid (AQ) into the solution A, stirring for 4-12 hours at room temperature, centrifuging and washing for 3 times, and dispersing the precipitate into 2-20 mL of pure water again to obtain a solution B; 0.05-0.5 mL of 30-60 mM HAuCl is added into the solution B 4 Stirring the solution for 0.5-1 hour at room temperature, adding 0.5-5 mL of 2-20 mM ascorbic acid solution, continuously stirring for 4-12 hours at room temperature, centrifuging, washing and drying to obtain a CNHs/AQ/AuNPs composite material; and dispersing the obtained CNHs/AQ/AuNPs composite material in 2-20 mL of pure water to obtain a CNHs/AQ/AuNPs solution.
Preferably, in the step (2), the CNHs/AQ/AuNPs solution is used in an amount of 4-8 μ L, and the concentration is 0.05-5 mg/mL.
Preferably, in the step (3), the dosage of the hDNA is 4-8 muL, and the concentration is 1-8 muM; the storage time is 4-12 hours under the room temperature condition.
Preferably, in the step (4), the dosage of the MCH is 4-8 muL, and the concentration is 0.01-10 mM; the incubation time is 0.5-2 hours under the room temperature condition.
Preferably, in the step (5), the dosage of the MB-Apt1 is 4-8 muL, and the concentration is 1-8 muM; the temperature is 37 ℃, and the reaction time is 1-2 hours.
Preferably, in the step (6), the dosage of the Fc-Apt2 is 4-8 muL, and the concentration is 1-8 muM; the temperature is 37 ℃, and the reaction time is 1-2 hours.
The working method of the sensor prepared by the invention for detecting malathion and omethoate specifically comprises the following steps:
(1) the prepared plurality of sensors MB-Apt1&Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE is added with different concentrations ofThe malathion and omethoate standard solutions correspond to one concentration for each sensor; after incubation at room temperature for a certain period of time, washing with Tris-HCl (pH 7.4) solution; in a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measured MB /I AQ And I Fc /I AQ Establishing a signal I of the ratio of the concentration of malathion to the concentration of omethoate MB /I AQ And I Fc /I AQ A standard linear curve of (d);
wherein, I MB For MB oxidation current, I Fc Is Fc oxidation current, I AQ Is AQ oxidation current;
(2) dropwise adding the sample solution to be detected to a sensor MB-Apt1&Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE, incubating at room temperature for a certain time, and washing with Tris-HCl (pH 7.4) solution; in a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measured MB /I AQ And I Fc /I AQ And (2) substituting the standard linear curve in the step (1) to realize high-precision simultaneous detection of the malathion and the omethoate of the samples to be detected.
Preferably, in the step (1), the concentration range of the malathion is 3 x 10 -12 ～3×10 -9 g/mL, omethoate concentration range of 1X 10 -11 ～1×10 -8 g/mL。
Preferably, in the steps (1) and (2), the incubation is carried out for 0.5-2 hours at room temperature.
The working principle of the sensor prepared by the invention is as follows:
firstly, sequentially modified CNHs/AQ/AuNPs, hDNA, MCH, MB-Apt1 and Fc-Apt2 are assembled on the surface of a glassy carbon electrode to form a double-stranded DNA structure based on hairpin DNA, and then a larger MB oxidation current (I) is generated MB ) Fc oxidation current (I) Fc ) And AQ oxidation current (I) AQ ). When malathion or omethoate is present, its specific binding to the aptamer results in stripping of MB-Apt1 or Fc-Apt2 from the electrode surface, resulting in I MB And I Fc Is reduced by AQ Remain unchanged. Thus, by measuring the ratio signal I MB /I AQ And I Fc /I AQ The simultaneous detection of the target substances malathion and omethoate is realized.
Compared with the prior art, the invention has the beneficial effects that:
(1) the CNHs/AQ/AuNPs composite material prepared by the invention can generate an internal standard signal I AQ And amplifying the target response signal I MB And I Fc 。
(2) The invention constructs the sensing interface by utilizing the specific complementary hybridization of the hairpin DNA and the 2 aptamers, and the cross influence among the 2 targets can not be generated.
(3) The ratio electrochemical aptamer sensor for simultaneously detecting malathion and omethoate, which is prepared by the invention, has the characteristics of high sensitivity, good selectivity, high precision and the like, and has potential application prospects in the field of agricultural product quality detection.
Drawings
FIG. 1 is a schematic diagram of the construction and detection process of the ratiometric electrochemical sensor.
FIG. 2(A) shows ACV responses corresponding to different concentrations of MAL and OMT, in which the concentration of MAL is 3 × 10 -12 ,1×10 -11 ,3×10 -11 ,1×10 -10 ,3×10 -10 ,1×10 -9 And 3X 10 -9 g/mL; the OMT concentration is 1 × 10 -11 ,3×10 -11 ,1×10 -10 ,3×10 -10 ,1×10 -9 ,3×10 -9 And 1X 10 -8 g/mL; (B) is a ratio signal I MB /I AQ And I Fc /I AQ Standard linear curves with logarithm of MAL, OMT concentration, respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
A method for preparing a ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate, wherein the construction and detection process of the ratiometric electrochemical aptamer sensor is shown in figure 1.
The method specifically comprises the following steps:
(1) polishing a glassy carbon electrode GCE with the diameter of 3mm by using aluminum oxide powder with the diameter of 0.3 mu m and aluminum oxide powder with the diameter of 0.05 mu m in sequence, performing ultrasonic treatment in ethanol and water in sequence to remove surface residues, and airing at room temperature;
(2) dripping 6 mu L of CNHs/AQ/AuNPs solution with the concentration of 1mg/mL on the electrode treated by the step (1), and airing at room temperature;
wherein, the preparation steps of the CNHs/AQ/AuNPs solution are as follows:
firstly, dispersing 5mg of carbon nanohorns in 10mL of water, and stirring for 0.5 hour at room temperature to obtain a solution A; adding 0.35mg of anthraquinone-2-carboxylic Acid (AQ) into the solution A, stirring for 12 hours at room temperature, centrifuging and washing for 3 times, and dispersing the precipitate into 10mL of pure water again to obtain a solution B; 0.2mL of 60mM HAuCl was added to solution B 4 Stirring the solution for 0.5 hour at room temperature, adding 2mL of 10mM ascorbic acid solution, continuously stirring for 8 hours at room temperature, centrifuging, washing and drying to obtain a CNHs/AQ/AuNPs composite material; and dispersing the obtained CNHs/AQ/AuNPs composite material in 5mL of pure water to obtain a CNHs/AQ/AuNPs solution.
(3) Modifying 6 mu L of hDNA with the concentration of 2 mu M on the electrode treated in the step (2), standing for 8 hours at room temperature, and fixing the hDNA on the surface of the electrode by utilizing a gold-sulfur bond;
(4) modifying Mercaptohexanol (MCH) at a concentration of 1mM at 6. mu.L on the electrode treated in step (3), and incubating at room temperature for 1 hour to block non-specific binding sites of gold;
(5) modifying the electrode treated in the step (4) with 6. mu.L of methylene blue labeled malathion aptamer (MB-Apt1) with the concentration of 2. mu.M, reacting at 37 ℃ for 1 hour, and assembling MB-Apt1 on the electrode by utilizing the complementary hybridization of the aptamer and hDNA;
(6) and (3) modifying the electrode treated in the step (5) with 6 mu L of ferrocene-labeled omethoate aptamer (Fc-Apt2) with the concentration of 2 mu M, reacting at 37 ℃ for 1 hour, and further assembling Fc-Apt2 on the electrode by utilizing complementary hybridization to obtain the ratiometric electrochemical aptamer sensor which is marked as MB-Apt1& Fc-Apt 2/MCH/hDNA/CNHs/AQ/AuNPs/GCE.
The prepared sensor MB-Apt1&Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE are sequentially dripped with malathion and omethoate standard solutions with different concentrations, wherein the concentration of MAL is 3 multiplied by 10 in sequence -12 ,1×10 -11 ,3×10 -11 ,1×10 -10 ,3×10 -10 ,1×10 -9 And 3X 10 -9 g/mL, OMT concentration 1X 10 -11 ,3×10 -11 ,1×10 -10 ,3×10 -10 ,1×10 -9 ,3×10 -9 And 1X 10 -8 g/mL; after incubation at room temperature for 40 minutes, washing was performed using a Tris-HCl (pH 7.4) solution. In a three-electrode system, the washed electrode is used as a working electrode, an Ag/AgCl (saturated KCl) electrode is used as a reference electrode, a platinum wire is used as a counter electrode, a phosphate buffer solution (pH 7.4) is used as an electrolyte, an electrochemical Alternating Current Voltammetry (ACV) curve is scanned, and a ratio signal I is measured MB /I AQ And I Fc /I AQ Establishing a signal I of the ratio of the concentration of malathion to the concentration of omethoate MB /I AQ And I Fc /I AQ Standard linear curve of (2).
From FIG. 2(A), it can be seen that as the concentration of MAL and OMT increases, I MB And I Fc Respectively, are gradually decreased.
From FIG. 2(B), it can be seen that the ratio I MB /I AQ The standard linear curve between the logarithm of the concentration of MAL is I MB /I AQ ＝-0.161-0.122LogC MAL [g/mL](R 2 0.997), the linear range is 3 pg/mL-3 ng/mL, and the detection limit is 1.3 pg/mL; ratio I Fc /I AQ The standard linear curve between the logarithm of the concentration and the OMT is I Fc /I AQ ＝-0.279-0.098LogC OMT [g/mL](R 2 0.998), the linear range is 10pg/mL to 10ng/mL, and the detection limit is 2.8 pg/mL.
The obtained sensor is applied to the labeling detection of malathion and omethoate with different concentrations in the lettuce which is an actual sample, and the detection process is as follows:
the sample solution was added dropwise to the sensor MB-Apt1&After incubation on Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE for 40 min at room temperature, washing was performed with Tris-HCl (pH 7.4) solution. In a three-electrode system, the rinsed electrode was used as a working electrode, an Ag/AgCl (saturated KCl) electrode was used as a reference electrode, a platinum wire was used as a counter electrode, a phosphate buffer solution (pH 7.4) was used as an electrolyte, an electrochemical ac voltammetry (ACV) curve was scanned, and a ratio signal I was measured FB /I AQ And I Fc /I AQ Substituting into the standard linear curve I MB /I AQ ＝-0.161-0.122LogC MAL [g/mL]And I Fc /I AQ ＝-0.279-0.098LogC OMT [g/mL]And calculating to obtain the concentration information of the malathion and the omethoate in the lettuce.
To verify the accuracy of the constructed sensors, the same lettuce samples were tested using high performance liquid chromatography-mass spectrometry (HPLC-MS). The results of the two methods are shown in table 1.
TABLE 1 detection results of MAL and OMT in lettuce samples respectively by the sensor constructed by the invention and HPLC-MS
The results in the table 1 show that the sensor constructed by the invention can realize high-sensitivity and high-precision simultaneous detection of malathion and omethoate.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A preparation method of a ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate is characterized by comprising the following steps of:
(1) sequentially polishing the glassy carbon electrode GCE by using alumina powder with different particle sizes, sequentially performing ultrasonic treatment in ethanol and pure water to remove surface residues, and airing at room temperature;
(2) dropwise adding the prepared carbon nanohorn/anthraquinone-2-carboxylic acid/gold nanoparticle composite material, namely CNHs/AQ/AuNPs, onto the electrode treated in the step (1), and airing at room temperature;
(3) modifying hairpin DNA, namely hDNA, on the electrode treated in the step (2), storing for a period of time at room temperature, and fixing the hDNA on the surface of the electrode by utilizing a gold-sulfur bond;
(4) modifying mercaptohexanol MCH on the electrode treated in the step (3), and incubating for a period of time at room temperature to block non-specific binding sites of gold;
(5) modifying the methylene blue marked malathion aptamer MB-Apt1 on the electrode treated in the step (4), reacting for a period of time under a certain temperature condition, and assembling MB-Apt1 on the electrode by utilizing complementary hybridization of the methylene blue marked malathion aptamer MB-Apt1 and hDNA;
(6) modifying ferrocene-labeled omethoate aptamer Fc-Apt2 on the electrode treated in the step (5), reacting for a period of time under a certain temperature condition, and further assembling Fc-Apt2 on the electrode by utilizing complementary hybridization to obtain the ratiometric electrochemical aptamer sensor which is marked as MB-Apt1& Fc-Apt 2/MCH/hDNA/CNHs/AQ/AuNPs/GCE.
2. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (1), the diameter of the glassy carbon electrode is 3 mm; the alumina powders used had particle sizes of 0.3 μm and 0.05 μm in this order.
3. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (2), the preparation method of the carbon nanohorn/anthraquinone-2-carboxylic acid/gold nanoparticle composite material comprises the following steps:
(1) dispersing 1-10 mg of carbon nanohorns in 2-20 mL of water, and stirring for 0.5-2 hours at room temperature to obtain a solution A;
(2) adding 0.1-1 mg of anthraquinone-2-carboxylic acid into the solution A, stirring for 4-12 hours at room temperature, centrifuging and washing for 3 times, and dispersing the precipitate into 2-20 mL of pure water again to obtain a solution B;
(3) 0.05-0.5 mL of 30-60 mM HAuCl is added into the solution B 4 Stirring the solution for 0.5-1 hour at room temperature, adding 0.5-5 mL of 2-20 mM ascorbic acid solution, continuously stirring for 4-12 hours at room temperature, centrifuging, washing and drying to obtain a CNHs/AQ/AuNPs composite material;
(4) and dispersing the CNHs/AQ/AuNPs composite material in 2-20 mL of pure water to obtain a CNHs/AQ/AuNPs solution.
4. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (2), the CNHs/AQ/AuNPs solution is used in an amount of 4-8 mu L, and the concentration is 0.05-5 mg/mL.
5. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (3), the dosage of hDNA is 4-8 mu L, and the concentration is 1-8 mu M; the storage time is 4-12 hours under the room temperature condition.
6. The method for preparing the ratiometric electrochemical aptamer sensor for simultaneously detecting malathion and omethoate according to claim 1, wherein in the step (4), the dosage of MCH is 4-8 muL, and the concentration is 0.01-10 mM; the incubation time is 0.5-2 hours under the room temperature condition.
7. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (5), the using amount of the MB-Apt1 is 4-8 mu L, and the concentration is 1-8 mu M; the temperature is 37 ℃, and the reaction time is 1-2 hours.
8. The method of preparing a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 1, characterized in that: in the step (6), the dosage of the Fc-Apt2 is 4-8 muL, and the concentration is 1-8 muM; the temperature is 37 ℃, and the reaction time is 1-2 hours.
9. A ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate made by the method of manufacture of any of claims 1 to 8.
10. A method of operating a ratiometric electrochemical aptamer sensor for simultaneous detection of malathion and omethoate according to claim 9, comprising the steps of:
(1) the prepared plurality of sensors MB-Apt1&Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE is sequentially dripped with malathion and omethoate standard solutions with different concentrations, and each sensor corresponds to one concentration; incubating for 0.5-2 hours at room temperature, and washing by using a Tris-HCl solution; in a three-electrode system, the electrode after washing is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, phosphate buffer solution is used as electrolyte, an electrochemical AC volt-ampere curve is scanned, and a ratio signal I is measured MB /I AQ And I Fc /I AQ Establishing a signal I of the ratio of the concentration of malathion to the concentration of omethoate MB /I AQ And I Fc /I AQ A standard linear curve of (d); wherein, I MB For MB oxidation current, I Fc To oxidize the Fc current、I AQ Is AQ oxidation current;
wherein the concentration range of the malathion is 3 multiplied by 10 -12 ～3×10 -9 g/mL, omethoate concentration range of 1X 10 -11 ～1×10 -8 g/mL；
(2) Dropwise adding the sample solution to be detected to a sensor MB-Apt1&Incubating Fc-Apt2/MCH/hDNA/CNHs/AQ/AuNPs/GCE for 0.5-2 hours at room temperature, and washing with Tris-HCl solution; in a three-electrode system, the electrode after washing is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire is used as a counter electrode, phosphate buffer solution is used as electrolyte, an electrochemical AC volt-ampere curve is scanned, and a ratio signal I is measured MB /I AQ And I Fc /I AQ And (3) substituting the standard linear curve in the step (1) to realize the simultaneous detection of the malathion and the omethoate of the sample to be detected.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003225672A (en) * | 2002-02-04 | 2003-08-12 | Sanyo Electric Co Ltd | Water treatment equipment |
| CN101398414A (en) * | 2008-10-15 | 2009-04-01 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| WO2017087659A1 (en) * | 2015-11-18 | 2017-05-26 | Platypus Technologies, Llc | Liquid crystal sensor |
| CN108732218A (en) * | 2018-05-21 | 2018-11-02 | 江苏理工学院 | A kind of electrochemical sensor measuring 2,4,6- trichlorophenol, 2,4,6,-Ts and its preparation and application |
| CN110514713A (en) * | 2018-10-23 | 2019-11-29 | 山东理工大学 | A kind of aptamers biosensor preparation method and application based on ferrocene as electron donor |
| CN111175364A (en) * | 2020-01-15 | 2020-05-19 | 江苏大学 | Preparation method of ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A |
| CN113624812A (en) * | 2021-08-20 | 2021-11-09 | 山东理工大学 | Preparation method of aptamer sensor based on copper-gold bimetallic core-shell structure nanoparticles |
| CN114096274A (en) * | 2019-07-12 | 2022-02-25 | 俄勒冈健康与科学大学 | Immunotherapeutic constructs and methods of use thereof |
-
2022
- 2022-07-08 CN CN202210806437.9A patent/CN115060777B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003225672A (en) * | 2002-02-04 | 2003-08-12 | Sanyo Electric Co Ltd | Water treatment equipment |
| CN101398414A (en) * | 2008-10-15 | 2009-04-01 | 上海市公安局刑事侦查总队 | Method for qualitatively screening 242 kinds of compounds by liquid phase chromatography-mass spectra at the same times |
| WO2017087659A1 (en) * | 2015-11-18 | 2017-05-26 | Platypus Technologies, Llc | Liquid crystal sensor |
| CN108732218A (en) * | 2018-05-21 | 2018-11-02 | 江苏理工学院 | A kind of electrochemical sensor measuring 2,4,6- trichlorophenol, 2,4,6,-Ts and its preparation and application |
| CN110514713A (en) * | 2018-10-23 | 2019-11-29 | 山东理工大学 | A kind of aptamers biosensor preparation method and application based on ferrocene as electron donor |
| CN114096274A (en) * | 2019-07-12 | 2022-02-25 | 俄勒冈健康与科学大学 | Immunotherapeutic constructs and methods of use thereof |
| CN111175364A (en) * | 2020-01-15 | 2020-05-19 | 江苏大学 | Preparation method of ratiometric electrochemical aptamer sensor for simultaneously detecting aflatoxin B1 and ochratoxin A |
| CN113624812A (en) * | 2021-08-20 | 2021-11-09 | 山东理工大学 | Preparation method of aptamer sensor based on copper-gold bimetallic core-shell structure nanoparticles |
Non-Patent Citations (4)
| Title |
|---|
| CHENGXI ZHU等: "Dual-ratiometric electrochemical aptasensor based on carbon nanohorns/anthraquinone-2-carboxylic acid/Au nanoparticles for simulatneous detection of malathion and omethoate" * |
| 刘媛媛;陈媛媛;张红艳;尤金坤;魏小红;余宇燕;: "纳米金-核酸适配体快速可视化检测药材中4种有机磷农药的方法研究" * |
| 姚子仪等: "基于适配体检测农药残留的技术进展" * |
| 王丽;叶华;桑宏庆;王丹丹;: "基于核酸适配体的荧光法检测水胺硫磷和丙溴磷" * |
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