CN111235234B - Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure - Google Patents
Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure Download PDFInfo
- Publication number
- CN111235234B CN111235234B CN202010093276.4A CN202010093276A CN111235234B CN 111235234 B CN111235234 B CN 111235234B CN 202010093276 A CN202010093276 A CN 202010093276A CN 111235234 B CN111235234 B CN 111235234B
- Authority
- CN
- China
- Prior art keywords
- malathion
- manganese dioxide
- dna
- cadmium sulfide
- shell structure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
Abstract
The invention discloses a photoelectrochemical malathion detection method based on an enzyme catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure. The method comprises the steps of mixing and incubating magnetic beads, capture DNA and malathion aptamers, utilizing the competitive combination of malathion and the aptamers to cause the separation of partial malathion aptamers from the magnetic beads, utilizing the capture DNA reserved on the magnetic beads to trigger RCA reaction to form a long single-stranded DNA chain, and in addition, forming a long single-stranded DNA chain by the capture DNA and S 2 -Au-BChE probe hybridization, assembling a large amount of BChE on a long single-chain DNA chain, and catalyzing ATCH hydrolysis by the BChE to generate TCh so as to realize MnO 2 And (3) decomposing NF @ CdS to reduce the photocurrent, so that a linear correlation relationship between the photocurrent change value and the concentration of the parathion is established. The method is based on the specific recognition of the aptamer and the target object, and simultaneously adopts an RCA reaction amplification mechanism, so that the detection with high sensitivity and high selectivity on the concentration of the target object can be realized.
Description
Technical Field
The invention relates to a detection method of malathion, in particular to a method for cracking core-shell MnO based on an enzyme catalysis product 2 A method for photoelectrochemical detection of malathion with a nanoflower @ CdS structure belongs to the field of photoelectrochemical biological analysis.
Background
Malathion is an efficient and low-toxic organophosphorus pesticide, has wide agricultural application, and almost has residues in various grain commodities (rice, corn, wheat, barley, sorghum and the like). In 2013, malathion with a high standard exceeding concentration is detected in frozen food of a large aquatic product company in Japan, which causes poisoning of eaters. Malathion belongs to the class 2A carcinogens according to the 2017 carcinogen list published by the world health organization international agency for research on cancer. Therefore, the rapid and sensitive detection of malathion has great hygienic significance.
In recent years, the traditional methods for detecting malathion are mainly Gas Chromatography (GC), high Performance Liquid Chromatography (HPLC), mass Spectrometry (MS), etc. (1 e.center, a.k.sakhi, l.s.haug, c.thomsen, development of an ion-pair liquid chromatography-high resolution method for Determination of organic phosphorus peptides In large-scale biochemical assays, journal of chromatography.a,1454 (2016) 32-41. [ 2 ] j.i.cacho, n.campylo, p.vindes, m.hertz-corpoba, in a specific ionic liquid discrete-liquid chromatography coupled to a gas chromatography-mass spectrometry for the Determination of inorganic phosphoric acids, journal of chromatography.A,1559 (2018) 95-101. [ 3 ] R.Su, D.Li, X.Wang, H.Yang, X.Shi, S.Liu, determination of inorganic phosphoric acids In a vessel by carbon nano tube assay, reinforced concrete In a biological chromatography coupled with a biological chromatography, journal of chromatography, 152.152.1022.. Although these methods can detect a plurality of substances simultaneously and have high accuracy and precision, they require expensive large-scale instruments and cannot be directly characterized, cumbersome pretreatment and certain operation skill requirements for operators are required, so that they have limitations in rapid detection and limit their wide application. The development of a novel malathion detection method with high sensitivity and high selectivity is particularly important for environmental monitoring, food safety and disease diagnosis.
Compared with the traditional detection method, the Photoelectrochemical (PEC) detection technology is favored because of the advantages of low price, wide detection range, low detection limit, simple operation, high sensitivity and the like. The PEC process refers to a process in which molecules, ions, and semiconductor materials generate electron-hole pairs by exciting electrons due to absorption of photons under the action of light, and simultaneously, the conversion of light energy into electric energy is achieved. For example, titanium dioxide sensitized with nitrogen-doped carbon quantum dots in The literature ([ 4 ] w. Cheng, z. Zheng, j. Yang, m. Chen, q. Yao, y. Chen, w. Gao, the visible light-driven and selected-powered photo electrochemical biosensor for organic pesticides detection, electrochemical Acta,296 (2019) 627-636.) is used as a photoelectric material and The enzymatic product choline (TCh) is used as an electron donor to enhance The separation of photo-generated electron-hole pairs and enhance The response of The signal amplification, thereby detecting organophosphorus pesticides, although this method provides a very good method for detecting pesticide residues with high selectivity and narrow detection range.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for photoelectrochemical detection of malathion, which is based on the specific identification of an aptamer and the malathion, adopts an RCA reaction amplification mechanism and utilizes a manganese dioxide nanoflower @ cadmium sulfide compound to improve the photocurrent intensity so as to realize high-sensitivity and high-selectivity photoelectricity detection of the malathion.
In order to realize the technical purpose, the invention provides a photoelectrochemical detection method of malathion based on an enzyme catalysis product cracked manganese dioxide nanoflower @ cadmium sulfide core-shell structure, which comprises the following steps:
1) Mixing and incubating avidin-modified magnetic beads, biotin-modified capture DNA and malathion aptamers, adding a standard malathion solution for incubation, dropping and reducing part of the malathion aptamers to generate single-chain biotin-modified capture DNA, performing rolling circle amplification reaction on the single-chain biotin-modified capture DNA to form long single-chain DNA, incubating a mixture obtained by the rolling circle amplification reaction with an auxiliary DNA-nanogold-butyrylcholinesterase signal probe to combine the two, and adding acetylcholine for incubation to obtain a mixed solution;
2) Dropwise adding the manganese dioxide nanoflower @ cadmium sulfide nanocomposite to the surface of an electrode, and carrying out photoelectric detection to obtain a background photocurrent response value;
3) Dropwise adding the mixed solution obtained in the step 1) to the surface of the electrode obtained in the step 2), incubating, and performing photoelectric detection to obtain a photocurrent response value;
4) Carrying out photoelectric detection on standard malathion solutions with different concentrations according to the steps 1) to 3) to obtain a series of photocurrent response values, and constructing a standard curve between the concentration of the malathion solution and the photocurrent response change value;
5) And carrying out photoelectric detection on the malathion solution to be detected according to the steps 1) to 3), obtaining a corresponding photoelectric current response value, and calculating the concentration of the malathion solution to be detected according to a standard curve.
According to the technical scheme, the manganese dioxide nanoflower @ cadmium sulfide core-shell structure is used as a photoelectric active material, and electrons are excited to emit from MnO under the irradiation of light 2 CB of (2) is transferred to CB of CdS and holes are transferred from VB of CdS to MnO 2 VB of (b), then by an electron donor (Na) 2 S and Na 2 SO 3 ) And occupation, electrons and holes are effectively separated, and the intensity of photocurrent is improved. MnO can be converted from TCh generated by BChE catalytic hydrolysis of ATCH 2 Decomposition of NF (nuclear) to Mn 2+ And release the CdS nanoparticles (shells) from the electrodes, resulting in a decrease in photocurrent.
According to the technical scheme, a signal amplification strategy is reasonably designed, the circular DNA is used as a template, the primer DNA is extended into a long single chain under the catalysis of enzyme, the long single chain can be hybridized with the functionalized auxiliary probe DNA, the excellent signal amplification effect can be realized, the detection range is widened, and the selectivity of the sensor is greatly improved by introducing the aptamer.
In a preferred scheme, the avidin modified magnetic beads, the biotin modified capture DNA and the malathion aptamer are mixed and incubated for 0.5 to 1.5 hours at the temperature of between 30 and 40 ℃. More preferably, the concentration of the avidin-modified magnetic beads is 5mg mL -1 (ii) a The concentration of the biotin-modified capture DNA (cDNA) is 0.5 to 1.0. Mu.M. The concentration of malathion aptamer is 0.5-1.0 mu M.
In a preferred embodiment, the process of rolling circle amplification reaction to form long single-stranded DNA: firstly, T4 DNA ligase and padlock DNA are reacted to form a DNA circular template, and then Phi29DNA polymerase is used for catalyzing dNTP to synthesize a long single-stranded DNA molecule. Padlock DNA (pDNA), forming a DNA circular template which is complementarily matched with the base of the primer sequence in the biotin-modified capture DNA with the aid of T4 DNA ligase, and extending cDNA along the circular template from the 5'-3' direction with the help of Phi29DNA polymerase to catalyze dNTP to synthesize a long single-stranded DNA molecule.
In a preferred scheme, the preparation method of the manganese dioxide nanoflower @ cadmium sulfide nanocomposite comprises the following steps: performing hydrothermal reaction on permanganate and divalent manganese salt to obtain manganese dioxide nanoflowers; dispersing the manganese dioxide nanoflowers into water, adding cadmium salt, a sulfur source and triethylamine, and generating cadmium sulfide nanoparticles on the surfaces of the manganese dioxide nanoflowers through a precipitation method. In a preferable scheme, after dissolving potassium permanganate and manganese sulfate in water, transferring the solution to a reaction kettle, and carrying out hydrothermal reaction for 20-40 min at the temperature of 130-150 ℃ to obtain manganese dioxide nanoflowers; dispersing the manganese dioxide nanoflower into an aqueous solution, adding thiourea, cadmium acetate and triethanolamine, heating to 130-150 ℃, and reacting for 28-48 h to obtain the manganese dioxide nanoflower @ cadmium sulfide. In a more preferable scheme, the preparation method of the manganese dioxide nanoflower @ cadmium sulfide nanocomposite comprises the following steps: mixing KMnO 4 And MnSO 4 ·H 2 Adding O into deionized water to form a uniform solution, then transferring the mixed solution into a reaction kettle, and reacting for 30min at 140 ℃; washing the obtained solid product with centrifugal water for several times, and then re-dispersing the solid product into an aqueous solution to obtain MnO 2 NF dispersion liquid; thiourea, cd (CH) 3 COO) 2 ·2H 2 Rapidly adding O and triethanolamine to the MnO prepared in step (a) 2 Heating NF dispersion liquid to 140 ℃, reacting for 36h, and alternately washing the obtained product with ethanol and water for three times to obtain MnO 2 NF @ CdS core-shell structure.
In a preferred embodiment, the preparation method of the auxiliary DNA-nanogold-butyrylcholinesterase signal probe comprises the following steps: quickly injecting chloroauric acid into the sodium citrate aqueous solution heated to boiling, keeping slight boiling, and reacting until the solution is wine red to obtain gold nanoparticles; and dispersing the gold nanoparticles into a mixed solution containing butyrylcholinesterase and amino-modified auxiliary DNA for reaction to form an auxiliary DNA-nanogold-butyrylcholinesterase signal probe. Further preferably, the amino group-modified helper DNA (S) 2 ) The concentration of (A) is 0.5 to 1.0. Mu.M. The concentration of butyrylcholinesterase is 10 mug mL -1 . Most preferred helper DNA-nanogold-DPreparation method of acylcholinesterase signal probe (S) 2 -Au-BChE signal probe): (a) HAuCl is quickly injected into a sodium citrate water solution heated to boiling 4 Keeping slight boiling, observing the color of the solution to change from light yellow to colorless gray until wine red, removing heat source, and storing at 4 ℃ in dark to obtain Au nanoparticles (Au NP); (b) Dispersing the Au NP prepared in the step (a) into BChE and amino modified auxiliary DNA (S) 2 ) In the mixed solution of (2), the mixture was shaken for 12 hours to effect the bonding of Au-N bond to form S 2 -Au-BChE signal probe.
In a preferred scheme, the reaction time of the gold nanoparticles with butyrylcholinesterase and amino-modified auxiliary DNA is 8-16 hours.
In the photoelectric detection process of the invention, na is adopted 2 S and Na 2 SO 3 The mixed solution is electrolyte, visible light is exciting light, and a three-electrode system is adopted to detect photocurrent. The Na is 2 S and Na 2 SO 3 In the mixed solution, na 2 The concentration of S is 0.1M 2 SO 3 The concentration of (3) was 0.02M.
According to the technical scheme, the manganese dioxide nanoflower @ cadmium sulfide nanocomposite is synthesized, and specifically, the cadmium sulfide nanoparticles are coated on the surfaces of manganese dioxide nanoflower particles, the manganese dioxide nanoflower structure is similar to a full petal, the specific surface area is large, organic combination with CdS nanoparticles is facilitated, the manganese dioxide nanoflowers and the cadmium sulfide nanoparticles generate an obvious synergistic effect, energy level matching can be achieved, separation of photo-generated electron hole pairs is effectively promoted, the photocurrent intensity is remarkably improved, and the detection sensitivity is improved.
The detection principle of the technical scheme of the invention is as follows: in the absence of malathion, the manganese dioxide nanoflower @ cadmium sulfide nanocomposite modified on the electrode interface generates a strong photocurrent signal, and in the presence of malathion, the electrode interface modified manganese dioxide nanoflower @ cadmium sulfide nanocomposite is competitively combined with malathion aptamers to cause Rolling Circle Amplification (RCA) of capture DNA (cDNA) modified by auxiliary biotin to generate long single-stranded DNA (ssDNA) under the catalysis of enzyme. The ssDNA and S 2 -butyrylcholinesterase (BChE) catalyzes Acetylcholine (ATCH) after hybridization of Au-BChE signaling probe) Hydrolyzing to generate TCh, and reducing part of manganese dioxide in the manganese dioxide nanoflower @ cadmium sulfide nanocomposite by the TCh so as to realize MnO 2 The decomposition of NF @ CdS leads cadmium sulfide nanoparticles to fall off from the electrode, so that the photocurrent is reduced, and the change value of the photoelectric signal and the concentration of the malathion form a linear correlation relationship in a certain range. Thereby. Can realize the high-efficiency and high-sensitivity detection of the malathion.
The invention provides a method for MnO based on enzyme mediation 2 The photoelectrochemical detection method for malathion by decomposing the NF @ CdS core-shell structure comprises the following steps:
(1) Construction of photoelectrochemical biosensor: mu.L of avidin-modified magnetic beads (Strep-MB, 5mg mL) -1 ) 10. Mu.L of biotin-modified auxiliary DNA (cDNA, 1. Mu.M) and 20. Mu.L of malathion aptamer (1. Mu.M) were mixed and incubated at 37 ℃ for 1 hour, and 10. Mu.L of 0.001-100ng mL of a buffer solution was added -1 Malathion competes with cDNA to reduce the cDNA into single strand, then padlock DNA (pDNA) is added, a DNA circular template which is complementarily matched with the base of a primer sequence in the cDNA is formed under the assistance of T4 DNA ligase, the cDNA is extended from the 5'-3' direction along the circular template under the assistance of Phi29DNA polymerase, and dNTP is catalyzed to synthesize a long single strand DNA molecule (namely rolling circle amplification reaction, RCA); the RCA product was then mixed with 100. Mu.L of S 2 After incubation with the Au-BChE signal probe, magnetic separation and washing, 50. Mu.L of 10mM ATCH was added, BChE catalyzed ATCH to dissociate choline (TCh) which can convert MnO into MnO 2 Decomposing the NF @ CdS core-shell structure to reduce the photocurrent, thereby obtaining the photoelectrochemical biosensor;
(2) Photoelectric chemical detection of malathion: adding malathion standard solutions with different concentrations in the step (1), wherein the quantity of the competitive aptamers is different, the quantity of cDNA capable of performing RCA reaction is different, and then different quantities of S can be combined 2 The concentration of TCh catalytically decomposed by the Au-BChE signal probe is different, so that the core-shell MnO is decomposed 2 The ability of NF @ CdS varies; 20 μ L of 20mg mL -1 MnO of 2 Dripping NF @ CdS dispersion liquid on the surface of the electrode, and adding Na with concentration of 0.1M 2 S and Na in a concentration of 0.02M 2 SO 3 The mixed solution is electrolyte, visible light is exciting light, a three-electrode system is adopted to detect photocurrent (namely background current), then 20 mu L of TCh solution generated by catalytic decomposition is dripped into an electrode and incubated for 40min, after cleaning, the photocurrent is detected under the same condition, and a standard curve is drawn according to the photocurrent response change value to the standard sample concentration; and replacing the malathion standard solution with the solution to be detected to carry out the detection, and obtaining a concentration result through a standard curve.
The RCA reaction described in the step (1) is carried out by the following steps:
reducing cDNA containing a primer sequence into a single strand, adding pDNA, forming a DNA circular template which is complementarily matched with the base of the primer sequence in the cDNA under the assistance of T4 DNA ligase, extending the cDNA along the circular template from the 5'-3' direction under the assistance of Phi29DNA polymerase, and catalyzing dNTP to synthesize a long single strand DNA molecule;
the core-shell MnO of step (1) 2 NF @ CdS was prepared by:
(a) Under continuous stirring, 1.0g of KMnO was first added 4 And 0.4g MnSO 4 ·H 2 O was added to 30mL of deionized water to form a homogeneous solution; then, the solution was transferred to a 50mL stainless steel autoclave and reacted at 140 ℃ for 30min, after cooling to room temperature, mnO was collected by centrifugation 2 NF, respectively washing with ultrapure water and ethanol, and re-dispersing into 10mL of aqueous solution;
(b) Subjecting 0.05g of MnO prepared in step (a) to 2 NF was dissolved in ultrapure water (20 mL) and Cd (CH) 3 COO) 2 ·2H 2 O (2.6mM, 5mL) and CH 4 N 2 S (1.8 mM,5 mL) and then 50wt% triethanolamine was added completely to the mixture; the mixture was stirred for 2h; the resulting mixture was placed in a 50mL autoclave and heated at 140 ℃ for 36h; finally, the obtained product was centrifuged and washed three times with ethanol and ultrapure water alternately.
S in the step (1) 2 -Au-BChE signal probe prepared by the following steps:
(a) 0.6mL of 0.035M aqueous sodium citrate solutionPouring into 20mL of water, heating to boil with stirring, and rapidly injecting 40 μ L of 4% HAuCl 4 Keeping slight boiling, observing the color of the solution to change from light yellow to colorless grey until the solution is wine red, removing a heat source, stopping stirring, and storing at 4 ℃ in a dark place to obtain Au nanoparticles (Au NP);
(b) Dispersing 10. Mu.L of the Au NP prepared in step (a) to 60. Mu.L of 10. Mu.g mL -1 BChE of (4) with 10. Mu.L of amino-modified helper DNA (S) at a concentration of 1. Mu.M 2 ) In the mixed solution of (2), mixing and oscillating for 12h to realize the combination of Au-N bonds to form S 2 -Au-BChE signal probe.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
1) The invention adopts special MnO 2 The NF @ CdS core-shell structure enhances current signals, wherein MnO is 2 The energy level matching between NF and CdS effectively promotes the separation of photo-generated electron-hole pairs and obviously improves the photocurrent intensity;
2) The invention improves the detection sensitivity by a signal amplification technology, takes circular DNA as a template, and extends primer DNA (complementary with part of the circular template) into a long single-stranded DNA under the catalysis of enzyme. The long-chain DNA can be associated with a large amount of S 2 Au-BChE signal probe complementation, and TCh generated by ATCH catalyzed by BChE decomposes MnO 2 NF @ CdS composite material, resulting in increased photocurrent variation.
3) The invention improves the photocurrent intensity by the specific recognition of the aptamer and the malathion and simultaneously adopting an RCA reaction amplification mechanism and utilizing the manganese dioxide nanoflower @ cadmium sulfide compound so as to realize the high-sensitivity and high-selectivity photoelectric detection of the malathion.
4) The invention is based on enzyme-mediated convection of MnO 2 A photoelectrochemical detection method for malathion by decomposing NF @ CdS core-shell structure is carried out at 0.001-100ng mL -1 In the concentration range, the photocurrent has a good linear relation with the logarithm of the concentration of the target substance, and the method has the advantages of wide detection range and low lower limit.
Drawings
FIG. 1 is a pair of core-shell MnO based on enzyme mediation 2 Decomposition of NF @ CdSThe principle schematic diagram of the photoelectrochemistry malathion detection method.
FIG. 2 (A) shows MnO 2 NF scanning electron microscope picture; FIG. 2 (B) shows core-shell MnO 2 Scanning electron microscope image of NF @ CdS composite material.
FIG. 3 shows core-shell MnO 2 Energy spectrum of NF @ CdS composite material.
FIG. 4 shows the results of the detection of the standard sample in example 1, A: example 1 photocurrent variation versus log linear plot of concentration of detection target (inset is corresponding photocurrent response curve), B: selective examination of example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific implementation examples, but the scope of the present invention is not limited thereby.
Example 1
(1) Under continuous stirring, 1.0g of KMnO was first added 4 And 0.4g MnSO 4 ·H 2 O was added to 30mL of deionized water to form a homogeneous solution; then, the solution was transferred to a 50mL stainless steel autoclave and reacted at 140 ℃ for 30min, after cooling to room temperature, mnO was collected by centrifugation 2 NF, and washed with ultrapure water and ethanol, respectively, and redispersed in 10mL of an aqueous solution.
(2) Adding 0.05g of MnO prepared in the step (1) 2 NF was dissolved in ultrapure water (20 mL) and then reacted with Cd (CH) 3 COO) 2 ·2H 2 O (2.6mM, 5mL) and CH 4 N 2 S (1.8 mM,5 mL) was mixed. At the same time, 50wt% triethanolamine was added completely to the mixture; the mixture was stirred for 2h; the resulting mixture was placed in a 50mL autoclave and heated at 140 ℃ for 36h; finally, the obtained product is subjected to core-shell MnO 2 Centrifuging NF @ CdS, and alternately washing with ethanol and ultrapure water for three times.
(3) 0.6mL of 0.035M aqueous sodium citrate solution are poured into 20mL of water, heated to boiling with stirring and 40. Mu.l of 4% HAuCl are injected rapidly 4 Keeping slight boiling, observing the color of the solution from light yellow to colorless gray until wine red, removing heat source, stopping stirring, storing at 4 deg.C in dark to obtain Au nanoparticles (Au NP)。
(4) Dispersing 10. Mu.L of the Au NP prepared in step (3) to 60. Mu.L of 10. Mu.g mL -1 BChE of (4) with 10. Mu.L of amino-modified helper DNA (S) at a concentration of 1. Mu.M 2 ) In the mixed solution of (2), mixing and oscillating for 12h to realize the combination of Au-N bonds to form S 2 -Au-BChE signal probe.
(5) 20 microliter of 20mg mL -1 MnO prepared in the step (2) 2 Dripping the dispersed liquid of NF @ CdS nano-composite on the surface of a clean glassy carbon electrode by using Na with the concentration of 0.1M 2 S and Na in a concentration of 0.02M 2 SO 3 The mixed solution is electrolyte, visible light is exciting light, and a three-electrode system is adopted to detect photocurrent (namely background current).
(6) mu.L of avidin-modified magnetic beads (Strep-MB, 5mg mL) -1 ) 10 μ L of biotin-modified helper DNA (cDNA, 1 μ M) and 20 μ L of malathion aptamer (1 μ M) were mixed and incubated at 37 ℃ for 1h, and 10 μ L of a 60ng mL solution was added -1 Malathion, compete with cDNA for aptamer sequence to reduce cDNA into single strand, magnetically separating and washing, then incubating T4 DNA ligase (15U) and padlock DNA (10 uL, 1 uM) in 1 Xligase buffer solution at 22 ℃ for 1h to form a DNA circular template, magnetically separating and washing again, catalyzing dNTP (5 uL, 10 mM) by Phi29DNA polymerase (20U) in 1 XPhi 29 buffer solution to synthesize a long single strand DNA molecule (namely rolling circle amplification reaction, RCA), namely mixing and incubating at 37 ℃ for 1h;
(7) S prepared in the step (4) 2 Mixing and oscillating the Au-BChE signal probe and the RCA reaction product at room temperature for 1h to combine the Au-BChE signal probe and the RCA reaction product; washing by magnetic separation, adding 50 μ L ATCH with concentration of 10mM, and incubating at 37 deg.C for 1h; dripping 20 mu L of upper layer solution on the ITO electrode with the background current measured in the step (5), incubating for 40min again, allowing part of the modified material to fall off from the electrode, washing with purified water, and performing photoelectric detection under the same condition to find that the intensity of photocurrent is reduced and the reduction amplitude has a certain relation with the concentration of malathion, thereby realizing sensitive detection of the malathion; replacing malathion standard solution with the solution to be detected to perform the detection, and obtaining a concentration result through a standard curve;
under the same conditions, dichlorvos (Dic) and chlorpyrifos (Chl) are respectively used as target objects, and the selectivity of the method is examined.
The DNA sequence used in example 1 [ purchased from Biotechnology (Shanghai) Ltd ] was as follows:
FIG. 1 is a diagram of an enzyme-mediated para-core-shell MnO in accordance with the present invention 2 A photoelectrochemical malathion detection process and a schematic diagram for decomposing NF @ CdS. FIG. 2 shows MnO 2 NF and core-shell MnO 2 Scanning electron microscope image of NF @ CdS composite material. Scanning electron micrograph showing MnO 2 NF has a complete hierarchical microsphere structure similar to full petal, and the combination of CdS nano-particles is promoted by a larger specific surface area, so that MnO is synthesized 2 NF @ CdS composite material. The EDS spectra results indicated that the material consisted primarily of sulfur, oxygen, manganese, cadmium, and gold elements (fig. 3). FIG. 4 shows the results of the measurement of the standard sample in example 1 (the corresponding photocurrent response curve is shown in the inset). In the range of 0.001-100ng mL -1 The photocurrent was well linearly related to the logarithm of the target concentration in the target concentration range (fig. 4A). Considering the requirement of practicability, the specificity and selectivity of the method on a specific target object are considered, and the influence of Dic and Chl as interferents on corresponding signals of the sensor is selected. The experimental results confirmed that the sensor has substantially no response to different interfering components and a significant response to a specific target, indicating that the sensor has good selectivity and specificity (fig. 4B).
The above description is an example of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (7)
1. A method for photoelectrochemical detection of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure is characterized by comprising the following steps: comprises the following steps:
1) Mixing and incubating avidin-modified magnetic beads, biotin-modified capture DNA and malathion aptamers, adding a standard malathion solution for incubation, dropping and reducing part of the malathion aptamers to generate single-chain biotin-modified capture DNA, performing rolling circle amplification reaction on the single-chain biotin-modified capture DNA to form long single-chain DNA, incubating a mixture obtained by the rolling circle amplification reaction with an auxiliary DNA-nanogold-butyrylcholinesterase signal probe to combine the two, and adding acetylcholine for incubation to obtain a mixed solution;
2) Dropwise adding the manganese dioxide nanoflower @ cadmium sulfide nanocomposite to the surface of an electrode, and carrying out photoelectric detection to obtain a background photocurrent response value;
3) Dropwise adding the mixed solution obtained in the step 1) to the surface of the electrode obtained in the step 2), incubating, and performing photoelectric detection to obtain a photocurrent response value;
4) Carrying out photoelectric detection on standard malathion solutions with different concentrations according to the steps 1) to 3) to obtain a series of photocurrent response values, and constructing a standard curve between the concentration of the malathion solution and the photocurrent response change value;
5) And carrying out photoelectric detection on the malathion solution to be detected according to the steps 1) to 3), obtaining a corresponding photocurrent response value, and calculating the concentration of the malathion solution to be detected according to a standard curve.
2. The photoelectrochemical detection method of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 1, characterized in that: mixing the avidin modified magnetic beads, the biotin modified capture DNA and the malathion aptamer, and incubating for 0.5-1.5 h at the temperature of 30-40 ℃.
3. The photoelectrochemical detection method of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 1, characterized in that: the process of rolling circle amplification reaction to form long single-stranded DNA: firstly, T4 DNA ligase and padlock DNA are utilized to react to form a DNA circular template, and then Phi29DNA polymerase is utilized to catalyze dNTP to synthesize a long single-stranded DNA molecule.
4. The method for photoelectrochemical detection of malathion based on enzymatic product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure as claimed in claim 1, wherein the method comprises the following steps:
the preparation method of the manganese dioxide nanoflower @ cadmium sulfide nanocomposite comprises the following steps: performing hydrothermal reaction on permanganate and divalent manganese salt to obtain manganese dioxide nanoflowers; dispersing the manganese dioxide nanoflowers into water, adding cadmium salt, a sulfur source and triethylamine, and generating cadmium sulfide nanoparticles on the surfaces of the manganese dioxide nanoflowers through a precipitation method.
5. The method for photoelectrochemical detection of malathion based on enzymatic product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 4, wherein the method comprises the following steps: dissolving potassium permanganate and manganese sulfate in water, transferring the solution to a reaction kettle, and carrying out hydrothermal reaction for 20-40 min at the temperature of 130-150 ℃ to obtain manganese dioxide nanoflowers; dispersing the manganese dioxide nanoflower into an aqueous solution, adding thiourea, cadmium acetate and triethanolamine, heating to 130-150 ℃, and reacting for 28-48 h to obtain the manganese dioxide nanoflower @ cadmium sulfide.
6. The photoelectrochemical detection method of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 1, characterized in that: the preparation method of the auxiliary DNA-nanogold-butyrylcholinesterase signal probe comprises the following steps: quickly injecting chloroauric acid into the sodium citrate aqueous solution heated to boiling, keeping slight boiling, and reacting until the solution is wine red to obtain gold nanoparticles; and dispersing the gold nanoparticles into a mixed solution containing butyrylcholinesterase and amino-modified auxiliary DNA for reaction to form an auxiliary DNA-nanogold-butyrylcholinesterase signal probe.
7. The photoelectrochemical detection method of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 6, characterized in that: the reaction time of the gold nanoparticles with butyrylcholinesterase and amino-modified auxiliary DNA is 8-16 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010093276.4A CN111235234B (en) | 2020-02-14 | 2020-02-14 | Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010093276.4A CN111235234B (en) | 2020-02-14 | 2020-02-14 | Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111235234A CN111235234A (en) | 2020-06-05 |
| CN111235234B true CN111235234B (en) | 2022-11-25 |
Family
ID=70878221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010093276.4A Active CN111235234B (en) | 2020-02-14 | 2020-02-14 | Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111235234B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607388A (en) * | 2020-06-23 | 2020-09-01 | 江苏省特种设备安全监督检验研究院 | Preparation method and application of graphene quantum dot-rare earth up-conversion compound |
| CN112098492B (en) * | 2020-09-11 | 2022-09-16 | 江西师范大学 | Method for photoelectrochemical detection of organophosphorus pesticide by bismuth oxybromide/bismuth sulfide semiconductor heterojunction based on biological induction generation |
| CN113267529B (en) * | 2021-05-12 | 2022-10-25 | 江西师范大学 | Temperature type biosensor and method for detecting target aptamer by using temperature type biosensor |
| CN113249377B (en) * | 2021-06-29 | 2021-09-24 | 中国农业大学 | Method for assembling and regulating morphology of double-chain functional nucleic acid nanoflower and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090046466A (en) * | 2007-11-06 | 2009-05-11 | 경북대학교 산학협력단 | A kit for detecting pesticide residues in crops and the detecting method thereof |
| WO2014203092A1 (en) * | 2013-06-19 | 2014-12-24 | Indian Institute Of Technology Madras | Sensors for detecting organophosphorous materials, and methods for their preparation |
| CN107870242A (en) * | 2017-10-12 | 2018-04-03 | 广东省生态环境技术研究所 | A kind of fluorescence detection reagent kit based on aptamer |
| CN110715912A (en) * | 2019-10-08 | 2020-01-21 | 河北大学 | Sulfur quantum dot/manganese dioxide nanosheet composite material and preparation method and application thereof |
-
2020
- 2020-02-14 CN CN202010093276.4A patent/CN111235234B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090046466A (en) * | 2007-11-06 | 2009-05-11 | 경북대학교 산학협력단 | A kit for detecting pesticide residues in crops and the detecting method thereof |
| WO2014203092A1 (en) * | 2013-06-19 | 2014-12-24 | Indian Institute Of Technology Madras | Sensors for detecting organophosphorous materials, and methods for their preparation |
| CN107870242A (en) * | 2017-10-12 | 2018-04-03 | 广东省生态环境技术研究所 | A kind of fluorescence detection reagent kit based on aptamer |
| CN110715912A (en) * | 2019-10-08 | 2020-01-21 | 河北大学 | Sulfur quantum dot/manganese dioxide nanosheet composite material and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| MnO2 Nanosheet-Carbon Dots Sensing Platform for Sensitive Detection of Organophosphorus Pesticides;Xu Yan 等;《Anal. Chem.》;20171214;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111235234A (en) | 2020-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111235234B (en) | Photoelectrochemistry malathion detection method based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure | |
| Renedo et al. | Recent developments in the field of screen-printed electrodes and their related applications | |
| Zhang et al. | Nanotechnology and nanomaterial-based no-wash electrochemical biosensors: from design to application | |
| CN108760852B (en) | Photoelectrochemical ochratoxin A detection method based on dual signal amplification | |
| CN110146580B (en) | Method for detecting l, 5-anhydroglucitol based on persimmon tannin composite nano material | |
| CN110470714B (en) | Electrochemical luminescence sensor based on DNA walker induced conformation transformation and signal amplification and application thereof | |
| CN108802133A (en) | A kind of preparation method and application of detection stomach neoplasms tumor markers interlayer type immunosensor | |
| CN109266332B (en) | Preparation method of ratiometric fluorescent probe for quantitatively detecting AChE and BChE in blood | |
| Xia et al. | Self-enhanced electrochemiluminescence of luminol induced by palladium–graphene oxide for ultrasensitive detection of aflatoxin B1 in food samples | |
| CN112098492B (en) | Method for photoelectrochemical detection of organophosphorus pesticide by bismuth oxybromide/bismuth sulfide semiconductor heterojunction based on biological induction generation | |
| Barua et al. | Fluorescence biosensor based on gold-carbon dot probe for efficient detection of cholesterol | |
| Li et al. | Photoelectrochemical biosensor based on BiVO4/Ag2S heterojunction coupled with Exo III-assisted silver nanoclusters amplification for tumor suppressor gene P53 | |
| Liang et al. | A facile and sensitive fluorescence assay for glucose via hydrogen peroxide based on MOF-Fe catalytic oxidation of TMB | |
| Meng et al. | The innovative and accurate detection of heavy metals in foods: A critical review on electrochemical sensors | |
| CN110174396B (en) | Colorimetric and electroluminescent dual-mode aptamer sensor and method for measuring malathion | |
| CN113322305B (en) | Based on gold nanocluster/MnO 2 Preparation and application of electrochemical luminescence sensor of nanoflower | |
| Gao et al. | Determination of magnesium ion in serum samples by a DNAzyme-based electrochemical biosensor | |
| CN114636746A (en) | Detect Pb2+Carboxyl ligand induced annihilation type ratio electrochemiluminescence aptamer sensing method | |
| Xiao et al. | Photoelectrochemical and quartz crystal microbalance dual-mode assay of protein tyrosine phosphatase 1B activity based on hollow CuO and TiO2 polyhedra | |
| CN110186912B (en) | Electrochemiluminescence aptamer sensor based on thiocholine co-reaction accelerator and method for determining chlorpyrifos | |
| CN113185697A (en) | Porphyrin-based MOFs mimic enzyme, and preparation method and application thereof | |
| CN115015342B (en) | Preparation method of metal ion doped boron nano-sheet compound used as exosome ratio electrochemical aptamer sensor | |
| Chen et al. | Biosensors and flow injection analysis | |
| CN115728374A (en) | Electrochemical rapid analysis method for forbidden pesticides in food | |
| CN110699422B (en) | Lactic acid detection method based on gold nanocluster fluorescence enhancement |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |