CN111235234A - Photoelectrochemical detection method for malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure - Google Patents
Photoelectrochemical detection method for malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure Download PDFInfo
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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 S2-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 MnO2And (3) decomposing the NF @ CdS to reduce the photocurrent, so that a linear correlation relationship between the photocurrent change value and the concentration of the phofos is established. The method is based onThe method is suitable for 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 product2A 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 carcinogen 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 malathion detection are mainly Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), and Mass Spectrometry (MS) (1. E.Cerique, A.K.Sakhi, L.S.Haug, C.Thomsen, development of an-patient liquid chromatography-high resolution method of organic phosphorus catalysis In large-scale biological chromatography, Journal of chromatography. A,1454 (32-41.) [ 2 ] J.I.CaCho, N.Camplo, P.Vinars, M.nano-Cordata, In situ biological chromatography, 2016. about.2. J.I.C.C., 1022(2016)141-152.). 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 fluorescence-drive and selected-powered photonic biosensor for organic phosphorus catalysis detection and on nitrogen-doped carbon quantum dots for The signal amplification, electrochemical Acta, 2019)627 and 636. The enzyme catalysis product choline (TCh) is used as an electron donor to enhance The separation of photo-generated electron-hole pairs and increase The photocurrent response to detect organophosphorus pesticides, although this method provides a very good method for detecting pesticide residues with a high selectivity and a 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 photocurrent 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 light2CB of (2) is transferred to CB of CdS and holes are transferred from VB of CdS to MnO2VB of (1), then by an electron donor (Na)2S and Na2SO3) 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 ATCH2Decomposition of NF (nuclear) to Mn2+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 0.5-1.5 h at the temperature of 30-40 ℃. More preferably, the concentration of the avidin-modified magnetic beads is 5mg mL-1(ii) a The concentration of the capture DNA (cDNA) modified by biotin is 0.5-1.0 μ M. The concentration of the 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 long single-stranded DNA molecules. Padlock DNA (pDNA), a DNA circular template which is complementarily paired with the primer sequence in the biotin-modified capture DNA is formed under the assistance of T4 DNA ligase, and cDNA is extended along the circular template from the 5 '-3' direction under the help of Phi29DNA polymerase, so that dNTP is catalyzed 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. According to 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 nanoflowers into an aqueous solution, adding thiourea, cadmium acetate and triethanolamine, heating to 130-150 ℃, and reacting for 28-48 hours to obtain the manganese dioxide nanoflowers @ cadmium sulfide. In a more preferable scheme, the preparation method of the manganese dioxide nanoflower @ cadmium sulfide nanocomposite comprises the following steps: mixing KMnO4And MnSO4·H2Adding 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 MnO2NF dispersion liquid; thiourea, Cd (CH)3COO)2·2H2Rapidly adding O and triethanolamine to the MnO prepared in step (a)2Heating NF dispersion liquid to 140 ℃, reacting for 36h, and alternately washing the obtained product with ethanol and water for three times to obtain MnO2NF @ 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 (B) is 0.5 to 1.0. mu.M. The concentration of butyrylcholinesterase is 10 mug mL-1. Preparation method (S) of most preferred auxiliary DNA-nanogold-butyrylcholinesterase signal probe2-Au-BChE signal probe): (a) HAuCl is quickly injected into a sodium citrate water solution heated to boiling4Keeping slight boiling, observing the color of the solution to change from light yellow to colorless gray until wine red, removing a heat source, and storing at 4 ℃ in a dark place 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 S2-Au-BChE signal probe.
In a preferable scheme, the reaction time of the gold nanoparticles, butyrylcholinesterase and amino-modified auxiliary DNA is 8-16 hours.
In the photoelectric detection process of the invention, Na is adopted2S and Na2SO3The mixed solution is electrolyte, visible light is exciting light, and a three-electrode system is adopted to detect photocurrent. The Na is2S and Na2SO3In the mixed solution, Na2The concentration of S is 0.1M, Na2SO3The concentration of (3) was 0.02M.
The technical scheme of the invention synthesizes the manganese dioxide nanoflower @ cadmium sulfide nanocomposite, and the specific structure is that 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, has a large specific surface area, is more favorable for realizing organic combination with CdS nanoparticles, and the manganese dioxide nanoflower and the cadmium sulfide nanoparticles generate obvious synergistic action, can be subjected to energy level matching, effectively promotes the separation of photo-generated electron-hole pairs, obviously improves the photocurrent intensity and improves the detection sensitivity.
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 competitively binds with malathion aptamer to cause Rolling Circle Amplification (RCA) of capture DNA (cDNA) modified by auxiliary biotin under the catalysis of enzyme to form long single-stranded DNA (ssDNA). The ssDNA and S2After the Au-BChE signal probe is hybridized, butyrylcholinesterase (BChE) catalyzes Acetylcholine (ATCH) to be hydrolyzed to generate TCh, and the TCh reduces part of manganese dioxide in the manganese dioxide nanoflower @ cadmium sulfide nanocomposite through reduction so as to realize MnO2By the decomposition of NF @ CdS, cadmium sulfide nanoparticles fall off from the electrode, so that the photocurrent is reduced, and the change value of a photoelectric signal and the concentration of 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 mediation2The photoelectrochemical malathion detection method for decomposing the NF @ CdS core-shell structure comprises the following steps of:
(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-1Malathion, competing with cDNA for aptamer sequence to reduce cDNA into single strand, adding padlock DNA (pDNA), forming a circular DNA template complementary to the base of primer sequence in cDNA with the aid of T4 DNA ligase, extending cDNA along the circular template from 5 '-3' direction with the aid of Phi29DNA polymerase to catalyze dNTP to synthesize one long single-stranded DNA molecule (rolling circle)Amplification reaction, RCA); the RCA product was then mixed with 100. mu.L of S2After incubation with Au-BChE signal probe, magnetic separation and washing, 50 μ L of 10mM ATCH was added, BChE catalyzes ATCH to decompose choline (TCh) which can convert MnO into MnO2Decomposing the NF @ CdS core-shell structure to reduce photocurrent to obtain 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 combined2The concentration of TCh catalytically decomposed by the Au-BChE signal probe is different, so that the core-shell MnO is decomposed2The ability of NF @ CdS is different; 20 μ L of 20mg mL-1MnO of2Dripping NF @ CdS dispersed liquid on the surface of an electrode, and adding Na with the concentration of 0.1M2S and Na in a concentration of 0.02M2SO3The 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 taken to be 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:
after the cDNA containing the primer sequence is reduced into a single strand, pDNA is added, a DNA circular template which is complementarily matched with the base of the primer sequence in the cDNA is formed under the assistance of T4 DNA ligase, and the cDNA is extended along the circular template from the 5 '-3' direction under the assistance of Phi29DNA polymerase, so that the dNTP is catalyzed to synthesize a long single-stranded DNA molecule;
core-shell MnO in step (1)2NF @ CdS was prepared by the following steps:
(a) under continuous stirring, 1.0g of KMnO was first added4And 0.4g MnSO4·H2O was added to 30mL of deionized water to form a homogeneous solution; the solution was then transferred to a 50mL stainless steel autoclave andreacting at 140 deg.C for 30min, cooling to room temperature, centrifuging to collect MnO2NF, 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) to2NF was dissolved in ultrapure water (20mL) and Cd (CH)3COO)2·2H2O (2.6mM, 5mL) and CH4N2S (1.8mM, 5mL) and then 50 wt% triethanolamine was added to the mixture; the mixture was stirred for 2 h; the resulting mixture was placed in a 50mL autoclave and heated at 140 ℃ for 36 h; 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 solution are poured into 20mL of water, heated to boiling with stirring and 40. mu.l of 4% HAuCl are injected rapidly4Keeping slight boiling, observing the color of the solution to change from light yellow to colorless gray 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-1BChE of (4) with 10. mu.L of amino-modified helper DNA (S) at a concentration of 1. mu.M2) In the mixed solution of (2), mixing and oscillating for 12h to realize the combination of Au-N bonds to form S2-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 MnO2NF @ CdS core-shell structure enhanced current signal, wherein MnO is2The 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 the circular DNA as a template, and extends the 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 S2Au-BChE signal probe complementation, and BChE catalyzes the TCh component generated by ATCHMnO resolution2NF @ CdS composite material, leading to 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 MnO2The photoelectrochemical detection method for malathion by decomposing NF @ CdS core-shell structure is carried out at 0.001-100ng mL-1In 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 mediation2A principle schematic diagram of a photoelectrochemical malathion detection method for decomposing NF @ CdS.
FIG. 2(A) shows MnO2NF scanning electron microscope picture; FIG. 2(B) shows core-shell MnO2And (3) a scanning electron microscope image of the NF @ CdS composite material.
FIG. 3 shows core-shell MnO2Energy 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: results of the selectivity 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 added4And 0.4g MnSO4·H2O 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 centrifugation2NF, and washed with ultrapure water and ethanol, respectively, and redispersed in 10mL of an aqueous solution.
(2) Will step with0.05g of MnO prepared in step (1)2NF was dissolved in ultrapure water (20mL) and then reacted with Cd (CH)3COO)2·2H2O (2.6mM, 5mL) and CH4N2S (1.8mM, 5mL) was mixed. At the same time, 50 wt% triethanolamine was added completely to the mixture; the mixture was stirred for 2 h; the resulting mixture was placed in a 50mL autoclave and heated at 140 ℃ for 36 h; finally, the obtained product is subjected to core-shell MnO2And after the NF @ CdS is centrifuged, washing the NF @ CdS with ethanol and ultrapure water for three times alternately.
(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 rapidly4Keeping slight boiling, observing the color of the solution to change from light yellow to colorless gray until the solution is wine red, removing a heat source, stopping stirring, and storing at 4 ℃ in a dark place to obtain the 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-1BChE of (4) with 10. mu.L of amino-modified helper DNA (S) at a concentration of 1. mu.M2) In the mixed solution of (2), mixing and oscillating for 12h to realize the combination of Au-N bonds to form S2-Au-BChE signal probe.
(5) 20 mu L of 20mg mL-1MnO prepared in the step (2)2Dripping the dispersed liquid of the NF @ CdS nano composite on the surface of a clean glassy carbon electrode by using Na with the concentration of 0.1M2S and Na in a concentration of 0.02M2SO3The 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 mixture of 60ng mL was added-1Malathion, competition with cDNA for aptamer sequence, reduction of cDNA to single strand, magnetic separation and washing, then in 1 Xligase buffer, T4 DNA ligase (15U) and padlock DNA (10 uL, 1 uM) at 22 deg.C for 1h, forming DNA circular template, again magnetic separation and washing, in 1 XPhi 29 buffer, Phi29DNA polymerase (20U) catalysis dNTP (5 uL, 10M M)M) synthesizing a long single-stranded DNA molecule (namely rolling circle amplification reaction, RCA), namely mixing and incubating for 1h at 37 ℃;
(7) s prepared in the step (4)2Mixing 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 1 h; dripping 20 mu L of upper layer solution on the ITO electrode which is measured with the background current 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, wherein the reduction of the photocurrent intensity is found, and the reduction amplitude and the concentration of malathion have a determined relationship, so that sensitive detection on the malathion is realized; 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 invention2A photoelectric chemical malathion detection process and a schematic diagram for decomposing NF @ CdS. FIG. 2 shows MnO2NF and core-shell MnO2And (3) a scanning electron microscope image of the NF @ CdS composite material. Scanning electron micrograph showing MnO2NF 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 synthesized2NF @ 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-1The photocurrent was well linearly related to the logarithm of the target concentration in the target concentration range (fig. 4A). Considering the requirement of practicality, we considerThe influence of Dic and Chl as interferents on the corresponding signals of the sensor is selected by examining the specificity and selectivity of the method on specific targets. 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: and 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 reacted to form a DNA circular template, and then Phi29DNA polymerase is used for catalyzing dNTP to synthesize long single-stranded DNA molecules.
4. 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 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 photoelectrochemical detection method of malathion based on enzymatic catalysis product cracking manganese dioxide nanoflower @ cadmium sulfide core-shell structure according to claim 4, characterized in that: 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 nanoflowers into an aqueous solution, adding thiourea, cadmium acetate and triethanolamine, heating to 130-150 ℃, and reacting for 28-48 hours to obtain the manganese dioxide nanoflowers @ 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.
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