CN110441369B - A chlorpyrifos electrochemical aptamer sensor based on flower-like SiO2 - Google Patents
A chlorpyrifos electrochemical aptamer sensor based on flower-like SiO2 Download PDFInfo
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- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 239000005944 Chlorpyrifos Substances 0.000 title claims abstract description 40
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 38
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims description 45
- 239000000377 silicon dioxide Substances 0.000 title claims description 24
- 235000012239 silicon dioxide Nutrition 0.000 title description 5
- 229910052681 coesite Inorganic materials 0.000 title description 2
- 229910052906 cristobalite Inorganic materials 0.000 title description 2
- 229910052682 stishovite Inorganic materials 0.000 title description 2
- 229910052905 tridymite Inorganic materials 0.000 title description 2
- 239000010931 gold Substances 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 230000000295 complement effect Effects 0.000 claims abstract description 9
- 229910052737 gold Inorganic materials 0.000 claims abstract description 8
- 239000002299 complementary DNA Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 23
- 239000012498 ultrapure water Substances 0.000 claims description 19
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000002105 nanoparticle Substances 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 7
- 239000011684 sodium molybdate Substances 0.000 claims description 7
- 235000015393 sodium molybdate Nutrition 0.000 claims description 7
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 6
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 238000005576 amination reaction Methods 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 230000008021 deposition Effects 0.000 claims description 3
- XQSBLCWFZRTIEO-UHFFFAOYSA-N hexadecan-1-amine;hydrobromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[NH3+] XQSBLCWFZRTIEO-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 230000009871 nonspecific binding Effects 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- -1 silica compound Chemical class 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 230000004544 DNA amplification Effects 0.000 abstract description 4
- 238000010276 construction Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 abstract description 2
- 229910004298 SiO 2 Inorganic materials 0.000 abstract 2
- 239000003446 ligand Substances 0.000 abstract 1
- 230000003321 amplification Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000447 pesticide residue Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001338 self-assembly Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 1
- 239000005875 Acetamiprid Substances 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000013335 mesoporous material Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/28—Electrolytic cell components
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
本发明涉及电化学适配体传感器的构建方法和检测方法，属于分析测试技术领域；特别是涉及一种基于花状氨基化介孔SiO2构建的毒死蜱电化学适配体传感器，包括电化学适配体传感器的制备步骤、使用该传感器测定毒死蜱的操作方法等；以DNA自身产生电流为识别信号，花状氨基化介孔SiO2通过纳米金负载大量适配体互补链以放大信号；构建的电化学适配体传感器，DNA放大策略简单，制备过程简捷，检测快速、特异性好、灵敏度高。
The invention relates to a construction method and a detection method of an electrochemical aptamer sensor, belonging to the technical field of analysis and testing; in particular to a chlorpyrifos electrochemical aptamer sensor constructed based on flower-like aminated mesoporous SiO 2 , comprising an electrochemical aptamer sensor. The preparation steps of the ligand sensor, the operation method of using the sensor to measure chlorpyrifos, etc.; the DNA self-generated current is used as the identification signal, and the flower-shaped aminated mesoporous SiO 2 is loaded with a large number of aptamer complementary chains through the nano-gold to amplify the signal; the constructed The electrochemical aptamer sensor has simple DNA amplification strategy, simple preparation process, rapid detection, good specificity and high sensitivity.
Description
Technical Field
The invention relates to a construction method and a detection method of an electrochemical aptamer sensor, belonging to the technical field of analysis and test; in particular to a SiO based on flower-shaped aminated mesoporous2The constructed chlorpyrifos electrochemical aptamer sensor comprises the steps of preparing the electrochemical aptamer sensor, using the sensor to measure the operation method of the chlorpyrifos and the like; flower-shaped aminated mesoporous SiO by using DNA self-generated current as identification signal2Amplifying signals by loading a large number of aptamer complementary strands (MSN-Au-cDNA) through nanogold; constructed electrochemical aptamer sensingThe preparation process is simple, the detection is rapid, the specificity is good, and the sensitivity is high.
Background
Chlorpyrifos is one of the important pesticides in the pesticide residue. Common methods for detecting chlorpyrifos include high performance liquid chromatography, gas chromatography, enzyme linked immunosorbent assay and the like. Although these methods can be used for sensitive detection of chlorpyrifos in real samples, they have limited their application in rapid detection due to the expensive, long and cumbersome instrumentation. The aptamer is a DNA/RNA fragment capable of specifically interacting with a molecule to be detected, and is widely used for constructing aptamer biosensors due to the advantages of convenience in synthesis, low cost, easiness in design, strong affinity and good specificity. The electrochemical aptamer sensor has the advantages of being rapid, stable, high in selectivity, suitable for online detection and the like, and therefore the electrochemical aptamer sensor is widely concerned in pesticide residue detection research. However, in electrochemical aptamer sensors, an identification signal is often generated by means of an electrochemical probe. The construction process is complicated, and the detection sensitivity is often influenced by the structure and the property of the probe. And because of the long range electron transfer involved, the signal transfer efficiency is low, affecting sensitivity. In recent years. It has been reported that DNA can be decomposed into phosphate, and that the phosphate can be reacted with sodium molybdate to generate heteropoly phosphomolybdate which has electrochemical activity, thereby generating an electric current signal (biosens, bioelectronic, 2016, 85, 220-225). Yangminghui reacted DNA directly with sodium molybdate to generate current, and an aptamer sensor was prepared for the detection of HER2 (anal. chem., 2017, 89: 2547-2552). Based on electrochemical aptamer sensors where the DNA itself generates a current, the signal intensity is determined by the amount of DNA on the electrode surface. Currently, DNA amplification techniques such as PCR, rolling-over reactions, self-assembly (anal. chem., 2017, 89: 10264-10269), and the like are commonly used. However, these amplification techniques have the disadvantages of complicated reaction process, long reaction time, etc.
Disclosure of Invention
The invention aims to overcome the defects in the research of the electrochemical aptamer sensor and construct the electrochemical aptamer sensor capable of being used for detecting pesticide residue chlorpyrifos with high sensitivity. The invention aims to solve the technical problem of the electrochemical signal amplification technology generated by DNA itself so as to simplify the construction process and shorten the detection time. The measure adopted by the invention is mainly that a large amount of DNA is loaded on the surface of the flower-shaped mesoporous silicon dioxide to amplify signals. The method comprises the steps of preparing flower-shaped aminated Mesoporous Silica (MSN), and loading chlorpyrifos aptamer complementary strand (cDNA) on a mesoporous material through nanogold to form an MSN-Au-cDNA compound; because the MSN surface has regular and orderly gaps which are diverged from the center to the outside, the specific surface area is increased, and a large amount of DNA can be loaded. The MSN-Au-cDNA generates a double-spiral structure through cDNA and an aptamer (Apt) loaded on the surface of the electrode so as to be connected into the electrode, so that the content of phosphate groups on the surface of the electrode is improved, and a signal is amplified; the constructed sensor can be used for detecting chlorpyrifos, and when no target exists, a large amount of DNA exists on the surface of an electrode, and a sensitive current signal can be generated after sodium molybdate is dripped; when chlorpyrifos exists, part of MSN-Au-cDNA falls off from the surface of the electrode, and the generated current signal is reduced; and the more the signal decreases as the concentration of acetamiprid increases. The invention provides a simple, convenient and feasible new method for detecting the residual chlorpyrifos.
Technical scheme of the invention
1. SiO based on flower-shaped aminated mesoporous2The constructed chlorpyrifos electrochemical aptamer sensor takes the current generated by DNA itself as an identification signal and loads a large amount of flower-shaped aminated mesoporous silicon dioxide compound (MSN-Au-cDNA) amplification signals of aptamer complementary chains; the chlorpyrifos can be quickly and specifically detected with high sensitivity;
2. the preparation method of the MSN-Au-cDNA compound comprises the following steps:
(1) adding 200 mu L of flower-shaped aminated Mesoporous Silica (MSN) of 5mg/mL into a conical flask, adding 12mL of gold nanoparticles, and stirring for 12 h; centrifuging at 8500rpm for 5min, and ultrasonically dispersing in 4mL of ultrapure water to obtain MSN-Au dispersion liquid;
(2) mixing 20 μ L and 10 μ L-7 mixing mol/L chlorpyrifos complementary strand cDNA with 180 mu L, MSN-Au dispersion liquid, shaking uniformly, and incubating for 12h at 4 ℃ to obtain an MSN-Au-cDNA compound;
3. the preparation method of the flower-shaped aminated Mesoporous Silica (MSN) comprises the following steps:
(1) flower-like mesoporous silica: 0.5g of hexadecyl ammonium bromide, 15.0mL of ultrapure water and 0.2g of urea are uniformly stirred in a three-neck flask; adding 0.46mL of isopropanol and 15.0mL of cyclohexane, continuously stirring uniformly, and sealing by using a sealing film; then adding 1.25mL of tetraethyl silicate, and stirring the mixed solution at a high speed for more than 30 min; condensing and refluxing for 16h under the condition of 70 ℃ water bath, ultrasonically washing the product for 3-5 times by using absolute ethyl alcohol, adding 2-4 drops of concentrated hydrochloric acid during washing, and dispersing the precipitate in 20mL of water for later use;
(2) amination of flower-like mesoporous silica: the mesoporous SiO is prepared2Ultrasonically dispersing the nano particles by using 38mL of absolute ethyl alcohol and 2mL of ultrapure water, and then transferring the nano particles into a round-bottom flask to be uniformly stirred; 200 mu.L of 3-aminopropyltriethoxysilane was quickly added dropwise to the solution, stirred well and added to N2Reacting for 12 hours at 70 ℃ under protection; washing the product after reaction with absolute ethyl alcohol and ultrapure water for 3 times in sequence, and ultrasonically dispersing in 20mL of water to prepare MSN;
4. the preparation method of the electrochemical aptamer sensor comprises the following steps:
(1) the treated glassy carbon electrode GCE was immersed in a solution containing 2.5mM HAuCl4150mM EDA and 0.5M H2SO4In the solution, constant potential deposition is carried out for 10min at 0.0V, and the nano gold modified glassy carbon electrode Au NPs/GCE is prepared;
(2) dripping 10 mu L of chlorpyrifos aptamer with the concentration of 1.0 mu M onto the surface of the Au NPs/GCE, and incubating for 12h at the temperature of 4 ℃ to obtain Apt/Au NPs/GCE;
(3) after the non-specific binding sites are blocked by the Apt/Au NPs/GCE through MCH, 5 mu L of MSN-Au-cDNA compound is dripped, and the mixture is incubated at 37 ℃ for 1h and then washed by high-purity water to obtain MSN-Au-cDNA/Apt/Au NPs/GCE;
5. the method for detecting chlorpyrifos comprises the following steps:
(1) dripping 10 mu L of chlorpyrifos standard solution with different concentrations on the surface of MSN-Au-cDNA/Apt/Au NPs/GCE, incubating for 60min at 37 ℃, and then washing with water;
(2) continuously dropwise adding 5 mu L of 10mM sodium molybdate solution on the surface of the electrode, and standing for 20min at normal temperature;
(3) immersing the prepared electrode into 0.5M sulfuric acid solution, and performing square wave volt-ampere Scanning (SWV) in a potential range of 0.0-0.5V; measuring and calculating the difference value between the current and the blank peak current, and drawing a working curve;
(4) replacing a chlorpyrifos standard solution with a sample solution to be detected, and measuring the peak current according to the methods of the steps (1), (2) and (3); and (5) calculating the content of the chlorpyrifos in the sample by a working curve method.
The invention has the advantages of
1. The flower-shaped mesoporous silica prepared by the invention is spherical, the surface of the flower-shaped mesoporous silica has regular and ordered gaps, the pores are dispersed outwards from the center, the specific surface area is increased, and more biological materials can be loaded, as shown in figure 1;
2. by utilizing the characteristics of the flower-shaped mesoporous silica, the DNA load is improved, and the current signal is amplified. The signal amplification technology of the invention is increased by 2 times compared with the signal of a bare electrode; the signal is also obviously improved compared with the signal obtained by using solid silicon dioxide (figure 2);
3. the DNA amplification technology of the invention overcomes the defects of common DNA amplification technologies, such as PCR, rolling reaction, self-assembly and the like. The preparation process of the sensor is simplified, and the method is simple and rapid;
4. the electrochemical sensor constructed based on the DNA self-generated current signal does not need an electrochemical probe, and has high signal conversion efficiency;
5. the invention combines the current signal generated by the DNA and the amplified DNA of the flower-shaped mesoporous silicon dioxide for the first time, and the constructed electrochemical aptamer sensor is applied to the detection of chlorpyrifos, and has the advantages of simple preparation, simple and convenient use, good stability and good reproducibility; the detection is rapid, and the sensitivity and the selectivity are good; the method can realize simple, rapid and high-sensitivity selective detection of the chlorpyrifos; linear range of 1.0X 10-6~1.0×10-12M, detection limit of 3.3X 10-14 M。
Drawings
FIG. 1 is a TEM image of silica
FIG. 2 shows different modified electrode dropsAfter adding sodium molybdate, the solution is at 0.5M H2SO4SWV curve of (1)
Wherein, 1- -Au NPs/GCE, 2- -Apt/Au NPs/GCE, 3- -SiO2-Au-cDNA/Apt/Au NPs/GCE,
4- -MSN-Au-cDNA/Apt/Au NPs/GCE, 5- -target/MSN-Au-cDNA/Apt/Au NPs/GCE.
FIG. 3 is a SWV and linear fit curve of the sensor at different concentrations of chlorpyrifos
Wherein, 1-9 respectively represent the concentration of chlorpyrifos: 10-6 , 10-7 ,10-8 ,10-9 ,10-10 ,10-11 ,10-12,10-13 ,0 M。
Fig. 4 is an abstract attached figure.
Detailed Description
For better understanding of the present invention, the technical solution of the present invention will be described in detail with specific examples, but the present invention is not limited thereto.
Example 1 preparation method of flower-like aminated Mesoporous Silica (MSN):
(1) synthesis of flower-like mesoporous silica: 0.5g of hexadecyl ammonium bromide, 15.0mL of ultrapure water and 0.2g of urea are uniformly stirred in a three-neck flask; adding 0.46mL of isopropanol and 15.0mL of cyclohexane, continuously stirring uniformly, and sealing by using a sealing film; then adding 1.25mL of tetraethyl silicate, and stirring the mixed solution at a high speed for more than 30 min; condensing and refluxing for 16h under the condition of 70 ℃ water bath, ultrasonically washing the product for 3-5 times by using absolute ethyl alcohol, adding 2-4 drops of concentrated hydrochloric acid during washing, and dispersing the precipitate in 20mL of water for later use;
(2) amination of flower-like mesoporous silica: the mesoporous SiO is prepared2Ultrasonically dispersing the nano particles by using 38mL of absolute ethyl alcohol and 2mL of ultrapure water, and then transferring the nano particles into a round-bottom flask to be uniformly stirred; 200 mu.L of 3-aminopropyltriethoxysilane was quickly added dropwise to the solution, stirred well and added to N2Reacting for 12 hours at 70 ℃ under protection; and washing the product after reaction with absolute ethyl alcohol and ultrapure water for 3 times in sequence, and ultrasonically dispersing in 20mL of water to prepare the MSN.
Example 2 preparation of MSN-Au-cDNA complexes:
(1) adding 200 mu L of flower-shaped aminated mesoporous silica MSN with the concentration of 5mg/mL into a conical flask, adding 12mL of gold nanoparticles, and stirring for 12 h; centrifuging at 8500rpm for 5min, and ultrasonically dispersing in 4mL of ultrapure water to obtain MSN-Au dispersion liquid;
(2) mixing 20 μ L and 10 μ L-7 mixing mol/L chlorpyrifos complementary strand cDNA with 180 mu L, MSN-Au dispersion liquid, shaking uniformly, and incubating for 12h at 4 ℃ to obtain an MSN-Au-cDNA compound;
chlorpyrifos aptamer complementary strand (cDNA) used: 5' NH2 -(CH2) 6-CGGGTGCCAAGCTTA-3'。
Example 3 preparation of gold nanoparticles
0.0015 g of sodium citrate and 20mL of ultrapure water were placed in a beaker, and 170. mu.L of 0.25 mM HAuCl was added thereto with rapid stirring4The solution was added 0.6 mL of 0.1M NaBH4Standing and aging the solution for 6 h.
Example 4 nanometer SiO2Preparation of solid spheres
Taking 10 mL of ammonia water, 25mL of ultrapure water and 16.5 mL of absolute ethyl alcohol, and uniformly stirring in a three-neck flask to obtain solution A; 45 mL of absolute ethyl alcohol and 4.5 mL of tetraethyl silicate were mixed and stirred uniformly in a three-neck flask to obtain solution B. And quickly pouring the solution B into the solution A, stirring at 1100 rpm for 30 s, then adjusting the rotation speed to 400 rpm, sealing with a sealing film, and stirring for 2 h. Centrifuging the mixture at 8500rpm for 5min, pouring out the supernatant, washing the precipitate with anhydrous ethanol for 3-5 times, washing with ultrapure water for 1-2 times, and dispersing the solid in 20mL of ultrapure water for later use.
Example 5 electrochemical aptamer sensor preparation method:
(1) the treated glassy carbon electrode GCE was immersed in a solution containing 2.5mM HAuCl4150mM EDA and 0.5M H2SO4In the solution, constant potential deposition is carried out for 10min at 0.0V, and the nano gold modified glassy carbon electrode Au NPs/GCE is prepared;
(2) dripping 10 mu L of chlorpyrifos aptamer with the concentration of 1.0 mu M onto the surface of the Au NPs/GCE, and incubating for 12h at the temperature of 4 ℃ to obtain Apt/Au NPs/GCE;
(3) after the non-specific binding sites are blocked by the Apt/Au NPs/GCE through MCH, 5 mu L of MSN-Au-cDNA compound is dripped, and the mixture is incubated at 37 ℃ for 1h and then washed by high-purity water to obtain MSN-Au-cDNA/Apt/Au NPs/GCE;
chlorpyrifos aptamer (Apt): 5' NH2-(CH2)6-CCTGCCACGCTCCGCAAGCTTAGGGTT ACGCCTGCAGCGATTCTTGATCGCGCTGCTGGTAATCCTTCTTTAAGCTTGGCACCCGCA TCGT-3'。
Example 6 method for detecting chlorpyrifos:
(1) dripping 10 mu L of chlorpyrifos standard solution with different concentrations on the surface of MSN-Au-cDNA/Apt/Au NPs/GCE, incubating at 37 ℃ for 60min, and then washing with water;
(2) continuously dropwise adding 5 mu L of 10mM sodium molybdate solution on the surface of the electrode, and standing for 20min at normal temperature;
(3) immersing the prepared electrode into 0.5M sulfuric acid solution, performing square wave volt-ampere Scanning (SWV) in a potential range of 0.0-0.5V, and recording experimental data; measuring the difference value (the peak current near 0.15V) DIp of the peak current before and after the chlorpyrifos is added; the experimental results of the linear range and detection limit of the sensor show that the linear range is 10-6 -10-13 M, linear regression equation DIp =0.63lgc +8.68, linear correlation coefficient 0.997, detection limit 3.3 × 10-14 M；
(4) Replacing a chlorpyrifos standard solution with a sample solution to be detected, and measuring the peak current according to the methods of the steps (1), (2) and (3); and (5) calculating the content of the chlorpyrifos in the sample by a working curve method.
Claims (3)
1. SiO based on aminated mesoporous2The preparation method of the chlorpyrifos electrochemical aptamer sensor is characterized in that the electrochemical aptamer sensor takes DNA self-generated current as an identification signal and loads a large amount of flower-shaped aminated mesoporous silica compound MSN-Au-cDNA amplified signals of aptamer complementary chains; the electrochemical aptamer sensor is prepared by the following steps:
(1) the treated glassy carbon electrode GCE was immersed in a solution containing 2.5mM HAuCl4150mM EDA and 0.5M H2SO4In the solution, constant potential deposition is carried out for 10min at 0.0V,preparing a nano gold modified glassy carbon electrode Au NPs/GCE;
(2) dripping 10 mu L of chlorpyrifos aptamer with the concentration of 1.0 mu M onto the surface of the Au NPs/GCE, and incubating for 12h at the temperature of 4 ℃ to obtain Apt/Au NPs/GCE;
(3) after the Apt/Au NPs/GCE seals the nonspecific binding site by MCH, 5 mu L of compound MSN-Au-cDNA is dripped, and the mixture is incubated at 37 ℃ for 1h and then washed by high-purity water to obtain MSN-Au-cDNA/Apt/Au NPs/GCE;
the MSN-Au-cDNA comprises the following preparation steps:
(1) adding 200 mu L of flower-shaped aminated mesoporous silica MSN with the concentration of 5mg/mL into a conical flask, adding 12mL of gold nanoparticles, and stirring for 12 h; centrifuging at 8500rpm for 5min, and ultrasonically dispersing in 4mL of ultrapure water to obtain MSN-Au dispersion liquid;
(2) mixing 20 μ L and 10 μ L-7mixing mol/L chlorpyrifos complementary strand cDNA with 180 mu L, MSN-Au dispersion liquid, shaking uniformly, and incubating for 12h at 4 ℃ to obtain an MSN-Au-cDNA compound;
the flower-shaped aminated mesoporous silica MSN is prepared by the following steps:
(1) synthesizing flower-shaped mesoporous silica: 0.5g of hexadecyl ammonium bromide, 15.0mL of ultrapure water and 0.2g of urea are uniformly stirred in a three-neck flask; adding 0.46mL of isopropanol and 15.0mL of cyclohexane, continuously stirring uniformly, and sealing by using a sealing film; then adding 1.25mL of tetraethyl silicate, and stirring the mixed solution at a high speed for more than 30 min; condensing and refluxing for 16h under the condition of 70 ℃ water bath, ultrasonically washing the product for 3-5 times by using absolute ethyl alcohol, adding 2-4 drops of concentrated hydrochloric acid during washing, and dispersing the precipitate in 20mL of water for later use;
(2) amination of flower-like mesoporous silica: the mesoporous SiO is prepared2Ultrasonically dispersing the nano particles by using 38mL of absolute ethyl alcohol and 2mL of ultrapure water, and then transferring the nano particles into a round-bottom flask to be uniformly stirred; 200 mu.L of 3-aminopropyltriethoxysilane was quickly added dropwise to the solution, stirred well and added to N2Reacting for 12 hours at 70 ℃ under protection; and washing the product after reaction with absolute ethyl alcohol and ultrapure water for 3 times in sequence, and ultrasonically dispersing in 20mL of water to prepare the MSN.
2. The use of the electrochemical aptamer sensor prepared by the preparation method according to claim 1 for detecting chlorpyrifos residue.
3. Use according to claim 2, characterized in that the steps are as follows:
(1) dripping 10 mu L of chlorpyrifos standard solution with different concentrations on the surface of MSN-Au-cDNA/Apt/Au NPs/GCE, incubating at 37 ℃ for 60min, and then washing with water;
(2) continuously dropwise adding 5 mu L of 10mM sodium molybdate solution on the surface of the electrode, and standing for 20min at normal temperature;
(3) immersing the prepared electrode into 0.5M sulfuric acid solution, and performing square wave volt-ampere scanning in a potential range of 0.0-0.5V; measuring and calculating the difference value with the blank peak current;
(4) replacing a chlorpyrifos standard solution with a sample solution to be detected, and determining according to the methods of the steps (1), (2) and (3); and (5) calculating the content of the chlorpyrifos in the sample by a working curve method.
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