CN110441369A - One kind being based on flower-shaped SiO2The chlopyrifos electrochemical aptamer sensor of building - Google Patents

Abstract

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 on the basis of flower-like aminated mesoporous SiO ₂ , including an electrochemical aptamer sensor The preparation steps of the ligand sensor, the operation method of using the sensor to detect chlorpyrifos, etc.; the current generated by DNA itself is used as the recognition signal, and the flower-like aminated mesoporous SiO ₂ is loaded with a large number of aptamer complementary chains through nano-gold to amplify the signal; the constructed The electrochemical aptamer sensor has a simple DNA amplification strategy, a simple preparation process, rapid detection, good specificity, and high sensitivity.

Description

An electrochemical aptamer sensor for chlorpyrifos based on flower-like SiO2

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 testing; in particular, to a chlorpyrifos electrochemical aptamer sensor constructed on the basis of flower-like aminated mesoporous SiO ₂ , including an electrochemical aptamer sensor The preparation steps of the ligand sensor, the operation method of using the sensor to detect chlorpyrifos, etc.; using the current generated by the DNA itself as the identification signal, the flower-like aminated mesoporous SiO ₂ is loaded with a large number of aptamer complementary strands (MSN-Au-cDNA ) to amplify the signal; the constructed electrochemical aptasensor has a simple preparation process, rapid detection, good specificity, and high sensitivity.

Background technique

Chlorpyrifos is one of the important pesticides in pesticide residues. Common methods for detecting chlorpyrifos include high performance liquid chromatography, gas chromatography, and enzyme-linked immunosorbent assay. Although these methods can be used for the sensitive detection of chlorpyrifos in actual samples, their application in rapid detection is limited due to the expensive instruments used, long time-consuming and cumbersome operations. Aptamers are DNA/RNA fragments that can specifically interact with the molecules to be tested. Due to their advantages of convenient synthesis, low cost, easy design, strong affinity, and good specificity, they are widely used to construct aptamers. biological sensor. Due to the advantages of fast, stable, high selectivity, and suitable for on-line detection, electrochemical aptasensors have attracted extensive attention in the research of pesticide residue detection. However, in electrochemical aptasensors, recognition signals are often generated by means of electrochemical probes. The construction process is cumbersome, and the detection sensitivity is often affected by the structure and properties of the probe. And because it involves long-distance electron transfer, the signal transfer efficiency is low, which affects the sensitivity. In recent years. It has been reported that DNA can be decomposed into phosphate radicals, and the phosphate radicals can react with sodium molybdate to generate electrochemically active phosphomolybdic acid heteropolyacids to generate current signals (Biosens. Bioelectron. 2016, 85, 220−225). Yang Minghui reacted DNA directly with sodium molybdate to generate an electric current, and prepared an aptasensor for the detection of HER2 (Anal. Chem., 2017, 89: 2547−2552). Based on the electrochemical aptasensor that DNA itself generates current, the signal strength depends on the amount of DNA on the electrode surface. At present, DNA amplification techniques are commonly used, such as PCR, rolling reaction, self-assembly (Anal. Chem., 2017, 89: 10264−10269), etc. However, these amplifying techniques all have defects such as complicated reaction process and long reaction time.

Contents of the invention

The purpose of the present invention is to construct an electrochemical aptasensor that can be used for highly sensitive detection of pesticide residue chlorpyrifos in view of the shortcomings in the research of the above-mentioned electrochemical aptasensor. The technical problem to be solved by the invention is the electrochemical signal amplification technology generated by DNA itself, so as to simplify the construction process and shorten the detection time. The measures taken in the present invention are mainly that a large amount of DNA is loaded on the surface of the flower-shaped mesoporous silica to amplify the signal. The present invention prepares flower-like aminated mesoporous silica (MSN), and loads chlorpyrifos aptamer complementary chain (cDNA) on the mesoporous material through nano-gold to form MSN-Au-cDNA complex; Regular and orderly voids that radiate from the center increase the specific surface area and can load a large amount of DNA. MSN-Au-cDNA generates a double helix structure through cDNA and the aptamer (Apt) loaded on the surface of the electrode to access the electrode, which increases the content of phosphate groups on the surface of the electrode and amplifies the signal; the constructed sensor can be used to detect chlorpyrifos. When there is no target, because there is a large amount of DNA on the surface of the electrode, a sensitive current signal will be generated after adding sodium molybdate; when chlorpyrifos exists, part of the MSN-Au-cDNA will fall off the surface of the electrode, and the current signal generated by it will decrease accordingly. ; And as the concentration of acetamiprid increased, the signal decreased more. The invention provides a simple and feasible new method for the detection of pesticide residue chlorpyrifos.

Technical scheme of the present invention

1. An electrochemical aptamer sensor for chlorpyrifos based on flower-like aminated mesoporous SiO ₂ , using the current generated by DNA itself as the recognition signal, loaded with a large number of flower-like aminated mesoporous aptamer complementary chains The complex (MSN-Au-cDNA) amplifies the signal; it can detect chlorpyrifos rapidly, highly sensitively and specifically;

2. The preparation method of the MSN-Au-cDNA complex:

(1) Take 200 μL, 5 mg/mL flower-like aminated mesoporous silica (MSN) and add 12 mL gold nanoparticles into a conical flask, stir for 12 h; centrifuge at 8500 rpm for 5 min, and ultrasonically disperse In 4 mL of ultrapure water, the MSN-Au dispersion was prepared;

(2) Mix 20 μL, 10 ^-7 mol/L chlorpyrifos complementary strand cDNA with 180 μL, MSN-Au dispersion, shake evenly, and incubate at 4°C for 12 h to obtain the MSN-Au-cDNA complex;

3. The preparation method of the flower-like aminated mesoporous silica (MSN):

(1) Flower-shaped mesoporous silica: Stir 0.5 g cetyl ammonium bromide, 15.0 mL ultrapure water and 0.2 g urea in a three-necked flask; add 0.46 mL isopropanol and 15.0 mL cyclohexane Then, add 1.25 mL of tetraethyl silicate and stir the mixture at high speed for more than 30 min; under the condition of 70℃ water bath, condense and reflux for 16 h, and ultrasonically wash the product with absolute ethanol for 3 -5 times, add 2-4 drops of concentrated hydrochloric acid when washing, and disperse the precipitate in 20mL water for later use;

(2) Amination of flower-shaped mesoporous silica: ultrasonically disperse the above mesoporous _SiO2 nanoparticles with 38 mL of absolute ethanol and 2 mL of ultrapure water, then transfer to a round-bottomed flask and stir evenly; Quickly add 200 μL of 3-aminopropyltriethoxysilane dropwise, stir evenly, and react at 70°C for 12 h under the protection of _N2 ; wash the reacted product three times with absolute ethanol and ultrapure water successively, and ultrasonically Disperse in 20 mL of water to prepare MSN;

4. The preparation method of the electrochemical aptasensor:

(1) The treated glassy carbon electrode GCE was immersed in a solution containing 2.5 mM HAuCl ₄ , 150 mM EDA and 0.5 MH ₂ SO ₄ , and was subjected to constant potential deposition at 0.0V for 10 min to prepare nano-gold modified glassy carbon electrode Au NPs /GCE;

(2) 10 μL, 1.0 μM chlorpyrifos aptamer was drop-coated onto the surface of the above-mentioned Au NPs/GCE, and incubated at 4°C for 12 h to obtain Apt/Au NPs/GCE;

(3) After Apt/Au NPs/GCE blocked the non-specific binding sites with MCH, 5 μL of MSN-Au-cDNA complex was drip-coated, incubated at 37°C for 1 h and washed with high-purity water to obtain MSN-Au-cDNA/Apt/ Au NPs/GCE;

5. The method for detecting chlorpyrifos as described:

(1) On the surface of MSN-Au-cDNA/Apt/Au NPs/GCE, drop 10 μL of chlorpyrifos standard solution of different concentrations, incubate at 37°C for 60 min and wash with water;

(2) Continue to drop 5 μL of 10mM sodium molybdate solution on the surface of the electrode, and let it stand at room temperature for 20 minutes;

(3) Immerse the electrode prepared above in 0.5M sulfuric acid solution, and perform a square wave voltammetry scan (SWV) in the potential range of 0.0 ~ 0.5 V; measure and calculate the difference with the blank peak current, and draw the working curve;

(4) Replace the standard solution of chlorpyrifos with the sample solution to be tested, and measure the peak current according to steps (1), (2) and (3); calculate the content of chlorpyrifos in the sample by the working curve method.

Beneficial effects of the present invention

1. The flower-shaped mesoporous silica prepared by the present invention is spherical, with regular and orderly pores on its surface, and the pores diverge from the center to the outside, which increases the specific surface area and can load more biological materials, as shown in Figure 1 shown;

2. Utilizing the characteristics of flower-shaped mesoporous silica, the DNA loading capacity is increased, and the current signal can be amplified. The signal amplification technology of the present invention is 2 times higher than the signal of the bare electrode; it is also significantly improved compared with the signal of using solid silicon dioxide (figure 2);

3. The DNA amplification technology of the present invention overcomes the defects of commonly used DNA amplification technologies, such as PCR, rolling reaction, self-assembly and the like. Simplifies the sensor preparation process, simple and fast;

4. The present invention is based on the electrochemical sensor constructed by the current signal generated by DNA itself, which does not require electrochemical probes and has high signal conversion efficiency;

5. For the first time, the present invention combines the current signal generated by DNA itself with the amplified DNA of flower-shaped mesoporous silica, and the constructed electrochemical aptamer sensor is applied to the detection of chlorpyrifos, which has the advantages of simple preparation, easy use, stability and heavy Good reproducibility; rapid detection, good sensitivity and selectivity; can realize simple, rapid, highly sensitive and selective detection of chlorpyrifos; linear range is 1.0×10 ^-6 ~1.0×10 ^-12 M, and the detection limit is 3.3 × ^10-14 M.

Description of drawings

Figure 1 is a TEM image of silica

Figure 2 is the SWV curves of different modified electrodes in 0.5MH ₂ SO ₄ after dropping sodium molybdate

Among them, 1--Au NPs/GCE, 2--Apt/Au NPs/GCE, 3--SiO ₂ -Au-cDNA/Apt/Au NPs/GCE,

4--MSN-Au-cDNA/Apt/Au NPs/GCE ,5--target/MSN-Au-cDNA/Apt/Au NPs/GCE.

Figure 3 is the SWV and linear fitting curve of the sensor at different concentrations of chlorpyrifos

Among them, 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.

Figure 4 is a summary drawing.

Detailed ways

In order to better understand the present invention, the technical solution of the present invention will be described in detail below 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: Stir 0.5 g cetyl ammonium bromide, 15.0 mL ultrapure water and 0.2 g urea in a three-necked flask; add 0.46 mL isopropanol and 15.0 mL Cyclohexane, continue to stir evenly, and seal with parafilm; then add 1.25 mL tetraethyl silicate, and stir the mixture at high speed for more than 30 min; under the condition of 70°C water bath, reflux for 16 h, and the product is ultrasonicated with absolute ethanol Wash 3-5 times, add 2-4 drops of concentrated hydrochloric acid during washing, and disperse the precipitate in 20 mL of water for later use;

(2) Amination of flower-shaped mesoporous silica: ultrasonically disperse the above mesoporous _SiO2 nanoparticles with 38 mL of absolute ethanol and 2 mL of ultrapure water, then transfer to a round-bottomed flask and stir evenly; Quickly add 200 μL of 3-aminopropyltriethoxysilane dropwise, stir evenly, and react at 70°C for 12 h under the protection of _N2 ; wash the reacted product three times with absolute ethanol and ultrapure water successively, and ultrasonically Disperse in 20 mL of water to prepare MSN.

Example 2 MSN-Au-cDNA complex preparation method:

(1) Take 200 μL, 5 mg/mL flower-like aminated mesoporous silica MSN, add 12 mL gold nanoparticles into a conical flask, stir for 12 h; centrifuge at 8500 rpm for 5 min, and ultrasonically disperse in 4 mL In ultrapure water, the MSN-Au dispersion was prepared;

(2) Mix 20 μL, 10 ^-7 mol/L chlorpyrifos complementary strand cDNA with 180 μL, MSN-Au dispersion, shake evenly, and incubate at 4°C for 12 h to obtain the MSN-Au-cDNA complex;

Chlorpyrifos aptamer complementary strand (cDNA): 5' NH ₂ -(CH ₂ ) ₆ -CGGGTGCCAAGCTTA-3'.

Example 3 Preparation of gold nanoparticles

Take 0.0015 g of sodium citrate and 20 mL of ultrapure water in a beaker, add 170 μL of 0.25 mM HAuCl ₄ solution under rapid stirring, then add 0.6 mL of 0.1M NaBH ₄ solution, and let it age for 6 h.

Example 4 Preparation of Nano SiO ₂ Solid Balls

Take 10 mL of ammonia water, 25 mL of ultrapure water and 16.5 mL of absolute ethanol in a three-necked flask and stir evenly as liquid A; take 45 mL of absolute ethanol and 4.5 mL of tetraethyl silicate in a three-necked flask, mix and stir evenly as liquid B. Quickly pour liquid B into liquid A, stir at 1100 rpm for 30 s, then adjust the speed to 400 rpm, seal with parafilm, and stir for 2 h. The mixture was centrifuged at 8500 rpm for 5 min, the supernatant was poured out, the precipitate was washed 3-5 times with absolute ethanol, and then washed 1-2 times with ultrapure water, and then the solid was dispersed in 20 mL ultrapure water for later use.

Example 5 Preparation method of electrochemical aptasensor:

(1) The treated glassy carbon electrode GCE was immersed in a solution containing 2.5 mM HAuCl ₄ , 150 mM EDA and 0.5 MH ₂ SO ₄ , and was subjected to constant potential deposition at 0.0 V for 10 min to prepare nano-gold modified glassy carbon electrode Au NPs /GCE;

(2) 10 μL, 1.0 μM chlorpyrifos aptamer was drop-coated onto the surface of the above-mentioned Au NPs/GCE, and incubated at 4°C for 12 h to obtain Apt/Au NPs/GCE;

(3) After Apt/Au NPs/GCE blocked the non-specific binding sites with MCH, 5 μL of MSN-Au-cDNA complex was drip-coated, incubated at 37°C for 1 h and washed with high-purity water to obtain MSN-Au-cDNA/Apt/ Au NPs/GCE;

Chlorpyrifos aptamer (Apt) used: 5' NH ₂ -(CH ₂ ) ₆ -CCTGCCACGCTCCGCAAGCTTAGGGTT ACGCCTGCAGCGATTCTTGATCGCGCTGCTGGTAATCCTTCTTTAAGCTTGGCACCCGCA TCGT-3'.

Embodiment 6 The method for detecting chlorpyrifos:

(1) On the surface of MSN-Au-cDNA/Apt/Au NPs/GCE, drop 10 μL of chlorpyrifos standard solution of different concentrations, incubate at 37°C for 60 min and wash with water;

(2) Continue to drop 5 μL of 10 mM sodium molybdate solution on the surface of the electrode, and let it stand at room temperature for 20 min;

(3) Immerse the electrode prepared above in a 0.5 M sulfuric acid solution, perform a square wave voltammetry scan (SWV) in the potential range of 0.0 to 0.5 V, and record the experimental data; measure the difference between the peak current before and after the addition of chlorpyrifos (0.15V nearby peak current) DIp; the experimental results of the linear range and detection limit of the sensor show that the linear range is 10 ^-6 -10 ^-13 M, the linear regression equation is DIp=0.63lgc+8.68, the linear correlation coefficient is 0.997, and the detection limit is 3.3×10 ^-14 M;

(4) Replace the standard solution of chlorpyrifos with the sample solution to be tested, and measure the peak current according to steps (1), (2) and (3); calculate the content of chlorpyrifos in the sample by the working curve method.

Claims (6)

1. one kind is based on the mesoporous SiO of amination₂The chlopyrifos electrochemical aptamer sensor of building, which is characterized in that certainly with DNA It is identification signal that body, which generates electric current, and load has the flower-shaped amination meso-porous titanium dioxide silicon compound MSN- of a large amount of aptamers complementary strands Au-cDNA amplified signal；It can quick, highly sensitive specific detection chlopyrifos.

2. MSN-Au-cDNA compound described in claim 1, which is characterized in that the preparation method comprises the following steps:

(1) take the flower-shaped amination mesoporous silicon oxide MSN of 200 μ L, 5 mg/mL that 12 mL Jenner's grain of rices are added in conical flask Son stirs 12 h；5 min are centrifuged under 8500 rpm revolving speeds, MSN-Au dispersion is made in 4 mL ultrapure waters in ultrasonic disperse Liquid；

(2) by 20 μ L, 10^-7 Mol/L chlopyrifos complementary strand cDNA is mixed with 180 μ L, MSN-Au dispersion liquid, and concussion is uniform, and 4 It is incubated for 12 h at DEG C, obtains MSN-Au-cDNA compound.

3. flower-shaped amination mesoporous silicon oxide MSN described in claim 2, which is characterized in that the preparation method is as follows:

(1) flower-shaped mesoporous silicon oxide synthesis: by 0.5 g cetyl ammonium bromide, 15.0 mL ultrapure waters and 0.2 g urea in It is stirred evenly in three-necked flask；0.46 mL isopropanol and 15.0 mL hexamethylenes are added thereto, continues to stir evenly, with sealing Film closing；Then 1.25 mL tetraethyl orthosilicates are added, by 30 min or more of mixed liquor high-speed stirred；It is cold under 70 DEG C of water bath conditions 16 h of solidifying reflux, by product dehydrated alcohol supersound washing 3-5 times, 2-4 drop concentrated hydrochloric acid is added in when washing, and sediment is dispersed in It is spare in 20 mL water；

(2) amination of flower-shaped mesoporous silicon oxide: by above-mentioned mesoporous SiO₂38 mL dehydrated alcohols of nanoparticle and 2 mL are super Pure water ultrasonic disperse, is then transferred in round-bottomed flask and stirs evenly；200 μ L 3- aminopropyl, three second is rapidly added dropwise into solution Oxysilane stirs evenly, in N₂Protect 12 h of lower 70 DEG C of reactions；Product after reaction is successively used into dehydrated alcohol and ultrapure water MSN is made in 20 mL water in washing 3 times, ultrasonic disperse.

4. electrochemical aptamer sensor described in claim 1, which is characterized in that the preparation method is as follows:

(1) the glass-carbon electrode GCE handled well is immersed and contains 2.5 mM HAuCl₄, 150 mM EDA and 0.5 M H₂SO₄Solution In, In Glassy Carbon Electrode Modified With Nano-gold Au NPs/GCE is made in 10 min of potentiostatic electrodeposition at 0.0V；

(2) 10 μ L, 1.0 μM of chlopyrifos aptamers drop coatings are hatched 12 h at 4 DEG C, obtained to the surface above-mentioned Au NPs/GCE Apt/Au NPs/GCE；

(3) after Apt/Au NPs/GCE closes nonspecific binding site with MCH, 5 μ L MSN-Au-cDNA compound of drop coating, It is cleaned after hatching 1 h at 37 DEG C with high purity water, obtains MSN-Au-cDNA/Apt/Au NPs/GCE.

5. the described in any item electrochemical aptamer sensors of claim 1-4 are for detecting chlopyrifos.

6. the method for detection chlopyrifos according to claim 5, which is characterized in that steps are as follows:

(1) on the surface MSN-Au-cDNA/Apt/Au NPs/GCE, it is added dropwise the chlopyrifos standard solution 10 μ L of various concentration, 37 It is washed with water after hatching 60 min at DEG C；

(2) continue that the 5 μ L of sodium molybdate solution of 10 mM is added dropwise in electrode surface, room temperature stands 20 min；

(3) sulfuric acid solution that electrode obtained above is immersed to 0.5 M carries out square wave volt-ampere in 0.0 ~ 0.5 V potential region Scanning；It measures and calculates the difference with blank peak current；

(4) testing sample solution is replaced into chlopyrifos standard solution, is measured by step (1), (2) and (3) method；With working curve Method seeks the content for calculating sample Chlorpyrifos.