Electrochemical aptamer sensor and method for quantitatively detecting heavy metal ion Hg < 2 + >

Abstract

The invention discloses an electrochemical aptamer sensor for quantitatively detecting heavy metal ions Hg < 2 + >. The electrochemical aptamer sensor is a three-electrode system sensor, and a working electrode of the electrochemical aptamer sensor is obtained by sequentially modifying the surface of an ITO (indium tin oxide) electrode with a compound of graphene aerogel and gold nanoparticles and hairpin DNA (deoxyribonucleic acid); wherein the hairpin DNA comprises oligonucleotide chains H2 and H3, the nucleotide sequences of the oligonucleotide chains H2 and H3 are respectively shown as SEQ ID NO: 2-3, the 5'end of H2 is modified with sulfydryl, and the 3 'end of H3 is modified with sulfydryl. The invention also discloses a method for quantitatively detecting the heavy metal ion Hg < 2 + > by adopting the electrochemical aptamer sensor. The detection method provided by the invention can detect the concentration of the heavy metal ion Hg < 2 + > in the sample, and has the advantages of high accuracy, high sensitivity, strong specificity, high stability, good reproducibility and the like.

Description

Technical Field

[0001] The present invention relates to the field of nano-biosensors and electrochemical detection technology, and in particular to an electrochemical aptamer sensor and a method for quantitatively detecting heavy metal ions Hg 2+ method. Background Art

[0002] With the continuous advancement of industrialization, heavy metal pollution has become a global environmental problem. Heavy metals are highly toxic and have bioaccumulative effects. For example, the typical heavy metal ion Cd 2+ , Pb 2+ , Cu 2+ , Hg 2+ They enter the human and animal bodies through the food chain, causing serious harm to health. 2+ It is one of the most toxic heavy metal ions. If it accumulates in the human body, it can cause a variety of diseases, such as immune dysfunction, kidney disease and cardiovascular disease. According to the World Health Organization (WHO, 2017), Hg in drinking water 2+ The maximum limit of Hg in milk is less than 0.02 mg / kg (10 nM). 2+ The maximum limit of Hg is less than 0.01 mg / kg (49.9 nM). Therefore, it is necessary to develop a rapid, accurate and sensitive method to detect Hg 2+ The approach is vital to ensuring food safety and environmental health.

[0003] Traditional detection of heavy metal ions Hg 2+ The methods include atomic absorption spectroscopy (AAS), surface enhanced Raman scattering (SERS), inductively coupled plasma mass spectrometry (ICPMS), atomic fluorescence spectroscopy (AFS), etc. Although these methods have the advantages of sensitivity and efficiency, they have the disadvantages of high equipment requirements, long pre-processing time and high operator requirements. Electrochemical aptamer sensors are favored by more and more researchers due to their low consumption, fast, stable and high sensitivity, but the actual samples have complex components and extremely low content, which puts higher requirements on electrochemical sensors. Summary of the invention

[0004] The present invention aims to overcome the defects of the prior art and provide a method for quantitatively detecting heavy metal ions Hg 2+ The invention discloses an electrochemical aptamer sensor and a corresponding detection method, which has the advantages of simple operation, high accuracy, high sensitivity, strong specificity, high stability and good reproducibility.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a method for quantitatively detecting heavy metal ions Hg 2+ An electrochemical aptamer sensor is a three-electrode system sensor, and the working electrode of the three-electrode system sensor is obtained by sequentially modifying a complex of graphene aerogel (GAs) and gold nanoparticles (AuNPs) and hairpin DNA on the surface of an ITO electrode; wherein the hairpin DNA comprises oligonucleotide chains H2 and H3, and the nucleotide sequences of the oligonucleotide chains H2 and H3 are respectively as shown in SEQ ID NO:2-3, and the 5' end of the oligonucleotide chain H2 is modified with a thiol group, and the 3' end of the oligonucleotide chain H3 is modified with a thiol group.

[0007] Further, the method for modifying the composite of graphene aerogel and gold nanoparticles on the surface of ITO electrode is:

[0008] Prepare a solution containing chloroauric acid (HAuCl 4 ·4H 2 O), graphene oxide (GO) and sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O) and subjecting the mixed solution to a hydrothermal reaction; after the reaction is completed, the product is collected and vacuum freeze-dried to obtain a composite of graphene aerogel and gold nanoparticles;

[0009] The composite of the graphene aerogel and the gold nanoparticles is prepared into a dispersion, which is drop-coated on the surface of the ITO electrode, and dried to obtain an ITO electrode modified with the composite of the graphene aerogel and the gold nanoparticles.

[0010] Furthermore, in the mixed solution, the concentration of graphene oxide is 0.1-1.5 mg / mL, the concentration of chloroauric acid is 0.1-1.5 mg / mL, and the concentration of sodium citrate is 0.05-1.5 mM. Furthermore, the concentration of graphene oxide is 1 mg / mL, the concentration of chloroauric acid is 1 mg / mL, and the concentration of sodium citrate is 0.1 M.

[0011] Further, the method for modifying the hairpin DNA H2 and H3 on the surface of the ITO electrode is:

[0012] A mixed solution of H2 and H3 was prepared, and the mixed solution was drop-coated on the surface of an ITO electrode modified with a composite of graphene aerogel and gold nanoparticles. After incubation, the electrode was washed and dried; and then the electrode was treated with 6-mercapto-1-hexanol to block unbound active sites on the electrode.

[0013] In a second aspect, the present invention provides a method for quantitatively detecting heavy metal ions Hg 2+The method adopts the aforementioned electrochemical aptamer sensor, and the method comprises the following steps:

[0014] a. subjecting the sample solution to be tested to ExoⅢ cyclic amplification treatment to obtain a mixed solution to be tested;

[0015] b. dropping the mixed solution to be tested onto the working electrode of the electrochemical aptamer sensor to react; then, continuing to drop a signal probe onto the working electrode to react;

[0016] c. Place the working electrode, counter electrode and reference electrode in an electrolyte, and use differential pulse voltammetry to obtain the absolute value of the peak current change; then calculate the heavy metal ion Hg in the sample solution to be tested based on the standard curve. 2+ concentration.

[0017] Furthermore, in step a, the cyclic amplification process of ExoⅢ comprises the following steps:

[0018] A solution containing DNA chain H1 and primer is prepared, and a sample solution to be tested and an Exo III solution are added thereto to perform a cyclic amplification reaction; then, the cyclic amplification reaction is terminated by heating, and then the temperature is lowered to allow the unreacted DNA chain H1 to reform into a hairpin structure, thereby obtaining a mixed solution containing DNA chain helper;

[0019] Wherein, the nucleotide sequences of H1, primer and helper are shown in SEQ ID NO: 1, 4-5 respectively.

[0020] Furthermore, in step b, the signal probe is a mixture containing DNA chains A1 and A2, the nucleotide sequence of A1 is 5'-CATACAAA-3', and the nucleotide sequence of A2 is 5'-GACCGGGC-3'; and the 3' end of A1 and the 5' end of A2 are both labeled with methylene blue.

[0021] Furthermore, in step c, the electrolyte is PBS buffer, the counter electrode is a platinum wire electrode, and the reference electrode is a calomel electrode.

[0022] Furthermore, in step c, the method for preparing the standard curve is:

[0023] i. Hg concentration in the range of 1fM-10nM 2+ The standard solution was subjected to the ExoⅢ cyclic amplification treatment;

[0024] j. Add the standard solution that has been cyclically amplified to the surface of the working electrode for reaction, wash and dry it, and then continue to add the signal probe for reaction, wash and dry it;

[0025] k. placing the working electrode, the counter electrode and the reference electrode in an electrolyte, and using differential pulse voltammetry to obtain the absolute value of the peak current change of methylene blue; the absolute value of the peak current change of methylene blue is the dependent variable, and the heavy metal ion Hg 2+ The logarithmic value of the concentration was used as the independent variable to prepare a standard curve.

[0026] Please see attached Figure 1 The working principle of the electrochemical aptamer sensor of the present invention is:

[0027] In the present invention, the hairpin DNA H1 and the auxiliary DNA primer have complementary sequences, and due to Hg 2+ Can specifically bind to thymine (T) to form stable T-Hg 2+ -T coordination compound, so when containing Hg 2+ When the solution is mixed with H1 and primer, primer and Hg 2+ It will bind to the nucleotide chain of H1 through hybridization reaction to form H1-Hg 2+ -primer hybrid chain. Then, exonuclease ExoⅢ is used to 2+ -primer hybrid chain is treated with enzymes to make the auxiliary DNA primer and Hg 2+ From H1-Hg 2+ -primer hybridization chain detaches, and obtains auxiliary DNA helper, which is a partial sequence in the hairpin DNAH1, and its content is similar to Hg 2+ Then, the separated primer and Hg 2+ The hybridization reaction and enzyme cleavage reaction continue to occur, thus achieving cyclic amplification and obtaining more auxiliary DNA helpers, thereby achieving the Hg 2+ The signal is amplified.

[0028] Next, when the solution after the cyclic amplification treatment is dripped onto the surface of the working electrode, since the auxiliary DNA helper has complementary sequences to the hairpin DNA H2 and H3 fixed on the electrode surface, the helper will bind to the H2 and H3 chains respectively through hybridization reaction, thereby being fixed on the surface of the working electrode. The DNA chains A1 and A2 in the signal probe are complementary to H2 and H3 respectively. When the signal probe solution is dripped onto the surface of the working electrode, A1 and A2 will bind to H2 and H3 respectively through hybridization reaction to form an H-type structure. At the same time, the A1 and A2 chains are labeled with methylene blue, which will generate current signals during differential pulse voltammetry measurements. And Hg in the solution 2+The higher the concentration, the more auxiliary DNA helpers bind to the surface of the working electrode, and the stronger the current signal generated. 2+ Quantitative detection of .

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The electrochemical aptamer sensor of the present invention can detect heavy metal ions Hg 2+ , and has a higher sensitivity. When the heavy metal ion Hg 2+ When it exists and its concentration is in the range of 1fM-10nM, a good linear relationship can be obtained, and the detection limit is as low as 0.16fM.

[0031] 2. The electrochemical aptamer sensor of the present invention has good specificity. The specificity experimental results show that the electrochemical aptamer sensor of the present invention has good specificity except for heavy metal ions Hg 2+ In addition, it will not be combined with other seven heavy metal ions (Mg 2+ , Pb 2+ , Ga 2 + 、Na + , K + 、Cd 2+ Cr 6+ )Specific binding.

[0032] 3. The electrochemical aptamer sensor of the present invention has good stability. The stability analysis results show that the sensor of the present invention still maintains 97.6%, 94.9% and 91.1% of the original stability after being stored at 4°C for 5 days, 10 days and 15 days.

[0033] 4. Detection of heavy metal ions Hg in milk samples using the electrochemical aptamer sensor of the present invention 2+ The results are consistent with those of commercial atomic absorption spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the detection principle of the electrochemical aptamer sensor of the present invention;

[0035] Figure 2 is the scanning electron microscopy (SEM) image of GAs-AuNPs;

[0036] Figure 3 is the transmission electron microscopy (TEM) image of GAs;

[0037] Figure 4 The electrochemical aptamer sensor of the present invention is sensitive to heavy metal ions Hg at different concentrations. 2+ Determined DPV diagram;

[0038] Figure 5 The electrochemical aptamer sensor of the present invention is used to detect heavy metal ions Hg at different concentrations. 2+ The linear relationship between the obtained peak current logarithmic values;

[0039] Figure 6 It is a histogram of electrochemical signals when the electrochemical aptamer sensor of the present invention is used to detect various heavy metal ions. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0043] Materials and reagents used in the present invention:

[0044] All DNA sequences, including hairpin DNA1 (hairpin DNA1, H1), hairpin DNA2 (hairpin DNA2, H2), hairpin DNA3 (hairpin DNA3, H3), primer DNA1 (primer DNA1, primer), helper DNA2 (helper DNA2, helper), and MB-labeled DNA (A1, A2) were purchased from Shanghai Sangon Biotechnology Co., Ltd. The oligonucleotide sequences are shown in Table 1.

[0045] Exonuclease III (Exo III) was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China);

[0046] Graphene oxide (GO) was purchased from Xianfeng Nanomaterials Technology Co., Ltd. (Jiangsu, China);

[0047] 6-Mercapto-1-hexanol ((SH)C 6 H 12 (OH)) was purchased from aladdin (Shanghai, China);

[0048] Chloroauric acid tetrahydrate (HAuCl 4·4H 2 O) purchased from Nanjing Chemical Reagent Co., Ltd.;

[0049] N,N-dimethylformamide (DMF), mercury ion standard solution (Hg 2+ ) was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China);

[0050] Sodium citrate (C 6 H 5 Na 3 O 7 ·2H 2 O), potassium ferrocyanide (K 3 [Fe(CN) 6 ]), potassium ferrocyanide (K 4 [Fe(CN) 6 ]), potassium chloride (KCl), acetone (C 3 H 6 O), ethanol (C 2 H 5 OH), concentrated sulfuric acid (H 2 SO 4 ), concentrated hydrochloric acid (HCl), sodium chloride (NaCl), magnesium chloride (MgCl 2 ) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China);

[0051] All the above chemical reagents were of analytical grade.

[0052] Table 1 Oligonucleotide sequences involved in the examples

[0053]

[0054]

[0055] MB is the abbreviation of methylene blue.

[0056] The instruments and equipment used in the present invention are:

[0057] Liquid crystal ultrasonic cleaner, from Kunshan Jielimei Ultrasonic Instrument Co., Ltd.; electric heating blast drying oven, from Hefei Youke Instrument Equipment Co., Ltd.; electronic analytical balance, from Huazhi (Fujian) Electronic Technology Co., Ltd.; electrochemical workstation, from Shanghai Chenhua Instrument Co., Ltd.

[0058] Example 1: Construction of a system for detecting heavy metal ions Hg 2+ Electrochemical aptasensors

[0059] The electrochemical aptamer sensor provided in this embodiment is a three-electrode system sensor, wherein: the counter electrode is a platinum wire electrode, the reference electrode is a saturated calomel electrode, and the working electrode is obtained by sequentially modifying the surface of the ITO electrode with graphene aerogel and gold nanoparticle complex (GAs-AuNPs), hairpin DNA H2 and H3. The construction method of the working electrode is described in detail below.

[0060] 1. Construction of the working electrode

[0061] (1) Construction of GAs-AuNPs / ITO electrode

[0062] Before use, ITO was ultrasonically cleaned with acetone, ethanol and pure water for 10 min respectively, and then blown dry to obtain a clean ITO electrode for use. The specific steps for constructing the GAs-AuNPs electrode are as follows:

[0063] (1) Weighing graphene oxide, adding deionized water, and ultrasonicating;

[0064] (2) HAuCl 4 ·4H 2 O (1 mg / mL) solution was mixed with graphene oxide aqueous solution, and then 1% sodium citrate was added and ultrasonicated;

[0065] (3) placing the mixed solution in a 100 mL autoclave, sealing and heating, cooling naturally to room temperature, and then placing it in a low-temperature refrigerator for pre-freezing; placing the pre-frozen material in a vacuum freeze dryer for freeze drying to obtain a porous GAs-AuNPs composite material;

[0066] (4) GAs-AuNPs powder was dissolved in DMF and ultrasonicated to prepare a 1.0 mg / mL GAs-AuNPs dispersion; then, a drop of the dispersion was applied to a bare electrode and dried naturally to prepare a GAs-AuNPs / ITO electrode.

[0067] Figure 2 This is a scanning electron microscope (SEM) photograph of GAs-AuNPs prepared in this example. Figure 3 This is a transmission electron microscope (TEM) photograph of the GAs prepared in this example.

[0068] (2) Modification of GAs-AuNPs / ITO electrode

[0069] 10 μL of a mixed solution of 5 μM hairpin DNA H2 and H3 was drop-coated on the surface of the GAs-AuNPs / ITO electrode and incubated at 37°C overnight. The electrode was then washed with PBS and treated with 2 mM MCH (6-mercapto-1-hexanol) for 2 h to block the unbound active sites. After further washing with PBS, the electrode was dried with nitrogen to obtain a GAs-AuNPs / ITO electrode modified with H2 and H3, which was the working electrode.

[0070] 2. Circular amplification reaction of ExoⅢ

[0071] First, dilute the two DNA chains H1 and primer to 10 μM and 5 μM respectively, then take 10 μL of 4 U / μL ExoⅢ and add it to 10 μL reaction buffer (the reaction buffer contains 10 mM NaCl and MgCl 2 ); Then, Hg 2+ The standard solution was added to the buffer solution, and the enzyme digestion and cyclic amplification reaction was carried out at 37°C for 80 minutes to obtain a DNA chain helper mixture; the mixed solution was then treated at 80°C for 10 minutes to inactivate ExoⅢ and terminate the cyclic amplification reaction; finally, it was kept at 4°C for 30 minutes to allow the unreacted HP1 to reform the hairpin structure and reduce interference with the experiment.

[0072] Example 2: Detection of heavy metal ions Hg using electrochemical aptamer sensors 2+

[0073] The electrochemical aptamer sensor prepared in Example 1 was used to detect heavy metal ions Hg at different concentrations. 2+ (1fM-10nM) to investigate the electrochemical aptasensor for heavy metal ions Hg 2+ The specific test steps are as follows:

[0074] 1. Prepare electrolyte: PBS buffer solution with pH = 7.4.

[0075] 2. Take heavy metal ions Hg with a concentration in the range of 1fM-10nM 2+ Solution, ExoⅢ cyclic amplification reaction was carried out according to the method in Example 1 to obtain a DNA chain helper mixed solution. 10 μL of the mixed solution was drop-coated on the surface of the working electrode, incubated at 37° C. for 2 h, and then the electrode surface was washed with PBS for several times and dried with nitrogen.

[0076] 3. Take 10 μL of the mixture of DNA chains A1 and A2, drop it on the surface of the working electrode, continue to incubate at 37°C for 2 hours, then wash it with PBS several times and blow dry it with nitrogen.

[0077] 4. After the above treatment, the working electrode, platinum wire electrode (counter electrode) and saturated calomel electrode (reference electrode) were placed in the electrolyte, and differential pulse voltammetry (DPV) was used to obtain the Hg concentrations of heavy metal ions at different concentrations. 2+ The current-potential response curve of the solution ( Figure 4 ).

[0078] like Figure 5 As shown in the figure, in the range of 1fM-10nM, after deducting the background current, the change value of the peak current (Δi p =i 0 -i p ) and heavy metal ions Hg 2+ The concentration logarithm shows a good linear relationship, and the linear regression equation obtained by fitting is ΔI = 0.3743lgC Hg 2+ +1.6974, the linear correlation coefficient was 0.9932, and the detection limit was 0.16fM.

[0079] Example 3: Specificity of electrochemical aptasensor

[0080] Take the heavy metal ion Hg at a concentration of 1pM 2+ Mg 2+ , Pb 2+ , Ga 2+ 、Na + , K + 、Cd 2+ Cr 6+ The solutions were respectively detected using the electrochemical aptamer sensors prepared in Example 1 to investigate the specificity of the electrochemical aptamer sensor prepared in Example 1.

[0081] The electrolyte is the same as in Example 2. The specific test steps are:

[0082] 1. According to the same method as in Example 1, each heavy metal ion solution was subjected to ExoⅢ cyclic amplification reaction to obtain a mixed solution containing DNA chain helper.

[0083] 2. Take 10 μL of the mixed solution after the cyclic amplification reaction, drop it onto the surface of the working electrode, incubate it at 37°C for 2 h, then wash the electrode surface with PBS and blow dry it with nitrogen.

[0084] 3. Take 10 μL of the mixture of DNA chains A1 and A2, drop it on the surface of the working electrode, continue to incubate at 37°C for 2 hours, then wash it with PBS several times and blow dry it with nitrogen.

[0085] 4. After the above treatment, the working electrode, platinum wire electrode (counter electrode) and saturated calomel electrode (reference electrode) were placed in the electrolyte and detected by differential pulse voltammetry (DPV) to obtain a bar graph of the electrochemical aptamer sensor detecting different heavy metal ions. The results are as follows: Figure 6 shown.

[0086] from Figure 6 It can be seen that the electrochemical aptamer sensor is sensitive to heavy metal ions Hg 2+ The electrochemical signal was obvious, but there was no obvious change for other heavy metal ions, which indicated that the electrochemical aptasensor was sensitive to heavy metal ions Hg 2+ Has good specificity.

[0087] Example 4: Reproducibility of electrochemical aptasensor

[0088] The electrolyte was the same as in Example 2. Five different working electrodes were prepared according to the method in Example 1. 2+ The measurement was performed and the RSD of the detection result was 2.75%.

[0089] The test results in this example show that the electrochemical aptamer sensor prepared by the present invention has good reproducibility.

[0090] Example 5: Stability of electrochemical aptasensor

[0091] After the electrochemical aptasensor prepared in Example 1 was stored at 4°C for 5, 10, and 15 days, it showed good sensitivity to 1 pM heavy metal ion Hg. 2+ The test results show that the response currents are 97.6%, 94.9% and 91.1% of the original values ​​respectively.

[0092] The test results of this example show that the electrochemical aptamer sensor prepared by the present invention has good stability.

[0093] Example 6: Detection of heavy metal ions Hg in milk samples using electrochemical aptamer sensors 2+

[0094] The test sample is a commercially available milk sample. The specific test method is as follows:

[0095] 1. Before electrochemical detection, the milk sample was pretreated. First, 9 mL of acetonitrile was added to 1 mL of whole milk, the mixed milk sample was shaken for 5 min, and then centrifuged at 8000 r / min for 10 min. The obtained supernatant was filtered through a 0.45 μm microporous membrane. Finally, the obtained supernatant was dried in a vacuum oven at 25 ° C, and 1 mL of ultrapure water was added to obtain a milk extract.

[0096] 2. Add different concentrations of Hg to milk extract 2+ , the electrochemical aptamer sensor prepared in Example 1 was used for measurement.

[0097] 3. Atomic absorption spectrometry (AAS) was used to detect Hg in milk samples. 2+ Conduct testing.

[0098] The test results are shown in Table 2 below.

[0099] Table 2 Heavy metal ions Hg in milk samples 2+ Test Results

[0100]

[0101] From the results in Table 2, it can be seen that the detection results of the aptamer sensor are basically consistent with the detection results of the commercial atomic absorption spectrometry, and the relative standard deviations are 3.37%, 2.68% and 1.41%, respectively, which shows that the electrochemical aptamer sensor prepared by the present invention has good practical value.

[0102] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A quantitative detection method for heavy metal ions Hg 2+ The electrochemical aptamer sensor is characterized in that The electrochemical aptamer sensor is a three-electrode system sensor, and the working electrode of the three-electrode system sensor is obtained by sequentially modifying the surface of an ITO electrode with a complex of graphene aerogel and gold nanoparticles and hairpin DNA; wherein the hairpin DNA comprises oligonucleotide chains H2 and H3, the nucleotide sequences of the oligonucleotide chains H2 and H3 are respectively shown in SEQ ID NO:2-3, and the 5' end of the oligonucleotide chain H2 is modified with a thiol group, and the 3' end of the oligonucleotide chain H3 is modified with a thiol group.

2. The electrochemical aptamer sensor according to claim 1, characterized in that: The method for modifying the composite of graphene aerogel and gold nanoparticles on the surface of ITO electrode is as follows: A mixed solution containing chloroauric acid, graphene oxide and sodium citrate is prepared, and the mixed solution is subjected to a hydrothermal reaction; after the reaction is completed, a product is collected, and after vacuum freeze-drying, a composite of graphene aerogel and gold nanoparticles is obtained; The composite of the graphene aerogel and the gold nanoparticles is prepared into a dispersion, which is drop-coated on the surface of the ITO electrode, and dried to obtain an ITO electrode modified with the composite of the graphene aerogel and the gold nanoparticles.

3. The electrochemical aptamer sensor according to claim 2, characterized in that: In the mixed solution, the concentration of graphene oxide is 0.1-1.5 mg / mL, the concentration of chloroauric acid is 0.1-1.5 mg / mL, and the concentration of sodium citrate is 0.05-1.5 mM.

4. The electrochemical aptamer sensor according to claim 1, characterized in that: The method for modifying hairpin DNA H2 and H3 on the surface of ITO electrode is as follows: A mixed solution of H2 and H3 was prepared, and the mixed solution was drop-coated on the surface of an ITO electrode modified with a composite of graphene aerogel and gold nanoparticles. After incubation, the electrode was washed and dried; and then the electrode was treated with 6-mercapto-1-hexanol to block unbound active sites on the electrode.

5. A quantitative detection method for heavy metal ions Hg 2+ The method is characterized in that Using the electrochemical aptamer sensor according to any one of claims 1 to 4, the method comprises the following steps: a. subjecting the sample solution to be tested to ExoⅢ cyclic amplification treatment to obtain a mixed solution to be tested; b. dropping the mixed solution to be tested onto the working electrode of the electrochemical aptamer sensor to react; then, dropping a signal probe onto the working electrode to react; c. Place the working electrode, counter electrode and reference electrode in an electrolyte, and use differential pulse voltammetry to obtain the absolute value of the peak current change; then calculate the heavy metal ion Hg in the sample solution to be tested based on the standard curve. 2+ concentration.

6. The method for quantitatively detecting heavy metal ions Hg according to claim 5. 2+ The method is characterized in that In step a, the cyclic amplification process of ExoⅢ comprises the following steps: A solution containing DNA chain H1 and primer is prepared, and a sample solution to be tested and an Exo III solution are added thereto to perform a cyclic amplification reaction; then, the cyclic amplification reaction is terminated by heating, and then the temperature is lowered to allow the unreacted DNA chain H1 to reform into a hairpin structure, thereby obtaining a mixed solution containing DNA chain helper; Wherein, the nucleotide sequences of H1, primer and helper are shown in SEQ ID NO: 1, 4-5 respectively.

7. The method for quantitatively detecting heavy metal ions Hg according to claim 5. 2+ The method is characterized in that In step b, the signal probe is a mixture containing DNA chains A1 and A2, the nucleotide sequence of A1 is 5'-CATACAAA-3', and the nucleotide sequence of A2 is 5'-GACCGGGC-3'; and the 3' end of A1 and the 5' end of A2 are both labeled with methylene blue.

8. The method for quantitatively detecting heavy metal ions Hg according to claim 5. 2+ The method is characterized in that In step c, the electrolyte is PBS buffer, the counter electrode is a platinum wire electrode, and the reference electrode is a calomel electrode.

9. The method for quantitatively detecting heavy metal ions Hg according to claim 5. 2+ The method is characterized in that In step c, the method for preparing the standard curve is: i. Hg concentration in the range of 1fM-10nM 2+ The standard solution was subjected to the ExoⅢ cyclic amplification treatment; j. dropping the standard solution after cyclic amplification treatment onto the surface of the working electrode for reaction, washing and drying, dropping the signal probe to continue the reaction, and then washing and drying; k. placing the working electrode, the counter electrode and the reference electrode in an electrolyte, and using differential pulse voltammetry to obtain the absolute value of the peak current change of methylene blue; the absolute value of the peak current change of methylene blue is the dependent variable, and the heavy metal ion Hg 2+ The logarithmic value of the concentration was used as the independent variable to prepare a standard curve.

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