An electrochemical aptamer sensor for detecting Cd<2+> based on HCR reaction and application thereof 2+ An electrochemical aptamer sensor for detecting Cd<2+> based on HCR reaction and application thereof

Abstract

The application belongs to the technical field of electrochemical sensing, and particularly discloses an electrochemical aptamer sensor for detecting Cd 2+ based on HCR reaction and application thereof. The electrochemical aptamer sensor comprises a reaction sheet and a detection solution, the detection solution comprises a hairpin probe H1 solution, a hairpin probe H2 solution, an AgNO3 solution and a NaBH4 solution, and the reaction sheet comprises a PET substrate and a working electrode, a graphite counter electrode and a silver / silver chloride reference electrode fixed on the PET substrate by silk screen printing; the working electrode is an Apt modified electrode, which is prepared through gold nanoparticle deposition and Apt solution incubation reaction steps. The electrochemical aptamer sensor captures Cd 2+ by coupling Apt on the electrode surface, triggers HCR reaction amplification signal through Apt which does not capture Cd 2+ , and effectively solves the signal interference problem by detecting the LSV signal of in-situ generated silver nanoclusters, so as to realize high sensitivity, high specificity and rapid detection of Cd 2+ .

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensing technology, specifically to a method for detecting Cd based on HCR reaction. 2+ Electrochemical aptamer sensors and their applications. Background Technology

[0002] Cadmium (Cd) is a heavy metal widely found in water, air, soil, and food. It easily accumulates in the human body through ingestion, inhalation, and skin contact. With a long half-life (10-30 years), it can damage the kidneys, liver, lungs, bones, reproductive system, and embryonic development, and may even cause cancer. The World Health Organization (WHO) stipulates that the concentration of Cd in drinking water should be within a certain limit. 2+ The maximum permissible concentration is 3 ng / mL. Therefore, an effective and sensitive Cd assay can be established in complex samples. 2+ The detection method is of great significance. Compared with traditional instrumental analysis methods that are expensive, require specialized operation, and involve complex processes, rapid and sensitive on-site detection methods have a competitive advantage.

[0003] Electrochemical technology offers advantages such as high selectivity, simple operation, low cost, and suitability for on-site monitoring. This technology performs qualitative or quantitative analysis of target substances by detecting changes in electrical parameters (current, potential, resistance, etc.). (Cd) 2+ Electrochemical detection of Cd commonly uses anodic stripping voltammetry, square wave voltammetry, and differential pulse voltammetry. During the detection process, Cd... 2+ Accumulated on the electrode surface, it is reduced to Cd under constant potential. 0 Then, Cd is scanned by voltage in the anodic direction. 0 Re-oxidized to Cd 2+ , generated with Cd 2+ The strong oxidation peak current is directly proportional to the level. However, the electrochemical signals of these methods are easily interfered with when applied to real samples, affecting the detection results. Summary of the Invention

[0004] To address the aforementioned problems, one objective of this invention is to provide a method for detecting Cd based on HCl reaction. 2+ An electrochemical aptamer sensor is proposed, using a gold nanoparticle-modified electrode prepared by gold deposition as the reaction substrate, employing aptamers (Apt) for specific recognition of biomolecules, and in-situ generated silver nanoclusters as electrochemical signal tags. This electrochemical adapter can achieve the detection of Cd... 2+ It can quickly detect and resolve interference problems, with high detection sensitivity and good specificity.

[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0006] A method for detecting Cd based on HCR reaction 2+The electrochemical aptamer sensor comprises a reaction sheet and a detection liquid, the detection liquid comprises a hairpin probe H1 solution, a hairpin probe H2 solution, an AgNO3 solution and a NaBH4 solution, and the reaction sheet comprises a PET substrate and a working electrode, a graphite counter electrode and a silver / silver chloride reference electrode fixed on the PET substrate by silk screen printing;

[0007] The working electrode is an Apt modified electrode, and the Apt modified electrode is prepared by the following steps:

[0008] S11. The chloroauric acid solution and the potassium sulfate solution are mixed in equal volumes and then added dropwise to the graphite electrode, gold nanoparticles are deposited on the electrode by electrochemical CV scanning, and the gold nanoparticle modified electrode is obtained by washing and drying to improve the conductivity and specific surface area;

[0009] S12. The Apt solution is added dropwise on the gold nanoparticle modified electrode for incubation reaction, and the thiol group at one end of the thiol-modified aptamer Apt is covalently coupled with the gold nanoparticles on the surface of the gold nanoparticle modified electrode through a gold-sulfur bond; after the incubation reaction, the un-coupled Apt is removed by washing with a phosphate buffer solution, and then the electrode is immersed in a mercaptohexanol solution for soaking to block the non-specific binding sites, and then the electrode is washed with a phosphate buffer solution and dried to obtain the Apt modified electrode.

[0010] The aptamer (Apt) is a single-stranded oligonucleotide, which is synthesized in vitro through the SELEX system and is obtained by affinity test screening, and has high selectivity and affinity for heavy metal ion targets due to its specific secondary structure. HCR is an isothermal enzyme-free nucleic acid amplification technology, and the principle is that the target sequence recognizes the cross-opening of two DNA hairpins H1 and H2 to form a DNA polymer nanowire, and the reaction process includes one after another trigger DNA switch, cross-opening and hybridization to produce a long double-stranded DNA, which has a structure composed of dozens to hundreds of repeating units, and the biological structure thus produced provides the possibility of producing more signal molecules to amplify the signal.

[0011] The present application is based on the HCR reaction and the silver nanocluster signal tag to construct an electrochemical aptamer sensor composed of a reaction sheet and a detection liquid. The sample solution to be detected is added dropwise to the Apt modified electrode on the reaction sheet, and the Apt coupled on the electrode can specifically recognize Cd 2+ , so that the conformation of the Apt changes; then the hairpin probe solution is added dropwise, and the Apt not capturing Cd 2+ can trigger the HCR reaction with H1 and H2, and long double-stranded DNA is formed on the electrode surface; after adding the AgNO3 solution and the NaBH4 solution, silver nanoclusters can be generated in situ in the C-rich sequence at the end of H1 in the long double-stranded DNA, and the electrochemical LSV signal of the silver nanoclusters is determined to realize the detection of Cd 2+Apt is able to select Cd 2+ The combination has good specificity, the HCR reaction and the silver nanocluster realize amplification detection of the electrochemical signal, greatly enhance the anti-interference ability and the detection sensitivity, and effectively solve the signal interference problem of the existing electrochemical detection technology. In addition, the electrochemical aptamer sensor is portable and can be used for rapid detection on site, and is suitable for various application scenarios.

[0012] Preferably, the sequence of the Apt is shown as SEQ ID No. 3, the sequence of the hairpin probe H1 is shown as SEQ ID No. 4, and the sequence of the hairpin probe H2 is shown as SEQ ID No. 5.

[0013] Preferably, in the step S11, the concentration of the chloroauric acid solution is 1-2mM, the concentration of the potassium sulfate solution is 5-20mM, and the dropwise adding amount of the mixed solution of chloroauric acid and potassium sulfate is 50-150μL.

[0014] Preferably, in the step S11, the scan rate of the CV scan is 0.05-0.2V / s, and the scan number is 20-50.

[0015] Preferably, in the step S12, the concentration of the Apt solution is 0.8-1.5μM, and the dropwise adding amount is 10-20μL; the incubation temperature on the electrode is 20-30℃, and the incubation time is 30-60min.

[0016] Preferably, in the step S12, the concentration of the mercaptohexanol solution is 1-2mM, and the soaking blocking time is 40-60min.

[0017] The second purpose of the present application is to provide the application of the above-mentioned electrochemical aptamer sensor in the detection of Cd 2+ , specifically in the detection of Cd 2+ in tea, vegetables and other foods.

[0018] S21. The Cd 2+ standard is diluted with ultrapure water to a gradient concentration, and the standard solution with the gradient concentration is added to the surface of the Apt modified electrode, and the Apt captures the Cd 2+ through conformational change, and then incubated at room temperature and washed with a phosphate buffer;

[0019] S22. The hairpin probe H1 solution and the hairpin probe H2 solution are sequentially added to the electrode after reaction, and the Apt not capturing the Cd 2+ triggers HCR with complementarity, and then incubated at room temperature and washed with a phosphate buffer;

[0020] S23. Add AgNO3 solution, generate silver nanoclusters in situ after incubation in dark, rinse the electrode with phosphate buffer after reaction, then detect the electrochemical LSV signal of silver nanoclusters on the reaction sheet, and fit the standard curve through the LSV peak intensity value;

[0021] S24. The sample solution to be detected is reacted and the electrochemical signal is detected according to the above steps, and the content of Cd 2+ is calculated according to the standard curve.

[0022] Preferably, in step S21, the dropwise addition amount of the standard solution is 10-20 μL, and the incubation time at room temperature is 20-40 min.

[0023] Preferably, in step S22, the dropwise addition amounts of the hairpin probe H1 solution and the hairpin probe H2 solution are 5-10 μL respectively, the concentrations are 1.5-2 μM respectively, and the incubation time at room temperature is 20-40 min.

[0024] Preferably, in step S23, the dropwise addition amount of the AgNO3 solution is 5-10 μL, the concentration is 5-10 mM; the dropwise addition amount of the NaBH4 solution is 5-10 μL, the concentration is 10-20 mM; and the in-situ generation reaction time of silver nanoclusters is 30-60 min.

[0025] The present application has the following beneficial effects:

[0026] 1. The electrochemical aptamer sensor of the present application captures Cd 2+ by coupling Apt on the electrode surface, triggers HCR reaction to amplify the signal by Apt that does not capture Cd 2+ , and greatly enhances the anti-interference ability and detection sensitivity by detecting the LSV signal of in-situ generated silver nanoclusters.

[0027] 2. The electrochemical aptamer sensor of the present application specifically binds Cd 2+ among many metal ions such as Cd 2+ , Pb 6+ , Cr 3+ , As + , K 2+ , Ca + , Na 2+ , Mg 3+ , Al 2+ , and the anti-interference ability is further enhanced.

[0028] 3. The electrochemical aptamer sensor of the present application is composed of a reaction sheet and a detection solution, is simple to prepare, portable, can be used for rapid detection on site, and is suitable for various application scenarios. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Cd 2+ Preparation principle and application schematic diagram of electrochemical aptamer sensor.

[0030] Figure 2 Cyclic voltammograms of gold nanoparticle modified electrodes with different deposition cycles (the deposition cycles of Au-SPE1 to Au-SPE6 are 10, 15, 20, 25, 50 and 100, respectively) in Example 2.

[0031] Figure 3 Impedance diagrams of gold nanoparticle modified electrodes with different deposition cycles (the deposition cycles of Au-SPE1 to Au-SPE6 are 10, 15, 20, 25, 50 and 100, respectively) in Example 2.

[0032] Figure 4 Scanning electron microscope diagram of gold nanoparticle modified electrode in Example 2.

[0033] Figure 5 Chronoamperogram of Apt modified electrode in Example 2.

[0034] Figure 6 Cd 2+ LSV diagram of standard solution.

[0035] Figure 7 Standard curve fitting diagram of Cd 2+ in Example 2. DETAILED DESCRIPTION

[0036] The application will be further described below in combination with the drawings and specific examples.

[0037] The principle diagram of the electrochemical aptamer sensor for detecting cadmium ions based on HCR reaction and application of the application is shown in Figure 1 A, specifically, a gold nanoparticle modified screen-printed electrode is prepared by gold deposition method, and a thiol-modified Cd 2+ aptamer Apt is coupled to the electrode surface through gold-sulfur bond covalent interaction; the Apt can specifically recognize Cd 2+ , so that the aptamer undergoes conformational change, and the Apt not capturing Cd 2+ can trigger a chain hybridization reaction (HCR) with H1 and H2 complementary trigger strands, forming long double-stranded DNA on the electrode surface; the C-rich sequence at the end of H1 can generate silver nanoclusters in situ, realizing signal amplification, and the detection of Cd 2+ is realized by measuring the change of silver nanocluster electrochemical LSV signal.

[0038] Example 1

[0039] This example is a screening experiment for Apt. First, three Apt samples with different sequences were selected, and then compared with Cd. 2+ Affinity testing was performed to determine the dissociation constant Kd. The specific Apt sequence and affinity test results are shown in the table below:

[0040]

[0041]

[0042] Finally, Apt selected SEQ ID No. 3 with the smallest Kd value based on the affinity test results. Then, based on the selected Apt sequence, hairpin probes H1 and H2 were designed. The sequence of hairpin probe H1 is shown in SEQ ID No. 4, and the sequence of hairpin probe H2 is shown in SEQ ID No. 5.

[0043]

[0044] Example 2

[0045] This embodiment utilizes HCR-based detection of Cd. 2+ Electrochemical aptamer sensor for detecting Cd in tea 2+ It includes the following steps:

[0046] S1. Aptamer sensor fabrication

[0047] The aptamer sensor includes a reaction plate and a detection liquid. The detection liquid includes a hairpin probe H1 solution, a hairpin probe H2 solution, an AgNO3 solution, and a NaBH4 solution. The reaction plate is as follows: Figure 1 As shown in Figure B, the device includes a PET substrate and a working electrode, a graphite counter electrode, and a silver / silver chloride reference electrode that are fixed on the PET substrate by screen printing; the working electrode is an Apt-modified electrode.

[0048] The Apt-modified electrode is prepared according to the following steps:

[0049] S11. Mix equal volumes of 1 mM chloroauric acid solution and 10 mM potassium sulfate solution, and add 100 μL to a graphite electrode with a diameter of 5 mm. Deposit gold nanoparticles by electrochemical CV scanning for 25 cycles at a scan rate of 0.1 V / s. Rinse and dry to obtain a gold nanoparticle modified electrode.

[0050] S12. 15 μL of the Apt solution (1 μM) was dropped on the gold nanoparticle modified electrode; after incubation at 25 °C for 30 min, the un-coupled Apts were removed by washing with phosphate buffer, and then the electrode was immersed in 2 mM mercaptohexanol solution for 60 min to block the non-specific binding sites, followed by washing with phosphate buffer and air-drying, to obtain the Apt modified electrode, which was stored in low temperature and away from light.

[0051] The scanning electron microscope (SEM) image of the gold nanoparticle modified electrode obtained in step S11 is shown in Fig. 2. Figure 4 As can be seen from Fig. 2, the electrode surface after gold deposition has a layer of gold nanoparticles uniformly distributed, proving the effective modification of gold nanoparticles. Figure 5 As can be seen from Fig. 3, the chronocoulometry of the Apt modified electrode has changed significantly, proving that the Apts have been successfully coupled to the electrode surface.

[0052] During the preparation of the Apt modified electrode, the present embodiment also performed electrochemical tests on the gold nanoparticle modified electrode under different deposition cycles in step S11, as shown in Fig. 4. Figures 2-3 As can be seen from Fig. 4, the gold nanoparticle modified electrode has a pair of clear reversible redox peaks, which belong to the oxidation and reduction of Fe(CN)6 3- and Fe(CN)6 4- respectively. The oxidation peak first increases and then decreases with the increase of the deposition cycle number, and the performance is best when the deposition cycle number is 25. With the increase of the deposition cycle number from 10 to 25, the electrode charge transfer resistance (Rct) value decreases from 62.4 Ω to 22.0 Ω. This is because the electron transfer of Fe 3+ / Fe 2+ is significantly accelerated due to the high electron conductivity of gold nanoparticles.

[0053] S2. Detection of the content of Cd 2+ in tea

[0054] S21. The Cd 2+ standard was diluted with ultrapure water to gradient concentrations (the concentration of the Cd 2+ solution was 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL), and 15 μL of the standard solution of gradient concentration was dropped on the surface of the Apt modified electrode, and the Apt captured the Cd 2+ by conformational change, and after incubation at room temperature for 30 min, the electrode was washed with phosphate buffer;

[0055] S22. 10 μL of the hairpin probe H1 solution (1.5 μM) and 10 μL of the hairpin probe H2 solution (1.5 μM) were sequentially dropped on the reacted electrode, and the Cd 2+Apt complementary trigger HCR, after incubation at room temperature for 30 min, wash with phosphate buffer solution;

[0056] S23. Add 10 μL AgNO3 solution (5 mM), avoid light incubation after adding 10 μL NaBH4 solution (10 mM) avoid light reaction 30 min, in situ generation of silver nanoclusters; after the reaction, the electrode is washed with phosphate buffer, and the electrochemical LSV signal of silver nanoclusters on the reaction sheet is detected, and the standard curve is fitted by LSV peak intensity value; electrochemical LSV signal and standard curve Figures 6-7 As shown in Fig. 5, it can be seen that when the concentration of Cd2+ increases from 0 ng / mL to 100 ng / mL, the LSV characteristic peak of Ag NCs becomes weak, the linear range obtained by fitting is 0.1-100 ng / mL, and the detection limit is 88 pg / mL;

[0057] S24. Add 3 mL nitric acid and 1 mL hydrogen peroxide (30%) to 0.5 g of crushed tea powder, then heat to 120℃ in a graphite digestion furnace for 90 min; after digestion, open the lid and heat to chase acid at 120℃, repeat three times, then dilute to 25 mL with water, filter the impurities and use as the sample solution to be tested; the sample solution to be tested is reacted and the electrochemical signal is detected according to the above steps, and the content of Cd 2+ is calculated according to the standard curve.

[0058] Example 3

[0059] This example uses an electrochemical aptamer sensor based on HCR reaction to detect Cd 2+ in tea, including the following steps: 2+

[0060] S1. Preparation of aptamer sensor

[0061] The aptamer sensor is basically composed of the same as in Example 2, except that the preparation parameters of the Apt modified electrode are different, which are as follows:

[0062] S11. Mix equal volumes of 1.5 mM chloroauric acid solution and 15 mM potassium sulfate solution, and take 50 μL and drop onto a graphite electrode with a diameter of 5 mm; deposit gold nanoparticles by electrochemical CV scanning for 25 cycles at a scanning rate of 0.05 V / s, and wash and dry to obtain a gold nanoparticle modified electrode;

[0063] ​S12. Add 15 μL of Apt solution (0.8 μM) to the gold nanoparticle modified electrode; after incubation at 20 °C for 40 min, rinse with phosphate buffer to remove uncoupled Apt, then immerse the electrode in 1.5 mM mercaptohexanol solution for 40 min to block non-specific binding sites, then rinse with phosphate buffer and air dry to obtain the Apt modified electrode, which is stored at low temperature and protected from light.

[0064] S2. Detection of Cd in tea leaves 2+ content

[0065] S21. Place Cd 2+ The standard was diluted with ultrapure water to a gradient concentration (Cd). 2+ The solutions had concentrations of 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL. 10 μL of these graded standard solutions were added dropwise to the surface of the Apt-modified electrode. Apt captured Cd through a conformational change. 2+ After incubating at room temperature for 20 minutes, wash with phosphate buffer.

[0066] 2.5 μL of hairpin probe H1 solution (2 μM) and 5 μL of hairpin probe H2 solution (2 μM) were sequentially added to the electrode after the reaction, and reacted with the uncaptured Cd. 2+ Apt complementary triggering of HCR was incubated at room temperature for 20 min and then washed with phosphate buffer.

[0067] S23. Add 5 μL AgNO3 solution (10 mM), incubate in the dark, then add 5 μL NaBH4 solution (20 mM) and react in the dark for 40 min to generate silver nanoclusters in situ. After the reaction, rinse the electrode with phosphate buffer and then detect the electrochemical LSV signal of the silver nanoclusters on the reaction sheet. Fit the standard curve by the LSV peak intensity value.

[0068] S24. React the same sample solution as in Example 2 according to the above steps and detect the electrochemical signal. Calculate Cd based on the standard curve. 2+ The content of [unspecified substance] was basically consistent with that in Example 2.

[0069] Example 4

[0070] This embodiment utilizes HCR-based detection of Cd. 2+ Electrochemical aptamer sensor for detecting Cd in tea 2+ It includes the following steps:

[0071] S1. Aptamer sensor fabrication

[0072] The aptamer sensor is basically the same as in Example 2, except that the preparation parameters of the Apt modified electrode are different, as follows:

[0073] S11. Mix equal volumes of 1.5 mM chloroauric acid solution and 15 mM potassium sulfate solution, and drop 120 μL onto a graphite electrode with a diameter of 5 mm; deposit gold nanoparticles by electrochemical CV scanning for 25 cycles at a scanning rate of 0.2 V / s, and then rinse and dry to obtain a gold nanoparticle modified electrode;

[0074] S12. Drop 15 μL of Apt solution (1.5 μM) onto the gold nanoparticle modified electrode; after incubation at 30°C for 60 min, rinse with phosphate buffer to remove uncoupled Apt, and then immerse the electrode in 1 mM mercaptohexanol solution for 60 min to block non-specific binding sites, and then rinse with phosphate buffer and dry to obtain an Apt modified electrode, which is stored at low temperature and away from light.

[0075] S2. Detect the content of Cd 2+ in tea

[0076] S21. Dilute the Cd 2+ standard to gradient concentrations (the concentrations of Cd 2+ solutions are 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL) with ultrapure water, and drop 20 μL of the standard solution with gradient concentrations onto the surface of the Apt modified electrode; the Apt captures Cd 2+ by conformational change, and after incubation at room temperature for 40 min, rinse with phosphate buffer;

[0077] S22. Drop 8 μL of hairpin probe H1 solution (1.8 μM) and 8 μL of hairpin probe H2 solution (1.8 μM) onto the reacted electrode in sequence, and trigger HCR with the Apt that does not capture Cd 2+ , and after incubation at room temperature for 40 min, rinse with phosphate buffer;

[0078] S23. Add 8 μL of AgNO3 solution (8 mM), and after incubation in the dark, add 8 μL of NaBH4 solution (15 mM) and react in the dark for 40 min to generate silver nanoclusters in situ; after the reaction, rinse the electrode with phosphate buffer, and then detect the electrochemical LSV signal of the silver nanoclusters on the reaction sheet, and fit the standard curve by the LSV peak intensity value;

[0079] S24. React and detect the electrochemical signal of the same sample solution to be tested as in Example 2 according to the above steps, and calculate the content of Cd 2+The content of [unspecified substance] was basically consistent with that in Example 2.

[0080] Example 5

[0081] This embodiment utilizes HCR-based detection of Cd. 2+ Electrochemical aptamer sensor for detecting Cd in vegetables 2+ It includes the following steps:

[0082] S1. Aptamer sensor fabrication

[0083] The composition of the aptamer sensor is basically the same as in Example 2, except that the preparation parameters of the Apt-modified electrode are different, as follows:

[0084] S11. Mix equal volumes of 2 mM chloroauric acid solution and 20 mM potassium sulfate solution, and add 150 μL to a graphite electrode with a diameter of 5 mm. Deposit gold nanoparticles by electrochemical CV scanning for 25 cycles at a scan rate of 0.1 V / s. Rinse and dry to obtain a gold nanoparticle modified electrode.

[0085] S12. Add 20 μL of Apt solution (1.5 μM) to the gold nanoparticle modified electrode; after incubation at 25 °C for 50 min, rinse with phosphate buffer to remove uncoupled Apt, then immerse the electrode in 2 mM mercaptohexanol solution for 40 min to block non-specific binding sites, then rinse with phosphate buffer and air dry to obtain the Apt modified electrode, which is stored at low temperature and protected from light.

[0086] S2. Detection of Cd in Shanghai Qingzhong 2+ content

[0087] S21. Place Cd 2+ The standard was diluted with ultrapure water to a gradient concentration (Cd). 2+ The concentrations of the solutions were 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL. 20 μL of the standard solutions with these gradient concentrations were added dropwise to the surface of the Apt-modified electrode. Apt captured Cd through a conformational change. 2+ After incubating at room temperature for 20 minutes, wash with phosphate buffer.

[0088] 2.5 μL of hairpin probe H1 solution (2 μM) and 5 μL of hairpin probe H2 solution (20 μM) were sequentially added to the electrode after the reaction, and reacted with the uncaptured Cd. 2+ Apt complementary triggering of HCR was incubated at room temperature for 40 min and then washed with phosphate buffer.

[0089] S23. Add 5 μL AgNO3 solution (10 mM), after incubation in the dark, add 5 μL NaBH4 solution (20 mM) to react in the dark for 50 min, in situ to generate silver nanoclusters; after the reaction is completed, the electrode is washed with phosphate buffer, and the electrochemical LSV signal of silver nanoclusters on the reaction sheet is detected, and the standard curve is fitted by the LSV peak intensity value;

[0090] S24. Add 3 mL nitric acid and 1 mL hydrogen peroxide (30%) to 0.5 g of Shanghai green powder, then heat to 120°C in a graphite digestion furnace for 90 min; after digestion is completed, open the lid and heat to chase acid at 120°C, repeat three times, then dilute to 25 mL with water, filter out impurities, and use as a sample solution to be tested; the sample solution to be tested is reacted and the electrochemical signal is detected according to the above steps, and the content of Cd 2+ is calculated according to the standard curve.

[0091] Example 6

[0092] In this example, solutions of Cd 2+ and 500 ng / mL of Pb 2+ , Cr 6+ , As 3+ , K + , Ca 2+ , Na + , Mg 2+ , Al 3+ metal ions with a concentration of 10 ng / mL were prepared, and the selectivity and anti-interference ability of the HCR electrochemical sensor were evaluated. The sample solution to be tested was added to the sensor according to the above steps, and the electrochemical signal was detected. Compared with the positive group with a concentration of 10 ng / mL of Cd 2+ , the negative group had no significant effect on the LSV signal. Therefore, the designed sensor platform has good specificity for Cd 2+ .

[0093] This specific embodiment is only an explanation of the present application and is not a limitation of the present application. Any changes made by those skilled in the art after reading the specification of the present application will be protected by the patent law as long as it is within the scope of the claims of the present application.

Claims

1. An electrochemical aptamer sensor for detecting Cd2+ based on HCR reaction, characterized in that: 2+ The reaction sheet and the detection solution are included; ​ The detection solution includes a hairpin probe H1 solution, a hairpin probe H2 solution, an AgNO3 solution and a NaBH4 solution; the sequence of the hairpin probe H1 is shown as SEQ ID No. 4, and the sequence of the hairpin probe H2 is shown as SEQ ID No. 5; The reaction sheet includes a PET substrate and a working electrode, a graphite counter electrode and a silver / silver chloride reference electrode fixed on the PET substrate by silk screen printing; the working electrode is an aptamer modified electrode, and the aptamer modified electrode is prepared by the following steps: S11. Mix equal volumes of chloroauric acid solution and potassium sulfate solution, and then drop them onto the graphite electrode; gold nanoparticles are deposited by electrochemical CV scanning; the gold nanoparticle modified electrode is obtained by washing and air-drying; S12. Drop the aptamer solution onto the gold nanoparticle modified electrode; the aptamer is coupled with the gold nanoparticles by incubation; the sequence of the aptamer is shown as SEQ ID No. 3; after the incubation reaction, the un-coupled aptamer is removed by washing with a phosphate buffer solution; then the electrode is immersed in a mercaptohexanol solution for soaking to block the non-specific binding sites; then the electrode is washed with a phosphate buffer solution and air-dried to obtain the aptamer modified electrode.

2. The HCR reaction based detection of Cd 2+ electrochemical aptamer sensor of claim 1, characterized in that: In step S11, the concentration of the chloroauric acid solution is 1-2 mM, the concentration of the potassium sulfate solution is 5-20 mM, and the dropwise amount of the mixed solution of chloroauric acid and potassium sulfate is 50-150 μL.

3. The HCR reaction based detection of Cd according to claim 1 2+ An electrochemical aptamer sensor for the detection of Cd2+ characterized in that: In step S11, the scanning rate of the CV scanning is 0.05-0.2 V / s, and the scanning number is 20-50.

4. The HCR reaction based detection of Cd according to claim 1 2+ An electrochemical aptamer sensor for the detection of Cd2+ characterized in that: In step S12, the concentration of the aptamer solution is 0.8-1.5 μM, and the dropwise amount is 10-20 μL; the incubation temperature on the electrode is 20-30°C, and the incubation time is 30-60 min.

5. The HCR reaction based detection of Cd2+ according to claim 1 2+ An electrochemical aptamer sensor for detecting Cd2+ is characterized by: In step S12, the concentration of the mercaptohexanol solution is 1-2 mM, and the soaking blocking time is 40-60 min.

6. Use of the electrochemical aptamer sensor according to any one of claims 1 to 5 for the detection of Cd 2+ characterized in that The method is as follows: S21. Cd 2+ The standard was diluted with ultrapure water to a gradient concentration, and the standard solution of the gradient concentration was added dropwise to the surface of the aptamer modified electrode. The aptamer captured Cd 2+ After incubation at room temperature, wash with phosphate buffer; S22. The hairpin probe H1 solution and hairpin probe H2 solution are added to the reacted electrode in turn, and the aptamer complementary to the un-captured Cd 2+ trigger HCR, and after incubation at room temperature, wash with phosphate buffer; S23. Add the AgNO3 solution, incubate in the dark, then add the NaBH4 solution to generate silver nanoclusters in situ, wash the electrode with a phosphate buffer solution after the reaction is completed, then detect the electrochemical LSV signal of the silver nanoclusters on the reaction sheet, and fit the standard curve by the LSV peak intensity value; S24. The sample solution to be tested is reacted according to the above steps and the electrochemical signal is detected, and the content of Cd is calculated according to the standard curve. 2+ S24. The sample solution to be tested is reacted according to the above steps and the electrochemical signal is detected, and the content of Cd is calculated according to the standard curve.

7. Use according to claim 6, characterized in that: In step S21, the dropwise amount of the standard solution is 10-20 μL, and the incubation time at room temperature is 20-40 min.

8. Use according to claim 6, characterized in that: In step S22, the dropwise amounts of the hairpin probe H1 solution and the hairpin probe H2 solution are 5-10 μL respectively, and the concentrations are 1.5-2 μM respectively; the incubation time at room temperature is 20-40 min.

9. Use according to claim 6, characterized in that: In step S23, the dropwise amount of the AgNO3 solution is 5-10 μL, and the concentration is 5-10 mM; the dropwise amount of the NaBH4 solution is 5-10 μL, and the concentration is 10-20 mM; the in-situ generation reaction time of the silver nanoclusters is 30-60 min.

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