An electrochemical aptamer sensor for simultaneous detection of Cd 2+ and Hg 2+ and its preparation method and application

Abstract

The application discloses an electrochemical aptamer sensor for simultaneously detecting Cd 2+ and Hg 2+ as well as a preparation method and application thereof. The electrochemical aptamer sensor comprises a multi-walled carbon nanotube and gold nanoparticle composite modified silk screen printing electrode, methylene blue-labeled MB-Apt is coupled to the electrode surface through gold-sulfur bond covalent action, and Cd 2+ detection is realized through MB electrochemical signal change; ssDNA and MB-Apt jointly capture Hg 2+ , silver nanoclusters are generated in-situ in the C-rich sequence of the ssDNA, and Hg 2+ detection is realized through electrochemical signal change of the silver nanoclusters; standard solutions with different concentrations are prepared by diluting Cd 2+ and Hg 2+ standard stock solutions, a standard curve of concentration and electrochemical signal is determined; after being pretreated, actual samples to be measured are added to the electrochemical biosensor, electrochemical signals are determined, and the concentration of the measured substance is calculated according to a linear model, so that specific, sensitive and rapid detection of the two heavy metal ions can be realized.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensing technology, specifically relating to a method for simultaneously detecting Cd. 2+ and Hg 2+ Electrochemical aptamer sensors, their preparation methods, and applications, specifically the detection of Cd in food using electrochemical aptamer sensors based on MB and silver nanocluster signals. 2+ and Hg 2+ Applications. Background Technology

[0002] Heavy metals accumulate in human organs through atmospheric circulation or the food chain, leading to chronic poisoning. Cadmium and mercury are typical heavy metals. Cadmium has a long half-life (10-30 years) and easily causes serious damage to multiple organs by disrupting gut microbiota, inducing intestinal inflammation, and causing cell damage. Mercury is one of the most common heavy metal contaminants in food and can cause irreversible damage to the central nervous system, leading to degenerative diseases. It is noteworthy that multiple heavy metal ions often coexist in food and biological systems; therefore, there is an urgent need to develop methods for the simultaneous quantitative detection of two or more heavy metal ions. Traditional instrumental methods for heavy metal detection include atomic absorption spectrometry, atomic fluorescence spectrometry, X-ray fluorescence spectrometry, and inductively coupled plasma mass spectrometry. These methods are complex to operate, costly, and cannot meet the requirements for real-time detection. In contrast, biosensors offer advantages such as low cost, high sensitivity, and rapid screening.

[0003] Electrochemical technology, with its mature theoretical foundation, simple operation, high sensitivity, and rapid detection process, has been widely used in biosensor analysis. Electrochemical biosensors combine the low detection limits of electrochemical sensors with the high specificity of biorecognition processes, offering advantages such as fast response, high sensitivity, ease of operation, and miniaturization. Multi-walled carbon nanotube and gold nanoparticle composites possess high mechanical strength and conductivity, effectively improving electrode conductivity and specific surface area. Methylene blue can provide electrochemical signals through redox reactions, while silver nanoclusters, due to their low toxicity, high biocompatibility, and excellent stability, can serve as a novel electrochemical signal probe with broad application prospects in electrochemical analysis. Currently, no electrochemical aptamer sensors based on methylene blue and silver nanocluster signal tags have been observed for the simultaneous detection of Cd. 2+ and Hg 2+ Related research. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first aspect of this invention provides a method for simultaneously detecting Cd. 2+ and Hg 2+A method for fabricating an electrochemical aptamer sensor was proposed, using methylene blue and in-situ generated silver nanoclusters as electrochemical signal tags, aptamers as specific biomolecule recognition agents, and a screen-printed electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite as the reaction substrate to construct a sensor for simultaneous detection of Cd. 2+ and Hg 2+ Electrochemical aptamer sensor.

[0005] A second aspect of the present invention provides a method for simultaneously detecting Cd. 2+ and Hg 2+ The electrochemical aptamer sensor was obtained by the above preparation method.

[0006] The third aspect of the present invention provides the above-described simultaneous detection of Cd. 2+ and Hg 2+ Applications of electrochemical aptamer sensors.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a method for simultaneously detecting Cd 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor includes the following steps:

[0009] (1) During the detection process, the biochemical reaction is carried out on the screen-printed electrode. The electrode is based on polyethylene terephthalate (PET). The working electrode, counter electrode and reference electrode are fixed on the substrate by screen printing. The working electrode and counter electrode are composed of graphite and polyacrylic acid derivatives, and the reference electrode is composed of silver / silver chloride paste.

[0010] (2) Polyethyleneimine-assisted dissolution and reduction to prepare multi-walled carbon nanotube and gold nanoparticle composites;

[0011] (3) The multi-walled carbon nanotube and gold nanoparticle composite was prepared to modify the screen-printed electrode to improve the conductivity and specific surface area, thus obtaining the multi-walled carbon nanotube and gold nanoparticle composite modified electrode.

[0012] (4) Methylene blue labeled Cd 2+ The thiol group at one end of the aptamer (MB-Apt) is covalently coupled to gold nanoparticles on the electrode surface via gold-sulfur bonds to obtain an MB-Apt modified electrode.

[0013] (5) The analyte and ssDNA were simultaneously added to the MB-Apt modified electrode, and silver nanoclusters were generated in situ at the C-rich sequence end of the ssDNA. The electrochemical signals of MB and silver nanoclusters were measured to achieve the control of Cd. 2+ and Hg 2+ The detection.

[0014] In some embodiments, in step (1), the working electrode diameter of the screen-printed electrode is 3.5 mm.

[0015] In some embodiments, in step (2), the preparation method of the multi-walled carbon nanotube and gold nanoparticle composite includes: dispersing multi-walled carbon nanotubes and polyethyleneimine in an aqueous solution, ultrasonically treating them to obtain a multi-walled carbon nanotube dispersion; adding chloroauric acid to the system, and reducing chloroauric acid to gold nanoparticles with the assistance of polyethyleneimine under water bath heating conditions; cooling to room temperature, centrifuging and washing, removing the supernatant, concentrating, and storing at low temperature in the dark.

[0016] In some embodiments, in step (2), the mass ratio of the multi-walled carbon nanotubes and polyethyleneimine dispersed in the aqueous solution is 1:20-25, preferably 1:20.

[0017] In some embodiments, the ultrasonic treatment time in step (2) is 20-40 min, preferably 30 min.

[0018] In some embodiments, in step (2), the mass ratio of chloroauric acid to multi-walled carbon nanotubes is 10-15:1, preferably 10:1.

[0019] In some embodiments, in step (2), the water bath heating temperature is 60-80℃, preferably 70℃; the heating time is 100-150min, preferably 120min.

[0020] In some embodiments, in step (2), the centrifugation speed is 8000-10000 rpm, preferably 8000 rpm; the centrifugation time is 10-15 min, preferably 15 min.

[0021] In some embodiments, in step (2), the concentration after concentration is 1.5-2 mg / mL, preferably 2 mg / mL.

[0022] In some embodiments, step (3) involves preparing the electrode modified by the multi-walled carbon nanotube and gold nanoparticle composite by: adding diluted multi-walled carbon nanotube and gold nanoparticle composite onto the electrode to completely cover the working electrode, and then drying it.

[0023] In some embodiments, in step (3), the amount of multi-walled carbon nanotube and gold nanoparticle composite added to the electrode is 10-20 μL, preferably 10 μL.

[0024] In some embodiments, in step (3), the concentration of the multi-walled carbon nanotube and gold nanoparticle composite after dilution is 20-50 ng / mL, preferably 50 ng / mL.

[0025] In some embodiments, the drying temperature in step (3) is 30-50°C, preferably 40°C.

[0026] In some embodiments, in step (4), the preparation method of the MB-Apt modified electrode includes: adding MB-Apt to the working electrode, incubating the reaction, rinsing with phosphate buffer to remove uncoupled MB-Apt, then immersing the electrode in mercaptohexanol solution to block non-specific binding sites, then rinsing with phosphate buffer and drying to obtain the MB-Apt modified electrode, which is then stored at low temperature and protected from light.

[0027] In some embodiments, in step (4), the concentration of MB-Apt is 0.8-1.2 μM, preferably 1 μM; the amount added is 10-20 μL, preferably 10 μL.

[0028] In some embodiments, in step (4), the incubation temperature of MB-Apt on the electrode is 20-30°C, preferably 25°C; the incubation time is 30-60 min, preferably 40 min.

[0029] In some embodiments, in step (4), the concentration of mercaptohexanol solution is 1-2 mM, preferably 2 mM; the soaking and sealing time is 40-60 min, preferably 60 min.

[0030] In some embodiments, in step (5), Cd 2+ and Hg 2+ The detection method includes: sequentially adding the test solution and ssDNA to the surface of an MB-Apt modified electrode, incubating at room temperature, washing with phosphate buffer, then adding AgNO3, incubating in the dark, and then adding NaBH4 solution to generate silver nanoclusters in situ. After the reaction, the electrode is rinsed with phosphate buffer, and the signals of MB and silver nanoclusters are measured using the electrochemical DPV method, corresponding to Cd. 2+ and Hg 2+ .

[0031] In some embodiments, in step (5), the amount of the test solution added is 5-10 μL, preferably 5 μL.

[0032] In some embodiments, in step (5), the amount of ssDNA added is 5-10 μL, preferably 5 μL; the concentration is 0.5-1 μM, preferably 0.8 μM.

[0033] In some embodiments, in step (5), the incubation time of the test solution and ssDNA on the electrode is 30-60 min, preferably 30 min.

[0034] In some embodiments, in step (5), the amount of AgNO3 added is 5-10 μL, preferably 5 μL; the concentration is 5-8 mM, preferably 5 mM.

[0035] In some embodiments, in step (5), the amount of NaBH4 solution added is 5-10 μL, preferably 5 μL; the concentration is 10-20 mM, preferably 20 mM.

[0036] In some embodiments, in step (5), the in-situ generation reaction time of silver nanoclusters is 30-60 min, preferably 60 min.

[0037] A second aspect of the present invention provides a method for simultaneously detecting Cd. 2+ and Hg 2+ An electrochemical aptamer sensor, which simultaneously detects Cd as described above. 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor was obtained.

[0038] A third aspect of the present invention provides a method for simultaneously detecting Cd as described above. 2+ and Hg 2+ Electrochemical aptamer sensors for detecting Cd heavy metal ions in food 2+ and Hg 2+ Applications in [the field].

[0039] In some embodiments, heavy metal ions Cd are detected in tea and vegetables. 2+ and Hg 2+ The method, through the simultaneous detection of Cd 2+ and Hg 2+ The realization of the electrochemical aptamer sensor includes the following steps:

[0040] (a) Preparation of Cd at different concentration gradients 2+ and Hg 2+ Standard solution, dropped simultaneously to detect Cd 2+ and Hg 2+ On an electrochemical aptamer sensor, the intensity of its electrochemical characteristic peak signal was measured, and a standard curve was fitted.

[0041] (b) After pretreatment of tea leaves and vegetables, add them dropwise to the solution while simultaneously detecting Cd. 2+ and Hg 2+ On an electrochemical aptamer sensor, Cd was determined according to a linear model. 2+ and Hg 2+ The concentration.

[0042] In some embodiments, in step (a), Cd 2+ and Hg 2+ The standard curve fitting method: Cd2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations, and the solutions of different concentrations were added dropwise to the solution for simultaneous Cd detection. 2+ and Hg 2+ On an electrochemical aptamer sensor, the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the peak intensity values ​​of DPV.

[0043] In some embodiments, in step (a), Cd 2+ The solutions were available at concentrations of 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL.

[0044] In some embodiments, in step (a), Hg 2+ The solutions were available at concentrations of 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL.

[0045] In some embodiments, in step (b), nitric acid and hydrogen peroxide are added to the pulverized tea powder and vegetables, and then digested in a graphite digestion furnace; after digestion, the solution is diluted with water, filtered to remove impurities, and used as the solution for detection, which is then added dropwise to the solution for simultaneous Cd detection. 2+ and Hg 2+ Electrochemical signals are detected using an electrochemical aptamer sensor, and Cd in tea and vegetables is calculated based on a standard curve. 2+ and Hg 2+ The content of.

[0046] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0047] I. The multi-walled carbon nanotube and gold nanoparticle composite modified screen-printed electrodes prepared in this invention, wherein MB-Apt can covalently bind with the multi-walled carbon nanotube and gold nanoparticle composite through gold-sulfur bonds, and MB-Apt can specifically recognize Cd. 2+ This leads to a conformational change in the aptamer, and the change in the MB electrochemical signal can be used to control Cd. 2+ The detection of Hg; 2+ When present, ssDNA and MB-Apt can communicate via Hg. 2+ Bridging to form a T-Hg-T structure to achieve Hg 2+ The detection of Hg was achieved by in-situ generation of silver nanoclusters in c-rich ssDNA sequences and the change in electrochemical signal of the silver nanoclusters.2+ The detection.

[0048] II. The multi-walled carbon nanotube and gold nanoparticle composite of this invention exhibits excellent electrical conductivity, which can effectively improve the conductivity and specific surface area of ​​screen-printed electrodes; MB-Apt captures Cd 2+ At this time, the aptamer undergoes a conformational change, which leads to a change in the electrochemical signal of MB, realizing Cd 2+ Quantification; ssDNA is only found in Hg 2+ In the presence of Hg, it will be fixed on the electrode surface by forming a T-Hg-T structure with MB-Apt. The electrochemical signal generated by the silver nanoclusters generated in situ from the C-rich sequence in ssDNA can effectively affect the Hg content. 2+ Quantitative detection is performed; the electrochemical aptamer sensor of this invention can realize Cd by changes in the electrochemical signals of MB and silver nanoclusters. 2+ and Hg 2+ Simultaneous detection has the advantages of simple preparation, portability, and speed.

[0049] Third, this invention provides a novel method for preparing electrochemical biosensors with high sensitivity, high specificity, and simultaneous detection of multiple heavy metals. The method is novel and simple, and can be applied in the food testing industry; for example, it can rapidly detect Cd in tea and vegetable samples. 2+ and Hg 2+ Its content has significant practical application value. Attached Figure Description

[0050] Figure 1 For the simultaneous detection of Cd in this invention 2+ and Hg 2+ A schematic diagram illustrating the fabrication principle of the electrochemical aptamer sensor.

[0051] Figure 2 This is a transmission electron microscope (TEM) image of the multi-walled carbon nanotube and gold nanoparticle composite in Example 1.

[0052] Figure 3 The image shows cyclic voltammetry diagrams of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites at different concentrations in Example 1.

[0053] Figure 4 Impedance diagrams of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites at different concentrations in Example 1.

[0054] Figure 5 The image shows the chronocoulogram of the MB-Apt modified electrode in Example 1.

[0055] Figure 6 For the detection of Cd in Example 1 2+ DPV diagram.

[0056] Figure 7 For the detection of Cd in Example 1 2+ The standard curve fitting plot.

[0057] Figure 8 For the detection of Hg in Example 1 2+ DPV diagram.

[0058] Figure 9 For the detection of Hg in Example 1 2+ The standard curve fitting plot. Detailed Implementation

[0059] The present invention will be further described below with reference to the embodiments.

[0060] like Figure 1 As shown, this invention simultaneously detects Cd. 2+ and Hg 2+ The preparation method of the electrochemical aptamer sensor includes the following steps:

[0061] (1) The biochemical reactions during the detection process are mainly carried out on the screen-printed electrodes. The electrodes are fixed on the substrate of PET, and the working electrode, counter electrode and reference electrode are fixed on the substrate by screen printing technology. The working electrode and counter electrode are composed of graphite and polyacrylic acid derivatives, and the reference electrode is composed of silver / silver chloride paste.

[0062] (2) Polyethyleneimine-assisted dissolution and reduction to prepare multi-walled carbon nanotube and gold nanoparticle composites;

[0063] (3) Multi-walled carbon nanotubes and gold nanoparticle composites are used to modify screen-printed electrodes to improve conductivity and specific surface area.

[0064] (4) Methylene blue labeled Cd 2+ The thiol group at one end of MB-Apt is covalently coupled to gold nanoparticles on the electrode surface through gold-sulfur bonds to obtain an MB-Apt modified electrode.

[0065] (5) The analyte and ssDNA were simultaneously added to the MB-Apt modified electrode, and silver nanoclusters were generated in situ at the C-rich sequence end of the ssDNA. The electrochemical signals of MB and silver nanoclusters were measured to achieve the control of Cd. 2+ and Hg 2+ The detection.

[0066] Example 1:

[0067] This embodiment utilizes simultaneous detection of Cd 2+ and Hg 2+ Electrochemical aptamer sensor for detecting Cd in tea 2+ and Hg2+ The process includes the following steps:

[0068] (1) Preparation of multi-walled carbon nanotube and gold nanoparticle composites:

[0069] Multi-walled carbon nanotubes and polyethyleneimine were dispersed in an aqueous solution at a mass ratio of 1:20. After ultrasonic treatment for 30 min, a dispersion of multi-walled carbon nanotubes was obtained. Chloroauric acid was added to the system at a mass ratio of 10:1 to multi-walled carbon nanotubes. The mixture was heated in a water bath at 70 °C for 120 min. Polyethyleneimine assisted in the reduction of chloroauric acid to gold nanoparticles. The mixture was then cooled to room temperature, centrifuged (8000 rpm, 15 min), washed three times, and the supernatant was removed. The solution was concentrated to 2 mg / mL and stored at low temperature in the dark.

[0070] (2) Preparation of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites:

[0071] The electrode was fabricated on a PET substrate, with a working electrode, a counter electrode, and a reference electrode fixed on the substrate using screen printing technology. The working electrode had a diameter of 3.5 mm. The prepared multi-walled carbon nanotube and gold nanoparticle composite was diluted to 50 ng / mL, and 10 μL was dropped onto the electrode to completely cover the working electrode. After drying at 40 °C, the electrode modified with the multi-walled carbon nanotube and gold nanoparticle composite was obtained.

[0072] (3) Preparation of MB-Apt labeled electrodes:

[0073] 10 μL of MB-Apt (1 μM) was added to the working electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite. After incubation at 25 °C for 40 min, the uncoupled MB-Apt was removed by rinsing with phosphate buffer. The electrode was then immersed in 2 mM mercaptohexanol solution for 60 min to block non-specific binding sites. After rinsing with phosphate buffer and drying, the MB-Apt modified electrode was obtained and stored at low temperature in the dark.

[0074] (4) Fitting Cd 2+ and Hg 2+ Standard curve:

[0075] Will Cd 2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations (Cd). 2+ The solution concentrations were 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; Hg 2+The solutions were prepared at concentrations of 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. 5 μL of the test solution at different concentrations and 5 μL of 0.8 μM ssDNA were sequentially added to the electrochemical aptamer sensor. After incubation at room temperature for 30 min, the electrodes were washed with phosphate buffer, followed by the addition of 5 μL of AgNO3 (5 mM). After incubation in the dark, 5 μL of NaBH4 (20 mM) was added, and the reaction proceeded for 60 min in the dark, resulting in the in-situ formation of silver nanoclusters. After the reaction, the electrodes were rinsed with phosphate buffer, and the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the DPV peak intensity values.

[0076] (5) Detection of Cd in tea 2+ and Hg 2+ Content:

[0077] Add 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) to 0.5 g of pulverized green tea powder, then digest in a graphite digestion furnace at 120 °C for 90 min. After digestion, dilute to 25 mL with water, filter to remove impurities, and use the resulting solution as the detection solution. Add this solution dropwise to the constructed electrochemical aptamer sensor, react according to the above steps, and detect the electrochemical signal. Calculate the Cd content in the green tea powder based on the standard curve. 2+ and Hg 2+ The content of.

[0078] from Figure 2 As can be seen, the multi-walled carbon nanotube and gold nanoparticle composite can be successfully synthesized in this embodiment. The multi-walled carbon nanotube has a diameter of about 10 nm, and the gold nanoparticles are tightly bound to the surface of the multi-walled carbon nanotube. This is because the high amino density and branched structure of polyethyleneimine make it suitable as a primer and reducing agent for the in-situ synthesis of gold nanoparticles.

[0079] from Figure 3 and 4 It can be seen that the unmodified screen-printed electrode has a pair of distinct reversible redox peaks, which belong to Fe(CN)6. 3- and Fe(CN)6 4- The oxidation and reduction of Fe nanoparticles showed that the oxidation peak increased with increasing nanoparticle concentration, stabilizing at 50 ng / mL. The charge transfer resistance (Rct) of the unmodified screen-printed electrode was 1669 Ω, while it was less than 400 Ω when the nanomaterial concentration was greater than 20 ng / mL. This is due to the high electronic conductivity of the multi-walled carbon nanotube and gold nanoparticle composite. 3+ / Fe 2+ Electron transfer is significantly accelerated.

[0080] from Figure 5 It can be seen that the timing coulomb plot changed significantly after MB-Apt modification, proving that MB-Apt was successfully coupled to the electrode surface.

[0081] from Figure 6 and 7 It can be seen that in Cd 2+ When the concentration increased from 0 ng / mL to 200 ng / mL, the DPV characteristic peak of MB changed from weak to strong. The linear range obtained by fitting was 0.1-200 ng / mL, and the detection limit was 94.01 pg / mL.

[0082] from Figure 8 and 9 It can be seen that in Hg 2+ When the concentration increased from 0 ng / mL to 20 ng / mL, the DPV characteristic peak of silver nanoclusters changed from weak to strong. The linear range obtained by fitting was 0.05-20 ng / mL, and the detection limit was 15.74 pg / mL.

[0083] Example 2:

[0084] This embodiment utilizes simultaneous detection of Cd 2+ and Hg 2+ Electrochemical aptamer sensor for detecting Cd in tea 2+ and Hg 2+ The process includes the following steps:

[0085] (1) Preparation of multi-walled carbon nanotube and gold nanoparticle composites:

[0086] Multi-walled carbon nanotubes and polyethyleneimine were dispersed in an aqueous solution at a mass ratio of 1:22. After ultrasonic treatment for 20 min, a dispersion of multi-walled carbon nanotubes was obtained. Chloroauric acid was added to the system at a mass ratio of 15:1 to multi-walled carbon nanotubes. The mixture was heated in a water bath at 60 °C for 100 min. Polyethyleneimine assisted in the reduction of chloroauric acid to gold nanoparticles. The mixture was then cooled to room temperature, centrifuged (9000 rpm, 12 min), washed three times, and the supernatant was removed. The mixture was concentrated to 1.5 mg / mL and stored at low temperature in the dark.

[0087] (2) Preparation of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites:

[0088] The electrode was fabricated on a PET substrate, with a working electrode, counter electrode, and reference electrode fixed on the substrate using screen printing technology. The working electrode had a diameter of 3.5 mm. The prepared multi-walled carbon nanotube and gold nanoparticle composite was diluted to 40 ng / mL, and 15 μL was dropped onto the electrode to completely cover the working electrode. After drying at 35 °C, the electrode modified with multi-walled carbon nanotube and gold nanoparticle composite was obtained.

[0089] (3) Preparation of MB-Apt labeled electrodes:

[0090] 15 μL of MB-Apt (0.8 μM) was added to the working electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite. After incubation at 20 °C for 60 min, the uncoupled MB-Apt was removed by rinsing with phosphate buffer. The electrode was then immersed in 1.5 mM mercaptohexanol solution for 60 min to block non-specific binding sites. After rinsing with phosphate buffer and drying, the MB-Apt modified electrode was obtained and stored at low temperature in the dark.

[0091] (4) Fitting Cd 2+ and Hg 2+ Standard curve:

[0092] Will Cd 2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations (Cd). 2+ The solution concentrations were 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; Hg 2+ The concentrations of the solutions were 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. 8 μL of the test solution at different concentrations and 8 μL of 0.5 μM ssDNA were sequentially added to the electrochemical aptamer sensor. After incubation at room temperature for 40 min, the electrode was washed with phosphate buffer, and then 8 μL of 6 mM AgNO3 was added. After incubation in the dark, 8 μL of 15 mM NaBH4 was added, and the reaction was carried out for 40 min in the dark, resulting in the in-situ formation of silver nanoclusters. After the reaction, the electrode was rinsed with phosphate buffer, and the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the DPV peak intensity values.

[0093] (5) Detection of Cd in tea 2+ and Hg 2+ Content:

[0094] Add 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) to 0.5 g of pulverized black tea powder, then digest in a graphite digestion furnace at 120 °C for 90 min. After digestion, dilute to 25 mL with water, filter to remove impurities, and use the resulting solution as the detection solution. Add this solution dropwise to the constructed electrochemical aptamer sensor, react according to the above steps, and detect the electrochemical signal. Calculate the Cd content in the black tea powder based on the standard curve. 2+ and Hg 2+ The content of.

[0095] Example 3:

[0096] This embodiment utilizes simultaneous detection of Cd 2+ and Hg 2+ Electrochemical aptamer sensor for detecting Cd in tea 2+ and Hg 2+ The process includes the following steps:

[0097] (1) Preparation of multi-walled carbon nanotube and gold nanoparticle composites:

[0098] Multi-walled carbon nanotubes and polyethyleneimine were dispersed in an aqueous solution at a mass ratio of 1:25. After ultrasonic treatment for 40 min, a dispersion of multi-walled carbon nanotubes was obtained. Chloroauric acid was added to the system at a mass ratio of 12:1 to multi-walled carbon nanotubes. The mixture was heated in a water bath at 80 °C for 150 min. Polyethyleneimine assisted in the reduction of chloroauric acid to gold nanoparticles. The mixture was then cooled to room temperature, centrifuged (8000 rpm, 15 min), washed three times, and the supernatant was removed. The mixture was concentrated to 1.8 mg / mL and stored at low temperature in the dark.

[0099] (2) Preparation of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites:

[0100] The electrode was fabricated on a PET substrate, with a working electrode, a counter electrode, and a reference electrode fixed on the substrate using screen printing technology. The working electrode had a diameter of 3.5 mm. The prepared multi-walled carbon nanotube and gold nanoparticle composite was diluted to 30 ng / mL, and 20 μL was dropped onto the electrode to completely cover the working electrode. After drying at 50 °C, the electrode modified with the multi-walled carbon nanotube and gold nanoparticle composite was obtained.

[0101] (3) Preparation of MB-Apt labeled electrodes:

[0102] 20 μL of MB-Apt (0.8 μM) was added to the working electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite. After incubation at 30 °C for 30 min, the uncoupled MB-Apt was removed by rinsing with phosphate buffer. The electrode was then immersed in 1 mM mercaptohexanol solution for 40 min to block non-specific binding sites. After rinsing with phosphate buffer and drying, the MB-Apt modified electrode was obtained and stored at low temperature in the dark.

[0103] (4) Fitting Cd 2+ and Hg 2+ Standard curve:

[0104] Will Cd 2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations (Cd). 2+ The solution concentrations were 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; Hg 2+ The concentrations of the solutions were 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. 10 μL of the test solution at different concentrations and 10 μL of ssDNA (0.5 μM) were sequentially added to the electrochemical aptamer sensor. After incubation at room temperature for 60 min, the electrode was washed with phosphate buffer, and then 10 μL of AgNO3 (8 mM) was added. After incubation in the dark, 10 μL of NaBH4 (10 mM) was added, and the reaction was carried out for 50 min in the dark, resulting in the in-situ formation of silver nanoclusters. After the reaction, the electrode was rinsed with phosphate buffer, and the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the DPV peak intensity values.

[0105] (5) Detection of Cd in tea 2+ and Hg 2+ Content:

[0106] Add 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) to 0.5 g of pulverized dark tea powder, then digest in a graphite digestion furnace at 120 °C for 90 min. After digestion, dilute to 25 mL with water, filter to remove impurities, and use the resulting solution as the detection solution. Add this solution dropwise to the constructed electrochemical aptamer sensor, react according to the above steps, and detect the electrochemical signal. Calculate the Cd content in the dark tea powder based on the standard curve. 2+ and Hg 2+ The content of.

[0107] Example 4:

[0108] This embodiment utilizes simultaneous detection of Cd 2+ and Hg 2+ Electrochemical aptamer sensor for detecting Cd in vegetables 2+ and Hg 2+ The process includes the following steps:

[0109] (1) Preparation of multi-walled carbon nanotube and gold nanoparticle composites:

[0110] Multi-walled carbon nanotubes and polyethyleneimine were dispersed in an aqueous solution at a mass ratio of 1:20. After ultrasonic treatment for 20 min, a dispersion of multi-walled carbon nanotubes was obtained. Chloroauric acid was added to the system at a mass ratio of 12:1 to multi-walled carbon nanotubes. The mixture was heated in a water bath at 75 °C for 100 min. Polyethyleneimine assisted in the reduction of chloroauric acid to gold nanoparticles. The mixture was then cooled to room temperature, centrifuged (8000 rpm, 12 min), washed three times, and the supernatant was removed. The solution was concentrated to 2 mg / mL and stored at low temperature in the dark.

[0111] (2) Preparation of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites:

[0112] The electrode was fabricated on a PET substrate, with a working electrode, a counter electrode, and a reference electrode fixed on the substrate using screen printing technology. The working electrode had a diameter of 3.5 mm. The prepared multi-walled carbon nanotube and gold nanoparticle composite was diluted to 50 ng / mL, and 10 μL was dropped onto the electrode to completely cover the working electrode. After drying at 40 °C, the electrode modified with the multi-walled carbon nanotube and gold nanoparticle composite was obtained.

[0113] (3) Preparation of MB-Apt labeled electrodes:

[0114] 10 μL of MB-Apt (1.2 μM) was added to the working electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite. After incubation at 25 °C for 40 min, the uncoupled MB-Apt was removed by rinsing with phosphate buffer. The electrode was then immersed in 1.8 mM mercaptohexanol solution for 50 min to block non-specific binding sites. After rinsing with phosphate buffer and drying, the MB-Apt modified electrode was obtained and stored at low temperature in the dark.

[0115] (4) Fitting Cd 2+ and Hg 2+ Standard curve:

[0116] Will Cd 2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations (Cd). 2+The solution concentrations were 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; Hg 2+ The concentrations of the solutions were 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. 5 μL of the test solution at different concentrations and 5 μL of ssDNA (1 μM) were sequentially added to the electrochemical aptamer sensor. After incubation at room temperature for 50 min, the electrodes were washed with phosphate buffer, followed by the addition of 5 μL of AgNO3 (7 mM) and incubation in the dark. Then, 5 μL of NaBH4 (20 mM) was added and the reaction was carried out for 45 min in the dark, resulting in the in-situ formation of silver nanoclusters. After the reaction, the electrode was rinsed with phosphate buffer, and the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the DPV peak intensity values.

[0117] (5) Detection of Cd in vegetables 2+ and Hg 2+ Content:

[0118] Add 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) to 0.5 g of pulverized Shanghai bok choy, then digest in a graphite digestion furnace at 120 °C for 90 min. After digestion, dilute to 25 mL with water, filter to remove impurities, and use the resulting solution as the detection solution. Add this solution dropwise to the constructed electrochemical aptamer sensor, react according to the above steps, and detect the electrochemical signal. Calculate the Cd content in the Shanghai bok choy based on the standard curve. 2+ and Hg 2+ The content of.

[0119] Example 5:

[0120] This embodiment utilizes simultaneous detection of Cd 2+ and Hg 2+ Electrochemical aptamer sensor for detecting Cd in vegetables 2+ and Hg 2+ The process includes the following steps:

[0121] (1) Preparation of multi-walled carbon nanotube and gold nanoparticle composites:

[0122] Multi-walled carbon nanotubes and polyethyleneimine were dispersed in an aqueous solution at a mass ratio of 1:25. After ultrasonic treatment for 30 min, a dispersion of multi-walled carbon nanotubes was obtained. Chloroauric acid was added to the system at a mass ratio of 15:1 to multi-walled carbon nanotubes. The mixture was heated in a water bath at 65 °C for 120 min. Polyethyleneimine assisted in the reduction of chloroauric acid to gold nanoparticles. The mixture was then cooled to room temperature, centrifuged (10,000 rpm, 10 min), washed three times, and the supernatant was removed. The mixture was concentrated to 1.5 mg / mL and stored at low temperature in the dark.

[0123] (2) Preparation of screen-printed electrodes modified with multi-walled carbon nanotube and gold nanoparticle composites:

[0124] The electrode was fabricated on a PET substrate, with a working electrode, a counter electrode, and a reference electrode fixed on the substrate using screen printing technology. The working electrode had a diameter of 3.5 mm. The prepared multi-walled carbon nanotube and gold nanoparticle composite was diluted to 20 ng / mL, and 20 μL was dropped onto the electrode to completely cover the working electrode. After drying at 45 °C, the electrode modified with the multi-walled carbon nanotube and gold nanoparticle composite was obtained.

[0125] (3) Preparation of MB-Apt labeled electrodes:

[0126] 12 μL of MB-Apt (1 μM) was added to the working electrode modified with a multi-walled carbon nanotube and gold nanoparticle composite. After incubation at 30 °C for 30 min, the uncoupled MB-Apt was removed by rinsing with phosphate buffer. The electrode was then immersed in 1.2 mM mercaptohexanol solution for 45 min to block non-specific binding sites. After rinsing with phosphate buffer and drying, the MB-Apt modified electrode was obtained and stored at low temperature in the dark.

[0127] (4) Fitting Cd 2+ and Hg 2+ Standard curve:

[0128] Will Cd 2+ and Hg 2+ The standard (1 mg / mL) was diluted with ultrapure water to different concentrations (Cd). 2+ The solution concentrations were 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; Hg 2+The concentrations of the solutions were 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. 8 μL of the test solution at different concentrations and 8 μL of 0.8 μM ssDNA were sequentially added to the electrochemical aptamer sensor. After incubation at room temperature for 40 min, the electrodes were washed with phosphate buffer, followed by the addition of 8 μL of 5 mM AgNO3. After incubation in the dark, 8 μL of 15 mM NaBH4 was added, and the reaction proceeded for 60 min in the dark, resulting in the in-situ formation of silver nanoclusters. After the reaction, the electrode was rinsed with phosphate buffer, and the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs The standard curve was fitted using the DPV peak intensity values.

[0129] (5) Detection of Cd in vegetables 2+ and Hg 2+ Content:

[0130] Add 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) to 0.5 g of pulverized Shanghai bok choy, then digest in a graphite digestion furnace at 120 °C for 90 min. After digestion, dilute to 25 mL with water, filter to remove impurities, and use the resulting solution as the detection solution. Add this solution dropwise to the constructed electrochemical aptamer sensor, react according to the above steps, and detect the electrochemical signal. Calculate the Cd content in the Shanghai bok choy based on the standard curve. 2+ and Hg 2+ The content of.

[0131] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for simultaneous detection of Cd 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, Includes the following steps: (1) During the detection process, the biochemical reaction is carried out on the screen-printed electrode. The electrode is based on polyethylene terephthalate. The working electrode, counter electrode and reference electrode are fixed on the substrate by screen printing. The working electrode and counter electrode are composed of graphite and polyacrylic acid derivatives, and the reference electrode is composed of silver / silver chloride paste. (2) Polyethyleneimine-assisted dissolution and reduction to prepare multi-walled carbon nanotube and gold nanoparticle composites; The preparation method of the multi-walled carbon nanotube and gold nanoparticle composite includes: dispersing multi-walled carbon nanotubes and polyethyleneimine in an aqueous solution, ultrasonicating to obtain a multi-walled carbon nanotube dispersion; adding chloroauric acid to the system, reducing chloroauric acid to gold nanoparticles with the assistance of polyethyleneimine under water bath heating conditions, cooling to room temperature, centrifuging and washing, removing the supernatant, concentrating, and storing at low temperature in the dark. (3) The multi-walled carbon nanotube and gold nanoparticle composite obtained in step (2) is used to modify the screen-printed electrode to improve the conductivity and specific surface area, thus obtaining the multi-walled carbon nanotube and gold nanoparticle composite modified electrode. (4) The thiol group at one end of the methylene blue labeled MB-Apt is covalently coupled to the gold nanoparticles on the electrode surface through gold-sulfur bonds to obtain the MB-Apt modified electrode. (5) The analyte and ssDNA are simultaneously added to the MB-Apt modified electrode, and then silver nanoclusters are generated in situ at the C-rich sequence end of the ssDNA. The electrochemical signals of MB and silver nanoclusters are measured to achieve the control of Cd. 2+ and Hg 2+ The detection of Cd; 2+ and Hg 2+ The detection method includes: the test solution and ssDNA are sequentially added to the surface of an MB-Apt modified electrode, incubated at room temperature, washed with phosphate buffer, AgNO3 is added, incubated in the dark, and then NaBH4 solution is added to generate silver nanoclusters in situ. After the reaction, the electrode is rinsed with phosphate buffer. The signals of MB and silver nanoclusters are measured by DPV using an electrochemical method, corresponding to Cd. 2+ and Hg 2+ ; The MB-Apt complex is covalently bound to multi-walled carbon nanotubes and gold nanoparticles via gold-sulfur bonds, and MB-Apt specifically recognizes Cd. 2+ This leads to a conformational change in the aptamer, and the change in the MB electrochemical signal enables the control of Cd. 2+ The detection of Hg; 2+ When present, ssDNA and MB-Apt communicate via Hg. 2+ Bridging to form a T-Hg-T structure to achieve Hg 2+ The detection of Hg was achieved by in-situ generation of silver nanoclusters in c-rich ssDNA sequences and the change in electrochemical signal of the silver nanoclusters. 2+ The detection.

2. The simultaneous detection of Cd according to claim 1 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, In step (1), the working electrode diameter of the screen printing electrode is 3.5 mm.

3. The simultaneous detection of Cd according to claim 1 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, In step (2), the mass ratio of the multi-walled carbon nanotubes and polyethyleneimine dispersed in the aqueous solution is 1:20-25; And / or the ultrasonic treatment time is 20-40 min; And / or the mass ratio of the chloroauric acid to the multi-walled carbon nanotubes is 10-15:1; And / or the water bath heating temperature is 60-80℃, and the heating time is 100-150min; And / or the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 10-15 min; And / or the concentration after concentration is 1.5-2 mg / mL.

4. The simultaneous detection of Cd according to claim 1 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, In step (3), the preparation method of the multi-walled carbon nanotube and gold nanoparticle composite modified electrode includes: dripping diluted multi-walled carbon nanotube and gold nanoparticle composite onto the electrode to completely cover the working electrode, and then drying it to obtain the electrode; wherein: And / or the amount of the multi-walled carbon nanotube and gold nanoparticle composite added to the electrode is 10-20 μL; And / or the concentration of the multi-walled carbon nanotube and gold nanoparticle composite after dilution is 20-50 ng / mL; And / or the drying temperature is 30-50℃.

5. The simultaneous detection of Cd according to claim 1 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, In step (4), the preparation method of the MB-Apt modified electrode includes: adding MB-Apt to the working electrode, incubating the reaction, rinsing with phosphate buffer to remove uncoupled MB-Apt, then immersing the electrode in mercaptohexanol solution to block non-specific binding sites, rinsing with phosphate buffer and drying to obtain the MB-Apt modified electrode, which is then stored at low temperature and protected from light; wherein the concentration of MB-Apt is 0.8-1.2 μM and the amount added is 10-20 μL. And / or the incubation temperature of the MB-Apt on the electrode is 20-30℃, and the incubation time is 30-60 min; And / or the concentration of the mercaptohexanol solution is 1-2 mM, and the soaking and sealing time is 40-60 min.

6. The simultaneous detection of Cd according to claim 1 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor is characterized by, In step (5), the amount of the test solution added is 5-10 μL; And / or the amount of ssDNA added is 5-10 μL, and the concentration is 0.5-1 μM; And / or the incubation time of the test solution and ssDNA on the electrode is 30-60 min; And / or the amount of AgNO3 added is 5-10 μL, and the concentration is 5-8 mM; And / or the amount of NaBH4 solution added is 5-10 μL, and the concentration is 10-20 mM; And / or the in-situ generation reaction time of the silver nanoclusters is 30-60 min.

7. A method for simultaneous detection of Cd 2+ and Hg 2+ An electrochemical aptamer sensor, characterized in that, Simultaneous detection of Cd as described in any one of claims 1 to 6 2+ and Hg 2+ The method for preparing an electrochemical aptamer sensor was obtained.

8. The simultaneous detection of Cd as described in claim 7 2+ and Hg 2+ Electrochemical aptamer sensors for detecting Cd heavy metal ions in food 2+ and Hg 2+ Applications in [the field].

9. The application according to claim 8, characterized in that, Detection of heavy metal ions (Cd) in tea and vegetables 2+ and Hg 2+ By simultaneously detecting Cd 2+ and Hg 2+ The realization of the electrochemical aptamer sensor includes the following steps: (a) Preparation of Cd at different concentration gradients 2+ and Hg 2+ Standard solution, dropped simultaneously to detect Cd 2+ and Hg 2+ On an electrochemical aptamer sensor, the intensity of its electrochemical characteristic peak signal was measured, and a standard curve was fitted. (b) After pretreatment of tea leaves and vegetables, the mixture is added dropwise to the solution where Cd is simultaneously detected. 2+ and Hg 2+ On an electrochemical aptamer sensor, Cd was determined according to a linear model. 2+ and Hg 2+ The concentration.

10. The application according to claim 9, characterized in that, In step (a), Cd 2+ and Hg 2+ The standard curve fitting method: Cd 2+ and Hg 2+ The standard was diluted to different concentrations with ultrapure water, and solutions of different concentrations were added dropwise to the simultaneously detected Cd. 2+ and Hg 2+ On an electrochemical aptamer sensor, the electrochemical DPV signal I of MB and silver nanoclusters was detected. MB and I AgNCs A standard curve was fitted using the DPV peak intensity values; where: Cd 2+ The solutions were available at concentrations of 0 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL. and / or Hg 2+ The solutions were available at concentrations of 0 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. In step (b), nitric acid and hydrogen peroxide are added to the pulverized tea powder and vegetables, and then digested in a graphite digestion furnace; after digestion, the solution is diluted with water, filtered to remove impurities, and used as the solution for detection, which is then added dropwise to the solution for simultaneous Cd detection. 2+ and Hg 2+ Electrochemical signals are detected using an electrochemical aptamer sensor, and Cd in tea and vegetables is calculated based on a standard curve. 2+ and Hg 2+ The content of.

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