A method for preparing a high-precision electrochemical sensing device

Abstract

The application belongs to the technical field of electrochemical sensors, and particularly relates to a preparation method of a high-precision electrochemical sensing device. 2+ The aptamer is connected with the binding site on the electrode to form Apt / AuNPs / GCE; then, the excess binding sites are blocked by using MCH to form MCH / Apt / AuNPs / GCE; at the same time, the Pb 2+ The single-stranded DNA which is semi-complementary to the aptamer is connected with AgNWS@ZIF-8 / MB through Ag-S binding at the -SH-3' end to form S1 / AgNWS@ZIF-8 / MB, and is self-assembled on the MCH / Apt / AuNPs / GCE through hybridization, the prepared electrochemical sensing device is used for detecting lead ions in fish meat, has the advantages of high sensitivity and accuracy, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, specifically relating to a method for preparing a high-precision electrochemical sensing device. Background Technology

[0002] Heavy metal pollution of water resources is a serious public health problem. Due to the toxicity of heavy metals themselves and their accumulation effect in living tissues, heavy metal elements accumulate in the human body through the food chain, and long-term intake can harm human health. Fish, as part of the food chain, are a common food on human tables and are consumed in huge quantities. Researchers frequently report Pb levels... 2+ It accumulates in aquatic products, especially fish. Pb 2+ Excessive intake can lead to damage to various organs and tissues in the human body, including the respiratory, digestive, and nervous systems; therefore, it is necessary to establish an effective, sensitive, and cost-effective method for detecting Pb. 2+ It is of great significance to ensure the safety of agricultural products and people's health.

[0003] Many spectroscopic and electrochemical analyses are now applied to Pb. 2+ Determination. Spectroscopic analysis, including atomic adsorption spectroscopy (AAS), atomic fluorescence spectroscopy (AFS), inductively coupled plasma optical emission spectroscopy (ICP-OES), and inductively coupled plasma mass spectrometry (ICP-MS), has been widely accepted as a detection method. While they can provide highly reliable readings, they fall short of the urgent need for simplified operation and reduced instrument costs. Electrochemical methods, due to their budget-friendly equipment and rapid response, are playing a more significant role in in-situ determinations. In particular, the synergy between microfabricated sensors, aptamer-based functional materials, and ratiometric strategies can amplify the methodological advantages to address practical analytical challenges such as complex backgrounds.

[0004] Aptamers, due to their specific secondary structures, exhibit high selectivity and affinity for targets derived from heavy metal ions, small molecules, or proteins. Beyond single-signal electrochemical analysis, aptamers are highly compatible with ratiometric strategy-based sensors designed to eliminate unwanted external interference. Ratiometric strategies are based on dual-signal sensing, typically employing either an on / off or on-off mode. In the on / off mode, the reference signal remains almost constant, independent of analyte variations. In contrast, in the on-off mode, two signals are activated in opposite directions in response to alternating target changes, thus ensuring considerable stability and reproducibility to environmental or operational disturbances. Studies have shown that electrochemical aptamer sensors are effective for Pb... 2+The detection of most Pb is prone to high errors, while ratiometric electrochemical aptamer sensors with dual electrical signal outputs have inherent calibration capabilities, which can greatly improve the accuracy and sensitivity of detection. However, the development of ratiometric electrochemical aptamer sensors still faces significant challenges, requiring further improvement in detection sensitivity to meet the requirements for ultra-low levels of Pb. 2+ The growing need for monitoring. Summary of the Invention

[0005] This invention provides a method for fabricating a high-precision electrochemical sensing device, which uses semi-complementary aptamer pairs and AgNWS@ZIF-8 / MB signal probes for lead ratio detection, thereby improving detection sensitivity and meeting the requirements for ultra-low Pb levels. 2+ The demand.

[0006] To achieve the above objectives, this invention applies the synergistic effect of semi-complementary aptamer pairs and a signal on / off ratio strategy on a glassy carbon electrode (GCE) for the detection of lead ions (Pb). 2+ This invention relates to the detection of lead ions in fish meat. Specifically, a ferrocene signal was added to the 5'-end of the aptamer, and a methylene blue signal was self-assembled on the aptamer complementary strand via AgNWS@ZIF-8, employing an Fc-MB-based ratiometric strategy.

[0007] The AgNWs@ZIF-8 is a beaded AgNWs@ZIF-8 core-shell nanochain prepared by in-situ growth of ZIF-8 on silver nanowires (Ag NWs). AgNWs@ZIF-8 has strong adsorption properties similar to ZIF-8, while compensating for the weak electrical conductivity of ZIF-8, thus expanding the intensity range of methylene blue (MB) signals to a certain extent.

[0008] The ratiometric method is based on the parallel use of two electrochemical probes. In addition to the classic signal probe Fc found in other electronic sensors, the present invention also includes another redox probe MB as a control, i.e., the internal control signal (Fc) and the conformational change signal (MB) constitute the ratiometric sensing system.

[0009] The specific modification process of this invention involves electrodepositing gold nanoparticles (AuNPs) onto a glassy carbon electrode (GCE), and then using an Au-S binding method to modify Pb. 2+ The aptamer (Apt) attaches to the binding site on the electrode, forming Apt / AuNPs / GCE. Then, 6-mercapto-1-hexanol (MCH) is used to block excess binding sites and orthogonalize the aptamer, forming MCH / Apt / AuNPs / GCE. Simultaneously, it reacts with Pb... 2+The single-stranded DNA (S1) semi-complementary to the aptamer is combined with AgNWS@ZIF-8 / MB through Ag-S at the -SH-3' end to form S1 / AgNWS@ZIF-8 / MB. The product can be self-assembled on MCH / Apt / AuNPs / GCE by hybridization, but can be easily removed by Pb 2+ As a competitor is expelled. When S1 / AgNWS@ZIF-8 / MB is expelled, the MB signal is weakened, and the ferrocene signal is enhanced because the conformation change of the aptamer chain shortens the distance between the ferrocene molecule and the electrode. Fc And I MB Pb 2+ shows opposite response changes, thereby constituting I Fc / I MB ratio system.

[0010] A preparation method of a high-precision electrochemical sensing device, the operation steps are as follows:

[0011] Step one, synthesis of beaded Ag NWs@ZIF-8 core-shell nanochain;

[0012] Under stirring, zinc nitrate hexahydrate and 2-methylimidazole are added to the silver nanowire solution to obtain a mixed solution, stirring is stopped, and the mixed solution is reacted under certain temperature conditions. After the reaction, the product is collected by centrifugation, the obtained product is washed several times with methanol, and finally vacuum dried to obtain the product, which is beaded Ag NWs@ZIF-8 core-shell nanochain, ready for use;

[0013] Step two, electrodeposition of gold nanoparticles;

[0014] (1) Dissolve NH4Cl in water to obtain an ammonium chloride solution, then mix the ammonium chloride solution with a chloroauric acid solution to obtain a precursor solution;

[0015] (2) Pretreatment of the glassy carbon electrode: first polish the glassy carbon electrode with polishing powder, then rinse it with pure water; then move the glassy carbon electrode into an ultrasonic water bath for cleaning, and then ultrasonically clean it with ethanol, HNO3 and distilled water in turn; test the electrode performance after cleaning to complete the pretreatment of the glassy carbon electrode;

[0016] (3) Use the pretreated glassy carbon electrode as the working electrode, and perform linear voltammetric scanning in the precursor solution prepared in step (1); the glassy carbon electrode after scanning is denoted as AuNPs / GCE;

[0017] Step three, aptamer incubation and MCH modification;

[0018] Dropping the aptamer (APT) on the AuNPs / GCE prepared in step two, and then incubating at room temperature, and then drying naturally, and then washing the electrode with PBS after drying, and then immersing the obtained electrode into 6-mercapto-1-hexanol (MCH), and then obtaining the modified electrode after immersion treatment;

[0019] Step four, modifying the probe;

[0020] Dispersing the Ag NWs@ZIF-8 into a methylene blue solution, and then shaking at room temperature overnight to obtain an Ag NWs@ZIF-8 / MB suspension;

[0021] Then adding a single-stranded DNA (S1) that is semi-complementary to the aptamer (APT) into the Ag NWs@ZIF-8 / MB suspension, and then obtaining a mixture after shaking at room temperature; centrifuging the prepared mixture, collecting the product, and then washing the product with a Tris-HCl buffer for several times, and then suspending the washed product in the Tris-HCl buffer to obtain an Ag NWs@ZIF-8 / MB / S1 suspension; and finally dropping the Ag NWs@ZIF-8 / MB / S1 suspension onto the modified electrode treated in step three, and then obtaining an electrochemical sensing device after incubation.

[0022] Preferably, the amount of the silver nanowire solution, zinc nitrate hexahydrate and 2-methylimidazole in step one is 0.5 mL: 25 mL: 25 mL; the concentration of the silver nanowire (Ag NWs) solution is 5 mg / mL, the concentration of the zinc nitrate hexahydrate is 25 mM, and the concentration of the 2-methylimidazole is 25 mM; the reaction condition is 30℃ for 2 h; the centrifugation condition is 8000 rpm for 10 min; the number of washing times is 3-5 times, and the vacuum drying temperature is 60℃.

[0023] Preferably, in (1) of step two, the concentration of the ammonium chloride solution is 2.5 M, and the volume concentration of the chloroauric acid solution is 10%; the concentration of the chloroauric acid in the precursor solution is 10 mM.

[0024] Preferably, in (2) of step two, the polishing powder is Al2O3, and the particle sizes are 0.3 μm and 0.05 μm in sequence; the electrode is cleaned in an ultrasonic water bath for 2-3 min for three times; and the specific steps for testing the electrode performance are as follows: recording a cyclic voltammogram of 1×10 -3 mol / L K3Fe(CN) 6 solution in 0.20 mol / L KNO3, wherein the scanning speed is 50 mV / s, the scanning range is 0.6- -0.1 V, and the peak point difference in the obtained cyclic voltammogram is 80 mV.

[0025] Preferably, in (3) of step two, the scanning range is 0-0.8 V, and the scanning time is 200 seconds.

[0026] Preferably, the concentration of the aptamer (APT) in step three is 5-8 μM, the drop coating amount is 4-8 μL; the room temperature incubation time is 2 h; the concentration of the 6-mercapto-1-hexanol is 1 mM, and the soaking treatment time is 2 h.

[0027] Preferably, the concentration of the methylene blue solution in step four is 1 mg / mL; the concentration of the Ag NWs@ZIF-8 / MB suspension is in the range of 1 mg / mL-5 mg / mL; the volume ratio of the Ag NWs@ZIF-8 / MB suspension to the single-stranded DNA is 20:1, the concentration of the single-stranded DNA is 5-8 μM, and the room temperature oscillation time is 60 min; the pH of the Tris-HCl buffer is 7.4, and the amount of the product after washing is suspended in the Tris-HCl buffer in a ratio of 0.02-0.5 g:1 mL.

[0028] The drop coating amount of the Ag NWs@ZIF-8 / MB / S1 suspension is 6 μL, and the incubation condition is room temperature or 37 degrees for 1 h.

[0029] The electrochemical sensing device prepared in the application is applied to detect lead ions, and is particularly applied to detect lead ions in fish meat, and the operation steps are as follows:

[0030] (1) First, prepare lead ion solutions with different concentrations, and the concentration of the lead ions is in the range of 0.2 μM-0.9 μM; then drop them on the surface of the electrochemical sensing device, and the drop amount is V1; perform differential pulse voltammetry scanning, observe the signal change of the electrode under DPV under different lead ion solution concentrations, record the internal control signal (Fc) and the conformation change signal (MB); process the data to obtain the ratio of the signal change value, and then construct a standard curve with the concentration logarithm;

[0031] (2) Unknown sample detection, the sample is treated to obtain a sample solution; the operation method is the same as step (a), except that the lead ion solution is replaced by the sample solution, and the ratio of the signal change value is obtained after detection, which is substituted into the standard curve in step (a) to calculate the lead ion concentration in the fish meat.

[0032] Preferably, the drop amount V1 in step (1) is 4-8 μL.

[0033] Preferably, the specific steps of the sample treatment in step (2) are as follows:

[0034]

[0035] ②The solid product is re-distributed in 10mM Tris-HCl buffer to obtain a mixed solution with a concentration of 5mg / mL, that is, the sample solution; it is stored at 4 DEG C until use; wherein the Tris-HCl buffer is composed of 10mM tris(hydroxymethyl)aminomethane (Tris), 100Mm sodium chloride (NaCl) and 1Mm disodium ethylenediaminetetraacetate (EDTA).

[0036] The beneficial effects of the present application are:

[0037] The present application provides a preparation method of high-precision electrochemical sensing device, in the ratio electrochemical sensing process, Pb 2+ The aptamer acts as a basic recognizer and generates a target signal Fc, based on the complementary pairing principle of the aptamer pair, S1@AgNWS@ZIF-8@MB is self-assembled on the sensor to provide a reference signal MB. Wherein the reference signal MB is inhibited due to the double-strand break caused by competitive Pb 2+ Absorption, the target signal Fc is enhanced due to the conformational change of the aptamer. Compared with single signal reading, the existing sensor provides more reliable ratio meter reading, and shows considerable analytical progress in LOD, selectivity and stability. The specific application of the synergistic effect of semi-complementary aptamer pair and signal on-off ratio strategy detects lead ions in fish meat; in the determination of Pb 2+ In fish meat samples, the results of the sensor are equivalent to those of ICP-MS and recovery test, which provides more reliable ratio meter reading and improves the detection sensitivity, and meets the growing demand for monitoring of ultra-low level Pb 2+ . BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a flow chart of the preparation of the sensor of the method of the present application;

[0039] Figure 2 It is the change rule of MB and Fc signals under different Pb 2+ Concentrations;

[0040] Figure 3 It is I Fc / I MB And Pb 2+ ​Logarithmic linear relationship between concentrations;

[0041] Figure 4 is the DPV curve of GCE (a), Apt / AuNPs / GCE (b), S1 / AgNWS@ZIF-8 / MB / MCH / Apt / AuNPs / GCE (c) in PBS buffer;

[0042] Figure 5 is the DPV change of GCE (a), Apt / AuNPs / GCE (b), S1 / AgNWS@ZIF-8 / MB / MCH / Apt / AuNPs / GCE (c) at different Pb 2+ concentrations;

[0043] Figure 6 is the electroactivity of bare GCE (a), ZIF-8 / GCE (b) and AgNWs@ZIF-8 / GCE (c);

[0044] Figure 7 is the linear relationship between the CV peak of ZIF-8 / GCE and the square root of the scan rate;

[0045] Figure 8 is the linear relationship between the CV peak of AgNWs@ZIF-8 / GCE and the square root of the scan rate. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is not intended to limit the present application, but rather to explain certain aspects, features, and embodiments of the present application.

[0047] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described herein. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict, the content of the present specification will control.

[0048] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0049] The DNA strand aptamer (APT) sequence used in the embodiment of the application is 5'-Fc-GGG TGG GTG GGT GGG T-C6-SH-3', and the complementary strand S1 sequence is 5'-ATGGACACCCTCCCACAACA-C6-SH-3'. The application is not based on a genetic sequence, and does not involve a sequence table; the DNA strand aptamer used is a primer, and the complementary strand S1 is a conventional reagent, which is purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0050] Embodiment 1

[0051] The Figure 1 The preparation method of the high-precision electrochemical sensing device is shown in the flowchart, and the implementation steps are as follows:

[0052] Step one, synthesis of beaded Ag NWs@ZIF-8 core-shell nanochain;

[0053] AgNWs were synthesized by polyol method using ethylene glycol to reduce AgNO3; 20 mL of ethylene glycol was heated in an oil bath at 150℃ for 1 hour, then 5 mL of AgNO3-ethylene glycol solution (concentration of 0.048 mol / mL) and 5 mL of PVP-ethylene glycol solution (concentration of 0.048 mol / mL) were added dropwise at the same time, 10-15 minutes later, the solution became turbid and turned gray, indicating the presence of silver nanowires, then the reaction was continued at 150℃ for 5 hours, after the reaction was completed, a gray precipitate was obtained, which was washed with ethanol and acetone for 3-5 times, and dried at 60℃ for 10-12 hours, to obtain silver nanowires;

[0054] 0.5 mL of silver nanowire solution (Ag NWs, 5 mg / mL) was added to a round-bottom flask, then 25.0 mL of zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 25 mM) and 25.0 mL of 2-methylimidazole (2-MI, 25 mM) were continuously added under magnetic stirring. The mixture was reacted at 30℃ for 2h without stirring; the product was collected by centrifugation at 8000 rpm for 10 min, washed with methanol for 3 times, and the obtained product was vacuum dried at 60℃ to obtain beaded Ag NWs@ZIF-8 core-shell nanochain.

[0055] For comparison, pure ZIF-8 was prepared by the same method, the difference being that Ag NWs were not added.

[0056] Step two, electrodeposition of gold nanoparticles;

[0057] NH4Cl was dissolved in water to prepare an ammonium chloride solution, and the concentration of the ammonium chloride solution was 2.5M; then 3 mL of chloroauric acid solution with a concentration of 10% was mixed with the ammonium chloride solution to obtain a precursor solution, wherein the concentration of the chloroauric acid was 10mM;

[0058] The glassy carbon electrode was pretreated: the glassy carbon electrode was polished with 0.3 μm and 0.05 μm polishing powder (Al2O3) in turn, and then the electrode surface was washed with pure water; the glassy carbon electrode was moved into an ultrasonic water bath for cleaning, and was cleaned in the ultrasonic water bath for 2-3 min each time for three times; then the glassy carbon electrode was cleaned with ethanol, HNO3 and distilled water in turn by ultrasonic cleaning; after cleaning, the electrode performance was tested, wherein the scanning speed was 50 mV / s, the scanning range was 0.6-0.1 V, and when the peak point difference in the obtained cyclic voltammogram was 80 mV, the pretreatment of the glassy carbon electrode was completed;

[0059] Finally, the pretreated glassy carbon electrode was used as a working electrode, and linear voltammetry scanning was performed in the precursor solution, the scanning range was 0-0.8 V, and the scanning time was 200 s; at this time, the glassy carbon electrode was recorded as AuNPs / GCE.

[0060] Step three, incubation and MCH modification of aptamer;

[0061] Aptamer (initial concentration 5 μM) was drop-coated on the AuNPs / GCE, the drop-coating amount was 6 μL, and the room temperature incubation was 2 h, and then natural drying was performed; the obtained electrode was recorded as Apt / AuNPs / GCE. The electrode was washed with PBS to remove non-specifically adsorbed DNA, and then the obtained electrode was immersed in 1 mM 6-mercapto-1-hexanol (MCH) for 2 h to obtain a modified electrode, which was recorded as MCH / Apt / AuNPs / GCE.

[0062] Step four, modification of probe;

[0063] Ag NWs@ZIF-8 was quantitatively dispersed into 1 mL methylene blue solution (MB, 1 mg / mL) to obtain AgNWs@ZIF-8 / MB suspensions with different concentrations (initial concentration 1 mg / mL), which were shaken at room temperature overnight. In this process, part of the MB molecules were loaded into the pores of Ag NWs@ZIF-8. Then, 50 μL of single-stranded S1 (initial concentration 5 μM) was added, and was shaken at room temperature for 60 min to assemble with AgNWs@ZIF-8 through Ag-S bond. The prepared Ag NWs@ZIF-8 / MB / S1 was centrifuged and washed with Tris-HCl buffer for several times, and was suspended in 1.0 mL of Tris-HCl buffer with pH 7.4 to obtain Ag NWs@ZIF-8 / MB / S1 suspension, which was stored at 4 °C for standby;

[0064] 6 μL of Ag NWs@ZIF-8 / MB / S1 suspension was drop-coated on the modified electrode during modification, and the room temperature or 37 °C incubation was 1 h to obtain an electrochemical sensing device, which was recorded as S1 / AgNWS@ZIF-8 / MB / MCH / Apt / AuNPs / GCE.

[0065] Further optimization of the modified electrode performance yielded the optimal conditions: Pb 2+ The optimal concentration level for aptamers and S1 is 6 μM Pb. 2+ The aptamer and 8 μM S1; after the aptamer reacted with the target ion for 120 minutes, Pb 2+ All reactions with its aptamer reached saturation, and a buffer solution pH of 7.0 provided a higher ratio signal than other settings; a concentration level of 3 mg / mL for Ag NWs@ZIF-8 was the optimal choice.

[0066] Under these optimal conditions, the lead ion concentration was controlled within the range of 0.02 μM to 0.9 μM, and the Pb concentration was gradually increased for the sensor. 2+ The incubation concentration was determined, and then differential pulse voltammetry was performed to obtain the signal variation pattern. Based on... Figure 2 It can be seen that an oxidation peak of MB appears at -0.22V, and a strong oxidation peak of Fc appears near 0.2V, and the oxidation peak increases with Pb. 2+ With increasing levels, MB showed a clear downward trend. During this process, Pb... 2+ The mutual adsorption between it and its aptamer leads to the loss of S1@AgNWs@ZIF-8@MB, resulting in a decrease in the reference signal MB, denoted as I in the electrochemical signal. MB The morphological change of the aptamer brings the Fc signal molecule closer to the electrode, thereby enhancing the Fc signal. This electrochemical signal is denoted as I. Fc .like Figure 3 Based on this dual-signal response mode, I Fc / I MB and Pb 2+ The logarithmic linear relationship between concentrations was well established as follows:

[0067] Lg(I Fc / I MB )=0.889C(Pb 2+ -0.424, R 2 =0.997; where C(Pb) 2+ () represents the lead ion concentration.

[0068] Meanwhile, the detection limit was confirmed to be 0.01 μM (S / N = 3, where S refers to sensitivity and N is noise).

[0069] When determining heavy metals in food, the sample needs to be digested to convert the metal element to be tested into metal ions.

[0070] The present embodiment selects fish meat as the sample, and 3 kinds of fish meat purchased in the local aquatic product market are taken as samples, specifically, grass carp, silver carp and sea bass, denoted as sample A, sample B and sample C. The target elements are extracted by microwave digestion (Mars 6 digester), and the sample processing steps are as follows:

[0071] ① The fish meat is washed with ultrapure water and uniformly cut, 2 g of sample is accurately weighed for each source and transferred into a polytetrafluoroethylene tube, and then 30 mL of acid mixture is supplemented. The acid mixture as a digestion reagent is composed of concentrated nitric acid and concentrated sulfuric acid, and the volume ratio is fixed at 1:1.

[0072] ② 10 mL of 30% hydrogen peroxide is added to the naturally cooled digestion product, and is completely dried at 200℃.

[0073] ③ The solid is re-distributed into 10 mM Tris-HCl buffer, and the ratio of solid product to buffer is fixed at 5 mg / mL, so as to obtain a sample liquid, and is refrigerated at 4℃ until use. The Tris-HCl buffer is composed of 10 mM tris(hydroxymethyl)aminomethane (Tris), 100 Mm sodium chloride (NaCl) and 1 Mm disodium ethylenediaminetetraacetate (EDTA).

[0074] Three samples (Sample 1, Sample 2, Sample 3) are randomly selected from each fish meat source. Table 1 shows the average readings of DPVs and ICP-MS from repeated measurements, which are repeated 5 times. The relative error of the method is 3.1-5.3% compared with ICP-MS. Subsequently, 0.4 μm Pb 2+ After adding to the actual sample, the recovery rate of the recommended method is 96.9-102.6%.

[0075] Table 1:

[0076]

[0077] The effect of the present application is verified by the following experiments:

[0078] In the step-by-step modification, the modified electrodes at different stages are detected by differential pulse voltammetry in PBS buffer. As shown in Figure 4 , the ferrocene signal is detected after APT incubation, and the methylene blue signal and the ferrocene signal are detected simultaneously after S1 self-assembly, indicating that the corresponding signal can be obtained for each main step. As shown in Figure 5 , when there is a target ion, both signals will change significantly, the signal intensity of MB will decrease and the signal intensity of Fc will increase, and when the lead ion concentration changes, the two signals will also change correspondingly.

[0079] To verify the analytical advantages of the Fc-MB-based ratiometric strategy, CVs of bare GCE, ZIF-8 / GCE and AgNWs@ZIF-8 / GCE were first tested, and then the electroactive areas of these electrodes were calculated according to the Randles-Sevcik equation. The formula used is

[0080] Ip = (2.69 x 10 5 ) N 3 / 2 AD 1 / 2 CV 1 / 2

[0081] where Ip represents the peak current, A represents the active area of the electrode, N is the number of transferred electrons in the system, D and C are the diffusion coefficient and concentration of K3[Fe(CN)6] 3- / 4- respectively, and V is the predetermined scan rate.

[0082] As shown in Figure 6 , for bare GCE, ZIF-8 / GCE and AgNWs@ZIF-8 / GCE, their electroactive areas are 0.05, 0.042 and 0.059 square centimeters respectively at a scan rate of 0.1 V / s -1 . The decrease in the electroactive area caused by ZIF-8 modification alone will affect the detection effect of the modified electrode, while the electroactive area of AgNWs@ZIF-8 / GCE is larger than that of ZIF-8 / GCE, indicating that the addition of Ag NWs not only provides the required Ag-S bond for the complementary single-stranded S1, but also enhances the electrical activity of the modified electrode.

[0083] As shown in Figure 7 and Figure 8 , the redox signal of AgNWs@ZIF-8 / GCE shows an upward trend when the scan rate is gradually increased in the range of 0.01-0.1 V / s, and a good linear relationship between the redox peak and the square root of the scan rate is established, indicating that a good diffusion control process occurs on AgNWs@ZIF-8 / GCE.

[0084] Through the above technical solution, we obtain a signal switch ratiometric electrochemical sensor based on a half-complementary aptamer pair carrying a signal molecule, which is used for natural Pb 2+ analysis. In the ratiometric electrochemical sensing process, the Pb 2+ aptamer acts as a basic recognizer and generates a target signal Fc. Based on the complementary pairing principle of the aptamer pair, S1@AgNWS@ZIF-8@MB self-assembles on the sensor to provide a reference signal MB. The reference signal MB provides a reference signal MB due to the competitive Pb 2+The double-strand break caused by absorption is inhibited, and the target signal Fc is enhanced due to the conformational change of the aptamer. Compared with single signal reading, the existing sensor provides more reliable ratio meter reading, and shows considerable analytical progress in LOD, selectivity and stability. In the determination of Pb 2+ in fish meat samples, the results of the sensor are comparable to those of ICP-MS and recovery test, and have good application prospect.

[0085] Description: The above examples are only used to illustrate the technical solutions described in the present application and not to limit the present application; therefore, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced by equivalents; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for preparing a high-precision electrochemical sensing device, characterized in that, Includes the following steps: Step 1: Synthesize beaded Ag NWs@ZIF-8 core-shell nanochains; Under stirring conditions, zinc nitrate hexahydrate and 2-methylimidazole were added to the silver nanowire solution to obtain a mixed solution. Stirring was stopped, and the mixed solution was reacted under certain temperature conditions. After the reaction, the product was collected by centrifugation. The product was washed several times with methanol and finally dried under vacuum to obtain the beaded Ag NWs@ZIF-8 core-shell nanochains for later use. Step 2: Electrodeposition of gold nanoparticles; (1) Dissolve NH4Cl in water to prepare ammonium chloride solution, and then mix the ammonium chloride solution with chloroauric acid solution to obtain precursor solution; (2) Pretreatment of glassy carbon electrode: First, polish the glassy carbon electrode with polishing powder, then rinse it with pure water; then move the glassy carbon electrode into an ultrasonic water bath for cleaning, and then use ethanol, HNO3 and distilled water for ultrasonic cleaning again in sequence. After cleaning, the electrode performance was tested to complete the pretreatment of the glassy carbon electrode. (3) Using the pretreated glassy carbon electrode as the working electrode, a linear voltammetric scan was performed in the precursor solution prepared in step (1). The glassy carbon electrode after the scan was denoted as AuNPs / GCE. Step 3: Aptamer incubation and MCH modification; The aptamer was drop-coated onto the AuNPs / GCE prepared in step two, incubated at room temperature and then dried naturally. After drying, the electrode was rinsed with PBS, and then the resulting electrode was immersed in 6-mercapto-1-hexanol. After immersion treatment, the modified electrode was obtained. Step four, modify the probe; Ag NWs@ZIF-8 was dispersed in a methylene blue solution and shaken overnight at room temperature to obtain an Ag NWs@ZIF-8 / MB suspension. Then, a single-stranded DNA semi-complementary to the aptamer was added to the Ag NWs@ZIF-8 / MB suspension, and the mixture was shaken at room temperature to obtain a mixture. The prepared mixture was centrifuged, the product was collected and washed several times with Tris-HCl buffer, and the washed product was resuspended in Tris-HCl buffer to obtain the Ag NWs@ZIF-8 / MB / S1 suspension. Finally, the Ag NWs@ZIF-8 / MB / S1 suspension was drop-coated onto the modified electrode treated in step three, and the electrochemical sensing device was obtained after incubation.

2. The method for preparing a high-precision electrochemical sensing device according to claim 1, characterized in that, In step one, the ratio of silver nanowire solution, zinc nitrate hexahydrate, and 2-methylimidazole is 0.5 mL: 25 mL: 25 mL; the concentration of the silver nanowire solution is 5 mg / mL, the concentration of zinc nitrate hexahydrate is 25 mM, and the concentration of 2-methylimidazole is 25 mM; the reaction conditions are: 30℃, 2 h; the centrifugation conditions are: 8000 rpm, 10 min; washing is performed 3-5 times; and vacuum drying is carried out at 60℃.

3. The method for preparing a high-precision electrochemical sensing device according to claim 1, characterized in that, In step 2(1), the concentration of ammonium chloride solution is 2.5M, and the volume concentration of chloroauric acid solution is 10%; the concentration of chloroauric acid in the precursor solution is 10mM.

4. The method for preparing a high-precision electrochemical sensing device according to claim 1, characterized in that, The polishing powder mentioned in step (2) is Al2O3, with particle sizes of 0.3 μm and 0.05 μm respectively; it is cleaned three times in an ultrasonic water bath, each time for 2-3 minutes; the specific steps for testing electrode performance are as follows: record 1×10 in 0.20 mol / L KNO3. -3 mol / L K3Fe(CN) 6 Cyclic voltammetry curves of the solution were used to test electrode performance, with a scan rate of 50 mV / s and a scan range of 0.6 to -0.1 V. The peak potential difference in the resulting cyclic voltammograms was 80 mV.

5. The method for preparing a high-precision electrochemical sensing device according to claim 1, characterized in that, The scanning range described in step (3) of step two is 0 to 0.8V, and the scanning time is 200 seconds.

6. The method for preparing a high-precision electrochemical sensing device according to claim 5, characterized in that, In step three, the concentration of the aptamer is 5–8 μM, the drop volume is 4–8 μL, the incubation time at room temperature is 2 h, the concentration of 6-mercapto-1-hexanol is 1 mM, and the soaking treatment time is 2 h.

7. The method for preparing a high-precision electrochemical sensing device according to claim 1, characterized in that, In step four, the concentration of the methylene blue solution is 1 mg / mL; the concentration range of the Ag NWs@ZIF-8 / MB suspension is 1 mg / mL-5 mg / mL; the volume ratio of the Ag NWs@ZIF-8 / MB suspension to single-stranded DNA is 20:1, the concentration of the single-stranded DNA is 5-8 μM, and the shaking time at room temperature is 60 min; the pH of the Tris-HCl buffer is 7.4, and the washed product is suspended in the Tris-HCl buffer at a ratio of 0.02-0.5 g: 1 mL. The amount of Ag NWs@ZIF-8 / MB / S1 suspension used for drop coating was 6 μL, and the incubation conditions were room temperature or 37 degrees Celsius for 1 h.

8. The electrochemical sensing device prepared by any one of claims 1-7 is used for detecting lead ions.

9. The use according to claim 8, characterized in that, The electrochemical sensing device is used to detect lead ions in fish meat, and the steps are as follows: (1) First, lead ion solutions of different concentrations were prepared, with the lead ion concentration range being 0.2 μM to 0.9 μM; then, the solutions were dropped onto the surface of the electrochemical sensing device, with a dropping amount of V1; differential pulse voltammetry was performed to observe the signal changes of the electrode under DPV at different lead ion solution concentrations, and the internal control signal and conformational change signal were recorded; the data were processed to obtain the ratio of signal change values, and then a standard curve was constructed by comparing it with the logarithm of the lead ion concentration. (2) Unknown sample detection: The sample is processed to obtain a sample solution; the operation method is the same as step (1), except that the lead ion solution is replaced with the sample solution. After detection, the ratio of the signal change values ​​is obtained, and the lead ion concentration in the fish meat can be calculated by substituting it into the standard curve in step (1).

10. The use according to claim 9, characterized in that, The amount of liquid added in step (1) is 4-8 μL; The specific steps of sample processing described in step (2) are as follows: ① The fish meat was washed with ultrapure water and evenly chopped as a sample. The sample was accurately weighed and transferred to a polytetrafluoroethylene tube. Then, an acid mixture was added to carry out the digestion reaction. After the reaction, hydrogen peroxide was added to the digestion product after natural cooling. After drying, a solid product was obtained. The acid mixture consisted of concentrated nitric acid and concentrated sulfuric acid in a fixed volume ratio of 1:

1. The ratio of sample, acid mixture, and hydrogen peroxide was 2g:30mL:10mL, with the mass fraction of hydrogen peroxide being 30%. The drying temperature was 200℃. ② The solid product was redistributed to 10 mM Tris-HCl buffer to obtain a mixed solution with a concentration of 5 mg / mL, which is the sample solution; wherein the Tris-HCl buffer is composed of 10 mM tris(hydroxymethyl)aminomethane, 100 mM sodium chloride and 1 mM disodium ethylenediaminetetraacetate.

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