Aptamer-based colorimetric and electrochemical dual-mode visual Pb(Ⅱ) electrochemical sensor

Abstract

The invention discloses a method for preparing a colorimetric and electrochemical dual-mode visualized Pb(Ⅱ) electrochemical sensor based on an aptamer, which belongs to the technical field of combining material science with electrochemical sensors. The invention adopts a functionalized aptamer to modify a glassy carbon electrode to realize dual-mode detection of Pb(Ⅱ) by the sensor. Pb(Ⅱ) plays a specific cleavage enzyme role to cleave the aptamer, so that the functionalized aptamer chain of Au@Pd NPs is bound to the electrode to realize the enhancement of the electrochemical signal, and the electrochemical detection of Pb(Ⅱ) is realized according to the change of the electrochemical signal intensity by using differential pulse voltammetry. The porous core-shell structure of Au@Pd NPs provides sufficient active sites for hydrogen peroxide, plays a role of catalase, catalyzes hydrogen peroxide, causes the oxidative discoloration of the substrate 3,3',5,5'-tetramethylbenzidine, and realizes the colorimetric detection of Pb(Ⅱ) according to the color change. The minimum detection limit of the Pb(Ⅱ) sensor prepared by the invention is 0.4 nM, and was successfully applied to the quantitative and colorimetric detection of Pb(Ⅱ) in edible fungi, laying the foundation for the safe and rapid detection of edible fungi.

Description

Technical Field

[0001] The invention relates to an electrochemical sensor and a preparation method and a use method of the sensor, belonging to the technical field of combining material science with electrochemical sensors. Background Art

[0002] Heavy metal pollution has become a global environmental problem. Heavy metals contained in exhaust gas, liquid and solid waste from industrial manufacturing, agricultural production and transportation enter the environment and human body through the food chain, causing serious cumulative harm. Edible fungi are widely distributed all over the world. Agricultural waste such as sawdust is the main growth matrix of edible fungi. They can use biopolymers (such as cellulose) as a source of nutrition. Therefore, due to heavy metal pollution in the environment, the heavy metal content in edible fungi is relatively high, which will cause serious harm to food safety.

[0003] At present, traditional heavy metal detection methods, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry (AFS) and X-ray fluorescence spectrometry (XRFS) all rely on large equipment, which is not only costly but also requires professional operation. Therefore, it is necessary to develop a heavy metal detection method with low cost, high selectivity and strong stability.

[0004] Electrochemical detection technology has the characteristics of short detection time, strong specificity and small instrument size, which is suitable for rapid on-site detection; metal nanoparticles are easy to synthesize, low cost, have excellent electrocatalytic properties and good biocompatibility, and the unique porous core-shell structure of gold-palladium bimetallic nanoparticles gives them a large specific surface area and a large number of hydrogen peroxide catalytic sites; in the presence of hydrogen peroxide, the substrate 3,3',5,5'-tetramethylbenzidine (TMB) transfers its electrons to the nanomaterial and can be further oxidized to a blue product (ox TMB); the combination of specific aptamers and electrochemical sensors can greatly improve the sensitivity of detecting heavy metal ions; deoxyribozyme 8-17 is a DNA metalloenzyme that catalyzes RNA ester exchange in the presence of divalent metal ions; Pb(II) deoxyribozyme consists of an enzyme chain and a substrate chain. In the presence of target Pb(II), the four substrate chains can be split into two segments, enhancing the specific detection of lead ions. Summary of the invention

[0005] The purpose of the present invention is to prepare a dual-mode visual Pb(II) electrochemical sensor based on an aptamer, which improves the specificity and selectivity of the electrochemical sensor in detecting heavy metal ions, combines visual prediction with accurate electrochemical quantification, and realizes efficient and specific detection of heavy metal lead.

[0006] The technical scheme of the present invention is as follows: a preparation method of a dual-mode visualized electrochemical Pb (II) sensor based on an aptamer, wherein the used aptamer S2 has a lead ion-specific recognition site and can be specifically sheared by the lead ions; firstly, silver nanoparticles are dripped onto a pretreated electrode surface to form a uniform surface layer, and then aptamer chains S1-S2 and a certain concentration of lead are dripped, and the lead ions cause S2 to be cracked, exposing S1 to the electrode surface, and then long chain S4 is dripped to hybridize with S1, and then S3 chains with gold-palladium bimetallic nanoparticles are dripped to hybridize with the remaining exposed part of S4, thereby constructing a complete sensor, and using differential pulse voltammetry to detect heavy metal lead under optimal conditions.

[0007] Construction of the sensing interface: (1) Cleaning and performance testing of the bare glassy carbon electrode before preparing the electrochemical sensor. If the potential difference between the oxidation peak and the reduction peak in the cyclic voltammetry curve is ≤100 mV and the oxidation peak and the reduction peak are symmetrical, the glassy carbon electrode can be used. (2) Evenly dispersed nanosilver solution is drop-coated on the surface of the cleaned bare glassy carbon electrode. After drying, evenly mixed aptamer chains S1-S2 and lead ions are drop-coated on the surface to cleave the aptamer chains in the presence of lead ions. Then, long chain S4 and aptamer chain S3 with gold-palladium bimetallic nanoparticles are drop-coated in turn to hybridize with S1. After complete drying, the electrochemical sensor is completed and stored at room temperature for later use.

[0008] Electrochemical detection of target ions: Differential pulse voltammetry is used to detect heavy metal lead ions using a three-electrode system under optimal experimental conditions. The type of heavy metal ions and the concentration of metal ions contained in the solution can be determined by analyzing the position and size of the dissolution current peak in the test results, and the heavy metal ion concentration information can be obtained based on this.

[0009] The preparation principle of the invention is as follows: coating a nanosilver solution on the surface of a glassy carbon electrode to form a uniformly distributed nanosilver surface layer, dripping aptamers S1-S2, modifying the 5' end of the aptamer S1 with a thiol group (-SH), connecting the aptamer S1 with the electrode by using an Ag-S bond, dripping lead ions, utilizing the shearing effect of the lead ions on S2 to cause it to be cleaved, exposing the substrate chain S1 to the electrode; dripping long chain S4 to hybridize with S1, and then dripping S3 chains with gold-palladium bimetallic nanoparticles to hybridize with the remaining exposed part of S4, thereby obtaining a constructed aptamer sensor; the electrochemical sensor prepared by the above steps is simple to operate, has high sensitivity, good anti-interference and stability, can realize rapid detection, has no special requirements for samples, and does not require complex pretreatment.

[0010] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a dual-mode visualized electrochemical sensor that can combine visual prediction and electrochemical quantification; provides visual judgment of heavy metal detection by the principle that gold-palladium bimetallic nanoparticles catalyze hydrogen peroxide to cause TMB oxidation and discoloration; improves the specificity and selectivity of electrochemical sensors for detecting heavy metal ions by using aptamers as recognition elements, and effectively improves the specificity of heavy metal detection; in addition, the preparation of this sensor can be used for the quantitative detection of heavy metal lead in edible fungi, laying a foundation for the development of portable devices for rapid detection of heavy metal pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Flowchart of electrochemical biosensor construction.

[0012] Figure 2 Scanning electron microscopy, transmission electron microscopy, energy spectrum elemental analysis, X-ray diffraction patterns and UV-visible absorption spectrum characterization diagrams.

[0013] Figure 3 Agarose gel electrophoresis characterization diagram.

[0014] Figure 4 Conductivity study of the sensor construction process.

[0015] Figure 5 Optimization of aptamer concentration and detection solution pH of electrochemical sensors.

[0016] Figure 6 Measure the differential pulse voltammetry curves and standard curves of lead ions with different concentrations.

[0017] Figure 7 Image of base solution color change under different lead ion concentrations.

[0018] Figure 8 Differential pulse voltammetry testing of the sensor construction process.

[0019] Fig. 9 Reproducibility study of electrochemical sensors.

[0020] Fig.10 Study on anti-interference and stability of electrochemical sensors. DETAILED DESCRIPTION

[0021] Example:

[0022] (1) The prepared silver nanoparticles were washed five times with anhydrous ethanol and ultrapure water, centrifuged at 13,000 rpm for 10 min, and the supernatant was removed. The remaining precipitate was diluted with ultrapure water and aged at 4°C for two days before use. Preparation of gold-palladium bimetallic nanoparticles: In a boiling water bath, 44.5 mg PdCl2 was dissolved in 25 mL 20 mM hydrochloric acid solution to prepare an H2PdCl4 aqueous solution. The H2PdCl4 aqueous solution (10 mM, 0.5 mL), HAuCl4 aqueous solution (10 mM, 0.1 mL) and cetylpyridinium chloride (0.02 g) were added to 5 mL ultrapure water and ultrasonicated to form a uniform suspension. The newly prepared ascorbic acid aqueous solution (0.1 M, 0.4 mL) was quickly injected into the mixed solution under stirring, and the mixture was allowed to stand at 35°C for 120 min. The nanomaterials (i.e., Au@PdNPs) were collected by centrifugation and washed twice with ultrapure water. The obtained Au@Pd NPs were redispersed in 2 mL ultrapure water, stored at 4°C for future use;

[0023] (2) Pretreatment of aptamer chains before modification: Before modification, the aptamer chains modified with thiol groups were placed in 10 mM TCEP for 60 min to unwind the disulfide bonds. A mixture (S1-S2) containing 1.0 μM enzyme chain and 1.0 μM substrate chain was prepared in 1.0 mL PBS (10 mM, pH 7.4). The mixture was incubated at 65°C for 10 min, cooled to room temperature, and reacted on a shaker at room temperature for 12 h. The obtained S1-S2 solution was stored at 4°C for further use. To obtain S3-labeled porous bimetallic nanoparticles, the prepared S3 and 0.3 mL of porous bimetallic nanoparticles were mixed in 1.2 mL PBS and gently shaken at room temperature for 6 h. After centrifugation, the obtained precipitate was dispersed in 1.0 mL PBS for target lead ion detection.

[0024] (3) Cleaning of glassy carbon electrode: Before modification of glassy carbon electrode, polish the suede to a mirror surface with 0.3 μm Al2O3. After polishing, wash with deionized water to remove surface dirt, then move to ultrasonic water bath for cleaning, 5 min each time, repeat 2 times, ultrasonically clean with anhydrous ethanol and deionized water, and dry under nitrogen environment;

[0025] (4) Test of glassy carbon electrode: In the presence of 0.1 M KCl and 5 mM [Fe(CN)6] 3- / 4- The performance of the glassy carbon electrode is tested by running a cyclic voltammetry curve in a 0.01 M PBS solution at a scanning speed of 50 mV / s and a scanning potential of -0.1 to 0.6 V; when the redox peak potential difference in the cyclic voltammetry curve is below 100 mV, the glassy carbon electrode can be further used, otherwise, the glassy carbon electrode should be processed again in step (2) until it meets the requirements;

[0026] (5) Add silver nanoparticles to the surface of the pretreated glassy carbon electrode, and then add aptamer chains S1-S2, lead ions, long chains S4, and S3 with gold-palladium bimetallic nanoparticles in sequence after drying. After the electrode surface is completely dry, store it at room temperature for later use;

[0027] (6) A three-level system was used to optimize experimental factors such as aptamer concentration and test solution pH using differential pulse voltammetry. The lead ion aptamer concentration was 400 nM and the test solution pH was 7.0.

[0028] (7) Under the optimal test conditions, differential pulse voltammetry was used to perform concentration gradient quantitative analysis. The base solution pH was 7.0, and the lead ion concentration range for the test was 1-1000 nM. The minimum detection limit of lead ions was 0.4 nM, and the regression equation was ΔI=0.006C+1.321 (C: nM, R 2 =0.990), see attached Figure 6 ;

[0029] (8) Seven electrochemical sensors prepared by adding lead (400 nM), calcium, magnesium, zinc, iron, nickel (4 μM) and mixed interfering ion pairs into the test system were tested under differential pulse voltammetry to detect their anti-interference properties; the sensors were stored in a 4°C refrigerator and tested every 3 days for a total of 15 days to observe their stability;

[0030] (9) The edible fungus sample was incinerated and dissolved in nitric acid. Ultrapure water was added to prepare the sample solution, which was mixed with the buffer solution in a 1:1 ratio. The pH was adjusted and the lead ion solution was added. The standard addition method was used to detect different concentrations of lead ions in oyster mushrooms, shiitake mushrooms and seafood mushrooms, and the actual sample was analyzed.

[0031] This electrochemical sensor can detect lead ions in edible fungi. The sensor has low cost, simple operation, strong anti-interference ability, good stability, and strong specificity. It can detect heavy metals from both visual and electrochemical aspects at the same time. It is non-toxic and has high detection sensitivity.

Claims

1. A method for preparing a colorimetric and electrochemical dual-mode visual Pb(II) electrochemical sensor based on an aptamer, characterized in that: 5 μL of silver nanoparticles were dripped onto the glassy carbon electrode to form a uniform nanosilver surface layer; the aptamer chain S1-S2 was dripped and connected to the electrode using a silver-sulfur bond; the enzyme chain of DNA metalloenzyme deoxyribozyme 8-17 and the substrate chain were used to form Pb(Ⅱ) deoxyribozyme, and after the addition of Pb(Ⅱ), the RNA ester exchange reaction was catalyzed to cleave S2 and expose the substrate chain S1 to the electrode; the long chain S4 was dripped to hybridize with S1, and then the S3 chain with Au@Pd NPs was dripped to hybridize with the exposed part of S4 to complete the construction of the sensor; the aptamer concentration was 400 nM, TMB was used as the substrate, a three-electrode system was used, and the pH value of the test base solution was 7.0.

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