Preparation method of dual-mode electrochemical biosensor for detecting lead in aquatic products

Abstract

The present invention discloses a preparation method of a dual-mode electrochemical biosensor capable of detecting lead ions in aquatic products. The electrochemical biosensor for lead-containing aquatic product detection of the present invention uses CeO2 / CS@AuNPs as a substrate material to improve the specific surface area and conductivity of the sensor, and can simultaneously bind an aptamer through an Au-S bond, utilize the recognition of cadmium ions by the aptamer, the catalytic properties of Ag / Fe2O3 nanozymes, the shearing of endonucleases, and the properties of DNA walkers to detect lead ions in aquatic products. The lead ions in the aquatic products to be detected can be quantified according to the current peak value in the detection result, and the presence of target ions in the sample to be detected can be qualitatively determined according to the change of the bottom liquid PBS. The advantages of the present invention are: the sensor realizes dual-mode detection of lead ions through color changes and electrical signal changes; signal amplification is realized through the endonuclease Nb.BbvCI, and the movement of the DNA walker is realized; complex pre-treatment is avoided, and heavy metals can be qualitatively and quantitatively determined separately.

Description

Technical Field

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

[0002] As the degree of environmental pollution increases, heavy metals in the environment are not only distributed in the soil and water, but also accumulated in aquatic products as heavy metals are deposited, and further accumulated in the human body through the food chain, causing certain harm to the human body; as people's economic ability and living standards improve, people's demand for aquatic products increases, and shellfish, fish, etc. become common foods on people's tables, which further aggravates the harm of heavy metals to the human body; heavy metal pollution is mostly point pollution, which cannot be decomposed by microorganisms and is easily enriched by organisms. Heavy metals enter the human body through the transmission of the food chain and damage human health; lead is a heavy metal element that seriously endangers human health. Since lead is cumulatively poisonous, only when the lead content in the human body reaches a certain level will it cause serious damage to the body's blood system and nervous system, especially the irreversible impact on children's health and intelligence. It is one of the common heavy metals in aquatic products; therefore, developing a convenient, efficient, fast and practical analytical method for heavy metal pollution, especially a method that can detect lead ions, is a current research hotspot. This work has important theoretical and practical significance for protecting the ecological environment, maintaining food safety, and improving food quality.

[0003] Heavy metal detection has become a hot topic of concern, and detection technology has been gradually improved. Common heavy metal detection technologies include atomic absorption spectroscopy, ultraviolet-visible spectrophotometry, microspectral analysis, atomic fluorescence, inductively coupled plasma mass spectrometry, etc., which have played an important role in the quantitative detection of heavy metals due to their high sensitivity; however, these analytical methods require the use of relatively expensive instruments, sample pretreatment is cumbersome, on-site detection is difficult, and professional technicians are required to operate, which further limits their application; therefore, it is particularly important to develop a rapid heavy metal detection method that is not only highly sensitive but also efficient and easy to operate; the electrochemical method is simple to operate, highly sensitive, and highly stable, and has a positive response to the detection of small molecules; the use of aptamers as recognition molecules further improves the specificity of detection while ensuring sensitivity; and the electrochemical aptamer sensor has the advantages of no need for pre-treatment of the sample to be tested, fast analysis speed, single-system inspection, high accuracy, strong specificity, and low cost.

[0004] The purpose of the invention is to provide an electrochemical biosensor that can overcome the above problems and is easy to prepare, simple to operate, highly sensitive, highly anti-interference, reproducible, selective and stable. The electrochemical biosensor can achieve signal amplification with the assistance of endonucleases, improve the sensitivity of detection, and can realize visual detection by combining the color development of TMB in the "nanozyme" catalytic base solution. A preparation method of an electrochemical biosensor is provided for detecting lead ions in aquatic products. Summary of the invention

[0005] Technical solution: Cerium oxide / mesoporous carbon@nano-gold composite materials are modified on the surface of pretreated glassy carbon electrode, and cyclic voltammetry and alternating current impedance method are used to record the current trend on the electrode surface; differential pulse voltammetry is used to record the current potential curve, and the size of the current peak can be used to analyze the amount of heavy metals contained in the electrode; when lead ions exist in the sensing system, the aptamer recognizes the target substance, the DNA walker combines with the Ag / Fe2O3 nanoprobe, and the probe is sheared under the action of the endonuclease to amplify the signal, the aptamer on the electrode surface is reduced, resulting in an increase in the electron transfer rate, and the sheared nanozyme Ag / Fe2O3 can catalyze TMB color development to achieve visual detection of target ions; the presence of lead ions can be judged by the color of PBS, and the lead ions in the system can be qualitatively and quantitatively determined based on the changes in the current signal.

[0006] The method for preparing an electrochemical aptamer sensor for detecting lead ions and mercury ions is characterized by: cleaning of a glassy carbon electrode (d=3 mm), construction of an electrochemical aptamer sensing interface and process characterization (preparation of nanosilver to modify the electrode), establishment of an electrochemical aptamer sensor working curve, detection of the performance of the electrochemical aptamer sensor, and detection of actual samples by the electrochemical aptamer sensor.

[0007] The preparation method of the electrochemical aptamer sensor for detecting lead ions is characterized by: optimizing the experimental conditions, mainly including the aptamer concentration, incubation practice, endonuclease concentration and base solution pH; the working curve of the prepared lead ion aptamer sensor is: Y=6.753Lgc Pb2+ +135.464(R 2 =0.994); the performance test of electrochemical aptamer sensor includes the determination of reproducibility, specificity, stability and recovery rate in actual aquatic products.

[0008] The preparation principle is as follows: the prepared cerium oxide / mesoporous carbon@nano-gold composite material is added as the base material on the electrode surface, followed by the modification of the mixed solution of the aptamer and the DNA walker, and then the Ag / Fe2O3 nanoprobe is coated, all of which are combined by Au-S bonds; when the target Pb 2+ When dropped onto the electrode, the aptamer recognizes Pb 2+The freed DNA walker walks on the electrode surface and complementarily pairs with the Ag / Fe2O3 nanoprobe bases. The cleavage site of the endonuclease Nb.BbvCI is exposed through pairing, and the cyclic amplification detection of the electrochemical signal of lead ions is realized under the action of the endonuclease. At the same time, with the increase of the cleaved Ag / Fe2O3 "nanozymes", the Ag / Fe2O3 nanomaterial can catalyze the color development of TMB, realizing the visual detection of lead ions, and thus completing the construction of the electrochemical aptamer sensor; the cerium oxide / mesoporous carbon@nanogold composite material used in this paper provides a good biological interface for the fixation of the target ion aptamer on the electrode surface, and at the same time improves the sensitivity of the sensor; the electrochemical biosensor prepared by the above steps is simple and sensitive, and has high stability, reproducibility and specificity, and the recovery rate meets the requirements.

[0009] To achieve the above objectives, a technical solution is adopted to achieve: a method for preparing a dual-mode electrochemical sensor for detecting lead heavy metals in aquatic products, characterized by: (1) the clarity, activation and performance testing of the glassy carbon electrode before preparing the aptamer sensor. If the peak potential difference in the test cyclic voltammetry curve is below 100 mV, and the oxidation peak and the reduction peak are symmetrical, the glassy carbon electrode can be used. Otherwise, it is necessary to return to the cleaning step until it meets the requirements; (2) the surface of the cleaned glassy carbon electrode is drop-coated with a uniformly dispersed cerium oxide / mesoporous carbon@nano-gold composite material, and then the aptamer, and the non-specific binding sites are blocked with 6-mercaptohexanol (MCH); after the preparation of the aptamer sensor is completed, it is placed in a refrigerator at 4°C for storage.

[0010] In order to achieve the above objectives, the following technical scheme is adopted: a method for preparing an electrochemical aptamer sensor for detecting lead ions and mercury ions, characterized in that: (1) the prepared aptamer sensor is scanned in a working base solution by cyclic voltammetry and differential pulse voltammetry to obtain an electrochemical signal of the aptamer sensor; (2) the target ion Pb 2+ The mixed solution of Pb and endonuclease Nb.BbvC was dropped onto the electrode. After a certain incubation time, cyclic voltammetry and electrochemical impedance spectroscopy were used to scan and further verify the feasibility of the sensor. (3) A series of concentrations of Pb 2+ (1) The standard solution was used for differential pulse voltammetry to further obtain the working curve, detection range and detection limit of the sensor; (2) A series of other common divalent metal ions were prepared for detection to verify the anti-interference ability of the prepared biosensor; (3) The stability and reproducibility of the sensor were further verified by differential pulse voltammetry; (4) The actual sample (scallop) was analyzed and the recovery rate was calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 SEM characterization of electrochemical sensors.

[0012] Figure 2 Electrochemical Characterization of Electrochemical Sensors.

[0013] Figure 3 Optimization of incubation time for electrochemical sensors.

[0014] Figure 4 Aptamer concentration optimization for electrochemical sensors.

[0015] Figure 5 Optimization of pH of the substrate solution for electrochemical sensors.

[0016] Figure 6 Optimization of endonuclease concentration for electrochemical sensors.

[0017] Figure 7 The differential pulse voltammetry curves of heavy metal lead ions with different concentrations were measured.

[0018] Figure 8 Study on the anti-interference ability of electrochemical sensors.

[0019] Fig. 9 Study on the stability of electrochemical sensors.

[0020] Fig.10 Reproducibility study of electrochemical sensors.

[0021] Fig.11 The electrochemical sensor measures the color curve of heavy metal lead.

[0022] Fig.12 Standard curve of electrochemical sensor measuring heavy metal lead. DETAILED DESCRIPTION

[0023] Example:

[0024] (1) Preparation of cerium oxide / mesoporous carbon@nanoporous gold composite material: 2 mL of 1% chloroauric acid was mixed with 200 mL of ultrapure water, and then the mixture was heated to boiling. After that, the mixture was taken out of the glass bottle, and 4.8 mL of 1% trisodium citrate was added while shaking. The mixture was then heated until the solution turned wine red, and then cooled to room temperature and the volume was adjusted to 200 mL for later use. Then, 4 mg of cerium oxide was dissolved in 4 mL of 0.2% mesoporous carbon, and an ivory white solution was obtained after ultrasonic dispersion, which was the CeO2 / mesoporous carbon solution. Equal amounts of nanogold and CeO2 / mesoporous carbon solution were thoroughly shaken and mixed at room temperature overnight to obtain CeO2 / mesoporous carbon@AuNPs for later use.

[0025] (2) Preparation of Ag / Fe2O3 nanomaterials: 0.5 g Fe(NO3)3·9H2O, AgNO3 and PVP were dissolved in 35 mL ethylene glycol and 5 mL dimethylformamide. After vigorous stirring, the mixture was heat treated at 160°C for 8 h in an oil bath, cooled to room temperature, centrifuged and washed several times with anhydrous ethanol and ultrapure water to obtain an Ag / Fe2O3 solution, which was stored at 4°C for later use.

[0026] (3) Preparation of Ag / Fe2O3 nanoprobes: Ag / Fe2O3 solution and cDNA were mixed in equal amounts and incubated at 37°C for 1 h to obtain Ag / Fe2O3 / cDNA probes;

[0027] (4) Cleaning of glassy carbon electrode: Before modification of glassy carbon electrode, 0.3 μm Al2O3 was used to polish the suede to a mirror surface. After polishing, the surface dirt was removed by washing with deionized water. Then, the electrode was moved to an ultrasonic water bath for cleaning for 5 min each time, and repeated twice. The electrode was ultrasonically cleaned with anhydrous ethanol and deionized water, and dried under nitrogen environment.

[0028] (5) Test of glassy carbon electrode: In the presence of 0.1M KCl and 5mM [Fe(CN)6] 3- / 4- The performance of the glassy carbon electrode is tested by running a cyclic voltammetry curve in a 0.01M 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 is further processed in step (4) until it meets the requirements;

[0029] (6) mixing equal amounts and concentrations of aptamers and DNA walkers and incubating them for a period of time;

[0030] (7) The prepared cerium oxide / mesoporous carbon@nano-gold composite material was modified on the surface of the polished working electrode. After the substrate material was fixed, the aptamer and DNA walker mixture was added dropwise, followed by the Ag / Fe2O3 / cDNA probe. After the aptamer was fully bound, MCH was added to block other unbound sites on the electrode, and finally the target ion Pb was added dropwise. 2+ and endonuclease Nb.BbvCI, and then test after the electrode surface is completely dry;

[0031] (8) Using a three-electrode system, differential pulse voltammetry was used to optimize the experimental conditions such as incubation time, aptamer concentration, and base solution pH in potassium ferrocyanide. The optimal conditions were: aptamer concentration of 100 nM, incubation time of 50 min, and base solution pH of 7.5;

[0032] (9) Interfering ions such as zinc ions, nickel ions, magnesium ions, mercury ions, iron ions, copper ions, cobalt ions, and silver ions were added to the detection system for detection. At the same time, in order to verify the stability of the sensor, the electrochemical signals of 0 days, 5 days, 10 days, 15 days, and 20 days were selected as a group of 3 electrodes for comparison. Under the same conditions, 36 electrodes were prepared to detect the same concentration of lead ions to verify the reproducibility of the sensor.

[0033] (10) After filtering the actual sample, add lead ion standard solution to test the actual sample.

[0034] This electrochemical sensor can simultaneously detect heavy metal lead ions in water. The sensor has low cost, simple operation, strong anti-interference ability, good stability, strong specificity, and can simultaneously detect two heavy metals qualitatively and quantitatively. It is non-toxic and has high detection sensitivity.

Claims

1. A method for preparing an electrochemical biosensor for detecting lead ions, characterized in that: Here are the steps: (1) Preparation of cerium oxide / mesoporous carbon@nano-gold composite material: 2 mL of 1% chloroauric acid was mixed with 200 mL of ultrapure water, and then the mixture was heated to boiling. After that, the mixture was taken out of the glass bottle, and 4.8 mL of 1% trisodium citrate was added while shaking. The mixture was then heated until the solution turned wine red, cooled to room temperature, and the volume was adjusted to 200 mL for later use; 4 mg of cerium oxide was then dissolved in 4 mL of 0.2% mesoporous carbon, and an ivory white solution was obtained after ultrasonic dispersion, which was the CeO2 / mesoporous carbon solution; equal amounts of nano-gold and CeO2 / mesoporous carbon solution were thoroughly shaken and mixed at room temperature overnight to obtain CeO2 / mesoporous carbon@AuNPs for later use; (2) Preparation of Ag / Fe2O3 nanomaterials: Preparation of Ag / Fe2O3 nanomaterials: 0.5 g of Fe(NO3)3·9H2O, AgNO3 and PVP were dissolved in 35 mL of ethylene glycol and 5 mL of dimethylformamide. After vigorous stirring, the mixture was heat treated at 160°C in an oil bath for 8 h, cooled to room temperature, centrifuged and washed several times with anhydrous ethanol and ultrapure water to obtain an Ag / Fe2O3 solution, which was stored at 4°C for later use. (3) Preparation of Ag / Fe2O3 nanoprobes: Ag / Fe2O3 solution and cDNA were mixed in equal amounts and incubated at 37°C for 1 h to obtain Ag / Fe2O3 / cDNA probes; (4) Cleaning the glassy carbon electrode; (5) Testing glassy carbon electrodes; (6) Mix and incubate equal amounts and concentrations of aptamers and DNA walkers; the aptamer concentration is 100 nM, the incubation time is 50 min, and the base solution pH is 7.5; (7) The surface of the working electrode is modified using the cerium oxide / mesoporous carbon@nano-gold composite material prepared in step (1). After the substrate material is fixed, the aptamer and DNA walker mixture in step (6) is added dropwise, and then the Ag / Fe2O3 / cDNA probe prepared in step (3) is added dropwise. After the aptamer is completely bound, MCH is added dropwise to block other unbound sites on the electrode, and finally the target ion Pb is added dropwise. 2+ and endonuclease Nb.BbvCI mixture, and the process is completed after the electrode surface is completely dry.

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