Silver-doped hollow carbon nanospheres electrochemical probe and preparation method thereof

Abstract

The application relates to a silver-doped hollow carbon nanosphere electrochemical probe and a preparation method thereof, and belongs to the technical field of electroanalysis chemical detection and carbon nanomaterials. The Ag@HCS composite material is prepared by using a SiO2 template method, polymerizing formaldehyde and m-diphenol to form a phenolic resin ball in the presence of silver nitrate, and then performing carbonization treatment and in-situ reduction of silver and removing the template. The Ag@HCS is used for HbA 1c The determination of HbA 1c is realized based on the silver-based electrochemical oxidation signal of the electrochemical probe after the modification of the nucleic acid aptamer. Experiments show that the electrochemical sensor constructed by the method has high sensitivity, a wide detection range, high specificity and good stability, and exhibits good feasibility in the detection of actual samples, and the method provides a method for synthesizing a stable, controllable and sensitive electrochemical probe.

Description

Technical Field

[0001] This invention belongs to the fields of carbon nanomaterials and electroanalytical chemistry, specifically relating to a silver-doped hollow carbon nanosphere and its controllable preparation, and its use as an electrochemical probe for the determination of glycated hemoglobin. Background Technology

[0002] Carbon-based nanomaterials possess excellent electronic conductivity, thermal stability, good mechanical strength, and biocompatibility. Coupled with their abundant sources and low cost, they have found wide application in electrocatalysis and electroanalysis. Researchers have employed various methods for preparing carbon-based materials, including chemical vapor deposition, hydrothermal carbonization, and precursor carbonization (H. Wang, Y. Shao, S. Mei, Y. Lu, M. Zhang, J. K. Sun, K. Matyjaszewski, M. Antonietti, J. Yuan, Chem Rev. 2020, 120, 9363.). In particular, polymer precursor carbonization, through temperature regulation to control the carbonization process, allows for the controllable preparation of carbon nanostructures.

[0003] Phenolic resin spheres, synthesized from resorcinol and / or its derivatives with aldehydes (such as formaldehyde and furfural) to form macromolecular polymer structures, have been used as novel carbon precursors due to their regular structure and controllable morphology (Z. Mao, L. Cao, F. Zhang, ACS Appl Mater Interfaces, 2018, 10, 28709-28718). By changing the template and carbonization conditions, carbon nanospheres with different morphologies and functions can be designed and prepared (W. Li, G. Wang, W. Sui, T. Xu, Z. Li, AMParcvez C. Si, Carbon, 2022, 196, 819). Because phenolic resin-derived carbon nanospheres possess advantages such as good biocompatibility, ease of functionalization, high conductivity, and simple preparation, they show great promise in sensing, medical imaging, and bioimaging. Furthermore, to endow carbon nanospheres with more functions, they are typically modified with metal dopants to impart electrochemical and high catalytic activity; and since phenolic resins contain a large number of hydroxyl groups, they can be used for in-situ reduction of metals, thus eliminating the need for additional reducing agents. In summary, the combination of mesoporous carbon materials and metals represents an excellent composite material with promising applications in the field of electrochemistry.

[0004] Based on phenolic resin spheres as carbon precursors, silver-doped carbon spheres are generated by in-situ reduction of metallic silver salts during carbonization. Due to the electrochemical oxidation activity of silver itself, there are no reports yet on using this composite material to construct electrochemical nanoprobes for electrochemical sensing. Summary of the Invention

[0005] Based on existing technology, the present invention aims to provide an electrochemical probe based on uniformly dispersed silver clusters (Ag@HCS) doped in hollow carbon nanospheres and its synthesis method; another objective is to provide the composite material for glycated hemoglobin (HbA1c) synthesis. 1c Applications in electrochemical sensors.

[0006] To achieve the objective of this invention, a SiO2 template method is used to polymerize formaldehyde and resorcinol in the presence of silver nitrate to form phenolic resin spheres. These spheres are then carbonized, and silver is reduced in situ. After template removal, Ag@HCS composite materials are prepared. Ag@HCS is then subjected to HbA... 1c After modification with nucleic acid aptamers, HbA1c is used as an electrochemical probe based on the electrochemical oxidation signal of silver. 1c The determination.

[0007] The following technical solution is specifically adopted:

[0008] (1) Add ethanol, water and tetraethyl silicate to a beaker to dissolve and disperse evenly, then add NH3·H2O to react and obtain SiO2 monomer solution.

[0009] (2) Add m-diphenol, formaldehyde and silver nitrate to the SiO2 monomer solution prepared in step (1) and react. After solid-liquid separation by centrifugation, dry and preserve the silver cluster-doped phenolic resin balls (Ag@RF@SiO2).

[0010] (3) The Ag@RF@SiO2 obtained in step (2) is carbonized at high temperature, and then the carbonized Ag@HCS@SiO2 is mixed with NaOH solution to remove the SiO2 template, thus obtaining Ag@HCS material.

[0011] (4) The obtained Ag@HCS material is mixed with HbA 1c The aptamers were mixed in phosphate buffer solution, shaken in the dark, and then separated and preserved to obtain apt-Ag@HCS.

[0012] (5) Place the carbon cloth electrode in a container containing HbA 1c The aminophenylboronic acid monomer solution was subjected to CV cycling, and then HbA was washed away with NaOH. 1c Molecularly imprinted electrodes were obtained.

[0013] (6) Add HbA at different concentrations to the molecularly imprinted electrode obtained in step (5). 1c Then, it is placed in a solution containing the apt-Ag@HCS probe, and sandwich HbA1c is captured by the nucleic acid aptamer. 1c Construction of the biosensor. In application, differential pulse voltammetry is used to measure HbA1c levels via the silver cluster oxidation peak of the Ag@HCS probe. 1c Quantitative determination.

[0014] Furthermore, in the polymerization process of the phenolic resin balls, the characteristic is that the molar ratio of formaldehyde to resorcinol in step (2) is 4.2:1.

[0015] Furthermore, in the polymerization process of the phenolic resin balls, the characteristic is that the mass of AgNO3 added in step (2) is 12.5% ​​of the amount of resorcinol.

[0016] Furthermore, the template removal in the Ag@HCS preparation process: in 1 mol L -1 The SiO2 template was removed by reacting in NaOH solution at 70℃ for 5 hours.

[0017] Furthermore, the high-temperature carbonization process in the Ag@HCS preparation process is carried out under Ar gas protection at a carbonization temperature of 700℃ for 5 hours.

[0018] Furthermore, the preparation process of the electrochemical probe apt-Ag@HCS is as follows: the amount of nucleic acid aptamer added to 2g Ag@HCS dispersion is 5OD, and the reaction time is 2h.

[0019] The innovation of this invention lies in the following: Silver nitrate is added during the polymerization of formaldehyde and resorcinol using a SiO2 template method, followed by carbonization to prepare hollow carbon nanospheres with controllable morphology supported on silver clusters. This Ag@HCS composite material, combined with a nucleic acid aptamer, serves as an electrochemical signal probe, enabling the construction of a sandwich-type electrochemical sensor on the surface of a molecularly imprinted electrode for HbA1c synthesis. 1c Highly sensitive and selective quantitative determination.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The Ag@HCS probe was prepared by one-pot polymerization and subsequent carbonization and template removal. The preparation process is simple and the morphology is controllable.

[0022] (2) The prepared Ag@HCS probe has a large specific surface area (see Appendix). Figure 2 a) Uniform and high silver cluster loading promotes electron conduction, provides amplified electrochemical signals, and exhibits good stability (see Appendix). Figure 5 d.

[0023] (3) HbA 1c The highly specific binding between the molecularly imprinted electrode and the apt-Ag@HCS probe endows the electrochemical sensor with high selectivity, exhibiting excellent specificity in the presence of ascorbic acid, uric acid, bilirubin, urea, triglycerides, and glucose, etc. (See Appendix) Figure 5 (c)

[0024] (4) The electrochemical sensor of this invention realizes HbA1c Highly sensitive and selective determination, with a detection limit of 0.35 μg / L. -1 This achieved the detection of HbA1c in serum samples. 1c Quantitative detection. Attached Figure Description

[0025] Figure 1 This is the technical roadmap of the present invention.

[0026] Figure 2 These are characterization images of the Ag@HCS composite material prepared in this invention using scanning electron microscopy (a), high-resolution transmission microscopy (bc), and energy dispersive spectroscopy (d, d1, d2, d3).

[0027] Figure 3 These are scanning electron microscope images of the carbon cloth electrode used in this invention before and after electropolymerization of aminophenylboronic acid.

[0028] Figure 4 (a) is an impedance characterization diagram during the fabrication of the sensor of the present invention. In (a) diagram, (1) is a conventional carbon cloth electrode, and (2) is a electrode containing HbA. 1c The electropolymerized carbon cloth electrode, (3) is used to remove HbA 1c Molecularly imprinted modified carbon cloth electrode, (4) is HbA 1c The carbon cloth electrode modified with molecular imprinting after recognition, (5) the electrode after capturing the Ag@HCS probe (illustration: Randel equivalent circuit model). (b), (c), and (d) are pH, number of electropolymerization cycles, and HbA1c, respectively. 1c Optimization of incubation time conditions.

[0029] Figure 5 The sensor of this invention is for HbA 1c The response is shown in the following figures: (a) is the measured differential pulse voltammetry curve; (b) is the standard curve of current versus concentration; (c) is the selectivity of the electrochemical sensor of the present invention; and (d) is the stability of the electrochemical sensor of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments. The following embodiments are only used to illustrate the present invention, but do not limit the scope of the present invention in any way.

[0031] Example 1: Preparation of electrochemical probe Ag@HCS and construction of its electrochemical sensing system

[0032] (1) Add 10 mL of ethanol, 60 mL of water and 3 mL of tetraethyl silicate to a beaker to dissolve and disperse evenly. At this time, the solution is transparent. Then add 3 mL of NH3·H2O and react for 15 minutes to obtain a SiO2 monomer solution. At this time, the solution is milky white.

[0033] (2) Continue stirring the SiO2 obtained in step (1), then add 0.4g of m-diphenol, 560μL of formaldehyde and 50mg of silver nitrate to the SiO2 monomer solution obtained in step (1) and react for 24h. The solution color turns pink. Finally, the silver cluster-doped phenolic resin balls (Ag@RF@SiO2) are dried and stored after solid-liquid separation by centrifugation.

[0034] (3) The Ag@RF@SiO2 obtained in step (2) was carbonized at 700℃ in an Ar atmosphere for 5 hours. Then the carbonized Ag@HCS@SiO2 was mixed with 50 mL of 1 mol L -1 The SiO2 template was removed by mixing NaOH solution at 70℃ for 5 h, yielding the electrochemical probe Ag@HCS.

[0035] (4) Disperse 2g of the prepared Ag@HCS in 50mL of 0.1M PB (pH 7.4) for later use, and add 5OD HbA 1c Nucleic acid aptamers (sequence 5′-SHC6 / GGC AGG AAG ACA AAC ACA TCG TCG CGG CCT TAG GAG GGGCGG ACG GGG GGG GGC GTT GGT CTG TGG TGC TGT). The mixture was shaken in the dark for 2 hours to allow the aptamers to bind to Ag@HCS via Ag-S bonds. The prepared apt-Ag@HCS was then washed with water, centrifuged, dried, and then dispersed in 10 mL of deionized water for later use.

[0036] (5) Place the carbon cloth electrode (0.5*1.5) in a container containing HbA 1c In an aminophenylboronic acid monomer solution (20 mmol L -1 Aminophenylboronic acid monomer, 100 mmol L -1 NaF and 400 μg HbA 1c 0.1 mmol L -1 HCl) CV cycle 25 times, then with 0.1 mol L -1 NaOH washes away HbA 1c The construction of a molecular film-modified carbon cloth electrode was completed.

[0037] (6) Add HbA at different concentrations to the carbon cloth electrode modified with the molecularly imprinted film obtained in step (5). 1c After incubating for approximately 12 minutes, add 1 mL of apt-Ag@HCS solution and allow to stabilize for 15 minutes. Then use 0.1 mol / L... -1 After washing with phosphate buffer, HbA1c was analyzed by recording differential pulse voltammetry curves from the Ag oxidation peak.1c Testing.

[0038] Application Example 1: The Ag@HCS electrochemical probe of this invention is applied to HbA1c. 1c Measurement

[0039] Potentiometric measurements were performed using a three-electrode system, with a carbon cloth electrode as the working electrode, and a carbon electrode and an Ag / AgCl electrode used as the counter and reference electrodes, respectively. At 0.1 mol L... -1 In a phosphate buffer solution (pH = 7.0), the voltammetric response signal of the sensor was recorded using differential pulse voltammetry within the range of -0.1V to 0.3V under conditions of pulse amplitude of 0.004V, pulse width of 0.05μs, and sampling width of 0.0167μs. The signal was obtained by measuring different concentrations of HbA1c. 1c Standard solution, calculate the addition of HbA 1c The peak current of the silver cluster in the probe molecule was used to plot a standard curve using peak current value versus concentration. This peak current I... P With HbA 1c Concentration (C) at 0.8 μg / L -1 ~78.4 μg L -1 The correlation coefficient R is directly proportional to the range of values. 2 The value was 0.98, and the detection limit was 0.35 μg / L. -1 This indicates that the electrochemical sensor is used for HbA1c. 1c The determination has high sensitivity.

[0040] Application Example 2: Performance Evaluation of This Electrochemical Sensor

[0041] To investigate the stability of the sensor of this invention, the same electrode was prepared and tested repeatedly eight times. The relative standard deviation was less than 3.5%, indicating that the prepared hybrid electrode is effective against HbA2+. 1c The assays exhibit excellent repeatability. To investigate the reproducibility of the sensor of this invention, measurements were performed from 10 μg mL... -1 HbA 1c The oxidation current response was estimated to have fabrication reproducibility relative to eight different electrodes prepared independently in the same manner, with a deviation of 3.2% from the standard. To investigate the long-term stability of the electrochemical assay, the prepared electrodes were retained for 14 days. After 14 days, the current decreased by only about 2%. These results indicate that the sensor exhibits good stability due to the stability and environmental compatibility of the Ag@HCS material used in the sensing system. Furthermore, the use of the electrochemical signal of the silver cluster as a probe ensures accurate and stable detection applications.

[0042] To evaluate the specificity of the sensor for glycated hemoglobin, ascorbic acid, uric acid, bilirubin, urea, triglycerides, and glucose were added to a 0.1 mol / L solution at pH 7.4. -1In phosphate buffer solution, when 6.5 μg / mL is added to a solution containing the aforementioned interfering substances... -1 HbA 1c At that time, the peak current is only HbA 1c The peak currents in the solution were consistent. Based on the experimental data, the sensing system constructed using the Ag@HCS electrochemical probe effectively monitored HbA1c in laboratory standard samples. 1c The assay showed high sensitivity and good specificity.

[0043] Application Example 3: HbA in a real sample 1c Measurement

[0044] To evaluate the detection of HbA by an electrochemical sensing system 1c To ensure practicality and simulate the real human body fluid environment, a spiked recovery experiment was conducted in serum. To avoid interference from real samples, 0.1 mol / L HCl was used before the assay. -1 Serum was diluted with phosphate buffer (pH = 7.4), followed by relevant electrochemical tests. As shown in Table 1, the recoveries ranged from 98.7% to 99.6%, fully meeting the requirements for quantitative determination. These results indicate that the electrochemical sensor possesses good accuracy and has potential for practical application.

[0045] Table 1. HbA in actual samples 1c Content determination (n=5)

[0046]

Claims

1. A silver-doped hollow carbon nanosphere electrochemical probe, characterized in that, It is prepared by the following method: (1) Add ethanol, water, and tetraethyl silicate to a beaker, dissolve and disperse evenly, then add The reaction yields a SiO2 monomer solution; (2) Add resorcinol, formaldehyde and silver nitrate to the SiO2 monomer solution prepared in step (1) and react. After solid-liquid separation by centrifuge, dry and preserve the silver cluster-doped phenolic resin balls (Ag@RF@SiO2). (3) The Ag@RF@SiO2 obtained in step (2) is carbonized at high temperature, and then the carbonized Ag@HCS@SiO2 is mixed with NaOH solution to remove the SiO2 template to obtain Ag@HCS material; (4) The obtained Ag@HCS material is mixed with HbA 1c The aptamers were mixed in phosphate buffer solution, shaken in the dark, and then separated and stored to obtain the apt-Ag@HCS probe.

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