阅读说明：本技术 一种电化学传感器及制备方法与其在检测人参皂苷Rg3中的应用 (Electrochemical sensor, preparation method and application thereof in detection of ginsenoside Rg3 ) 是由 王岱杰 杜欣 胡金春 崔莉 赵恒强 张�浩 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种电化学传感器及制备方法与其在检测人参皂苷Rg3中的应用,包括电极,电极表面附着复合材料和分子印迹聚合物,复合材料为多壁碳纳米管与碳化钛的复合物,分子印迹聚合物的模板为人参皂苷Rg3。其制备方法为将多壁碳纳米管与碳化钛加入至含有邻苯二甲酸二甘醇二丙烯酸酯的溶剂中进行复合获得多壁碳纳米管、碳化钛与邻苯二甲酸二甘醇二丙烯酸酯的复合材料,采用复合材料对电极表面进行修饰形成修饰电极；以邻苯二胺为功能单体,以人参皂苷Rg3为模板分子,在修饰电极表面进行电聚合形成分子印迹聚合物,即获得电化学传感器。本发明提供的电化学传感器用于检测人参皂苷Rg3具有成本低、响应快、灵敏度高等优点。(The invention discloses an electrochemical sensor, a preparation method and application thereof in detecting ginsenoside Rg3, wherein the electrochemical sensor comprises an electrode, a composite material and a molecularly imprinted polymer are attached to the surface of the electrode, the composite material is a composite of a multi-walled carbon nanotube and titanium carbide, and a template of the molecularly imprinted polymer is ginsenoside Rg 3. The preparation method comprises the steps of adding the multi-walled carbon nanotube and titanium carbide into a solvent containing diethylene glycol diacrylate phthalate for compounding to obtain a composite material of the multi-walled carbon nanotube, the titanium carbide and the diethylene glycol diacrylate phthalate, and modifying the surface of an electrode by adopting the composite material to form a modified electrode; and performing electropolymerization on the surface of the modified electrode by using o-phenylenediamine as a functional monomer and ginsenoside Rg3 as a template molecule to form a molecularly imprinted polymer, thus obtaining the electrochemical sensor. The electrochemical sensor provided by the invention has the advantages of low cost, quick response, high sensitivity and the like when being used for detecting the ginsenoside Rg 3.)

1. An electrochemical sensor is characterized by comprising an electrode, wherein a composite material and a molecularly imprinted polymer are attached to the surface of the electrode, the composite material is a composite of a multi-walled carbon nanotube and titanium carbide, and a template of the molecularly imprinted polymer is ginsenoside Rg 3.

2. The electrochemical sensor of claim 1 wherein the composite material is a composite of multi-walled carbon nanotubes, titanium carbide and diethylene glycol diacrylate phthalate;

or the molecularly imprinted polymer is poly-o-phenylenediamine.

3. The electrochemical sensor according to claim 1, wherein the mass ratio of the multi-walled carbon nanotubes to the titanium carbide is 2.5 to 3.5: 1;

or, the surface of the electrode is sequentially covered with the composite material layer and the molecularly imprinted polymer.

4. A preparation method of an electrochemical sensor is characterized in that a multi-walled carbon nanotube and titanium carbide are added into a solvent containing diethylene glycol diacrylate phthalate for compounding to obtain a composite material of the multi-walled carbon nanotube, the titanium carbide and the diethylene glycol diacrylate phthalate, and the surface of an electrode is modified by the composite material to form a modified electrode; and performing electropolymerization on the surface of the modified electrode by using o-phenylenediamine as a functional monomer and ginsenoside Rg3 as a template molecule to form a molecularly imprinted polymer, thus obtaining the electrochemical sensor.

5. The method for producing an electrochemical sensor according to claim 4, wherein the compounding method is ultrasonic treatment;

or, after compounding, centrifugal washing is carried out, and then water is added to prepare a composite material solution.

6. The method of claim 4, wherein the electrode is polished and cleaned before being modified;

or the electropolymerization buffer solution is acetic acid solution.

7. The method of claim 4, wherein the electropolymerization is carried out at a potential ranging from 0 to 1.2V and a scanning speedThe ratio is 50 to 150 mV. s^-1(ii) a Preferably, the number of polymerization cycles is 10 cycles;

or, the template is eluted by adopting a mixed solution of methanol and acetic acid only by electropolymerization; preferably, the elution time is 11.5-12.5 min;

or the mass ratio of the o-phenylenediamine to the ginsenoside Rg3 is 1: 0.9-1.1.

8. An application of the electrochemical sensor in detecting ginsenoside Rg3 is provided.

9. A detection method of ginsenoside Rg3 is characterized in that the electrochemical sensor is used as a working electrode, potassium ferricyanide is used as an electrochemical active probe, and electrochemical detection is carried out on a solution to be detected containing ginsenoside Rg 3.

10. A method for detecting ginsenoside Rg3 in claim 9, wherein three-electrode electrochemical detection is adopted;

alternatively, the electrochemical detection is performed using cyclic voltammetry and/or differential pulse voltammetry.

Technical Field

The invention belongs to the technical field of analysis and detection, and relates to an electrochemical sensor, a preparation method and application thereof in detection of ginsenoside Rg 3.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Along with the improvement of living standard of people, people pay more and more attention to the homology of medicines and foods. Ginsenoside Rg3 is a rare saponin extracted from radix Ginseng Rubra, and has pharmacological activities of resisting tumor, lowering blood sugar, relieving inflammation, and resisting oxidation. It has been shown in studies to play an important role in dietary supplementation and disease prevention as an active monomer in medicinal plants. Therefore, the detection of the content of the ginsenoside Rg3 in the edible and medicinal samples can not only evaluate the quality of the samples, but also monitor the adulteration of the samples.

In recent years, various analysis methods of ginsenoside Rg3 are established successively, such as a colorimetric method, a high performance liquid chromatography, a mass spectrometry method and the like, and the detection methods all achieve good effects. However, according to the research of the inventor, the methods have the defects of complex equipment, high instrument maintenance cost, tedious and time-consuming sample pretreatment process and the like. Therefore, it is necessary to establish a sensitive, rapid and simple quantitative analysis method for ginsenoside Rg 3.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide an electrochemical sensor, a preparation method and application thereof in detecting ginsenoside Rg3, and the electrochemical sensor for detecting ginsenoside Rg3 provided by the invention has the advantages of low cost, fast response, high sensitivity and the like.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in one aspect, the electrochemical sensor comprises an electrode, wherein a composite material and a molecularly imprinted polymer are attached to the surface of the electrode, the composite material is a composite of a multi-walled carbon nanotube and titanium carbide, and a template of the molecularly imprinted polymer is ginsenoside Rg 3.

In order to realize the purposes of low cost, quick response and high sensitivity, an electrochemical method is adopted for detection. Electrode materials and structures play a crucial role in achieving high detection sensitivity.

Titanium carbide (Ti)₃C₂T_x) The electrochemical sensor electrode material has the advantages of large active area, good conductivity and the like, but the two-dimensional structure of the electrochemical sensor electrode material limits the electrochemical performance and prevents the contact between template molecules and the active material. Therefore, the invention adopts the multi-walled carbon nano-tube and the titanium carbide for compounding, generates the advantages of cooperative and rapid electron transfer capability, large electrochemical area, good catalytic activity and the like, and is beneficial to amplifying electrochemical signals, thereby improving the sensitivity of the sensor.

However, selectivity is a bottleneck for electrochemical sensor applications. In order to solve the problem, the specificity of the electrochemical sensor on the ginsenoside Rg3 is increased by adopting a molecularly imprinted polymer with a template of the ginsenoside Rg 3.

On the other hand, the preparation method of the electrochemical sensor comprises the steps of adding the multi-walled carbon nanotube and titanium carbide into a solvent containing diethylene glycol diacrylate phthalate for compounding to obtain a composite material of the multi-walled carbon nanotube, the titanium carbide and the diethylene glycol diacrylate phthalate, and modifying the surface of an electrode by adopting the composite material to form a modified electrode; and performing electropolymerization on the surface of the modified electrode by using o-phenylenediamine as a functional monomer and saponin Rg3 as a template molecule to form a molecularly imprinted polymer, thus obtaining the electrochemical sensor.

In a third aspect, the electrochemical sensor is applied to detection of ginsenoside Rg 3.

In a fourth aspect, the method for detecting the ginsenoside Rg3 is to perform electrochemical detection on a solution to be detected containing the ginsenoside Rg3 by using the electrochemical sensor as a working electrode and using potassium ferricyanide as an electrochemical active probe.

The invention has the beneficial effects that:

1. in the electrochemical sensor provided by the invention, the advantages of capability of synergistically generating rapid electron transfer capability, large electrochemical area, good catalytic activity and the like of the composite material of the multi-walled carbon nanotube and the titanium carbide are arranged, and particularly the MWCNT-Ti₃C₂T_xthe-PDDA composite material is effective in providing a large electrochemically active area and excellent electrical conductivity. The molecular imprinting polymer taking the ginsenoside Rg3 as a template can simultaneously identify and quantify the Rg3, so that the molecular imprinting polymer has high selectivity and anti-interference performance.

2. The invention provides a composite material of multi-wall carbon nano-tube and titanium carbide (especially MWCNT-Ti) in an electrochemical sensor₃C₂T_x-PDDA composite material) and a molecularly imprinted polymer using ginsenoside Rg3 as a template, can have a good synergistic effect on the electrocatalytic oxidation of ginsenoside Rg3, so that the formed electrochemical sensor has high regeneration capability.

3. Experiments show that when the electrochemical sensor provided by the invention is used for detecting ginsenoside Rg3, the concentration of the ginsenoside Rg3 is 10-2000 mu g/mL^-1The concentration range and the response current show good linear relation, and the detection limit is 0.34 mu g/mL^-1The method can be applied to detection of the ginsenoside Rg3 in actual food samples, and the recovery rate is good.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows MIP-MWCNT-Ti of example 1 of the present invention₃C₂T_xSchematic diagram of the manufacturing process of the PDDA sensor.

FIG. 2 is a representation of the morphology, structure and electrochemical performance of the material prepared in example 1 of the present invention; MWCNT-PDDA (A), Ti₃C₂T_xPDDA (B) and MWCNT-Ti₃C₂T_x-transmission electron microscopy images of pdda (c) (scale bar, 200 nm); MWCNT-PDDA (D), Ti₃C₂T_x-PDDA(E)And MWCNT-Ti₃C₂T_x-scanning electron microscopy images of pdda (f) (scale bar, 1 μm); (G) the scan rate recorded at 50mV/s is 10mM K₃[Fe(CN)₆]The bare GCE (a) and Ti in (1)₃C₂T_xPDDA (b), MWCNT-PDDA (c) and MWCNT-Ti₃C₂T_x-cyclic voltammogram of pdda (d); (H) the scan rate recorded at 50mV/s is 10mM K₃[Fe(CN)₆]The bare GCE (a) and Ti in (1)₃C₂T_xPDDA (b), MWCNT-PDDA (c) and MWCNT-Ti₃C₂T_x-differential pulse voltammogram of pdda (d).

FIG. 3 shows MWCNT-Ti prepared in example 1 of the present invention₃C₂T_x-cyclic voltammogram of PDDA/GCE electropolymerization in o-phenylenediamine and ginsenoside Rg3 polymer solution; number of scanning cycles: 10; scanning rate: 100mV s^-1。

FIG. 4 shows MWCNT-Ti prepared in example 1 of the present invention₃C₂T_x-PDDA/GCE scanning electron microscope images; (A) MIP-MWCNT-Ti₃C₂T_x-PDDA; (B) MIP-MWCNT-Ti after removal of template molecules₃C₂T_x-PDDA；(C)MIP-MWCNT-Ti₃C₂T_x-1 mg mL of PDDA reabsorption^-1Ginsenoside Rg 3; (D) NIP-MWCNT-Ti₃C₂T_x-PDDA。

FIG. 5 shows MIP-MWCNT-Ti prepared in example 1 of the present invention₃C₂T_x-characterization of the electrochemical properties of the PDDA electrode; (A) cyclic voltammogram, MWCNT-Ti, of different modified electrodes₃C₂T_x-PDDA(a)，MIP-MWCNT-Ti₃C₂T_xPDDA (b), MIP-MWCNT-Ti after removal of template molecules₃C₂T_x-PDDA (c), reabsorption 1mg mL^-1MIP-MWCNT-Ti of ginsenoside Rg3₃C₂T_x-PDDA(d)，NIP-MWCNT-Ti₃C₂T_x-PDDA(e)；(B)MIP-MWCNT-Ti₃C₂T_xKinetic analysis of PDDA/GCE, scan rate in the range 10-150mV/s, indicating that the reaction modifying the electrode isDiffusion-controlled surface reactions; (C) the oxidation peak current (Ipa) and the reduction peak current (Ipc) are related to the square root of the scan rate (v [)^1/2) Is used.

FIG. 6 shows ginsenoside Rg 31 mg mL of different pairs of sensors prepared by the embodiment of the invention^-1(1)、1.5mg mL^-1(2)、2mg mL^-1(3) The DPV curve of (a); (A) MIP-GCE; (B) MIP-Ti₃C₂T_x-PDDA/GCE；(C)MIP-MWCNT-PDDA/GCE；(D)MIP-MWCNT-Ti₃C₂T_x-PDDA/GCE。

FIG. 7 is a graph of experimental optimization characterization according to the present invention; (A) the influence of the number of electropolymerization cycles on the current response; (B) the influence of the ratio of the template molecules to the functional monomers on the current response; (C) effect of elution time on current response.

FIG. 8 shows MIP-MWCNT-Ti prepared in example 1 of the present invention₃C₂T_xElectrochemical response of PDDA/GCE to ginsenoside Rg3 at different concentrations; (A) ginsenoside Rg3(50, 100, 300, 500, 700, 1000, 1500 and 2000 μ g mL) at different concentrations^-1) In MIP-MWCNT-Ti₃C₂T_x-DPV curve on PDDA/GCE electrode; (B)10 to 2000. mu.g mL^-1MIP-MWCNT-Ti under ginsenoside Rg3 concentration₃C₂T_x-a calibration curve of the DPV response current of the PDDA/GCE; error bars represent mean ± standard deviation (n ═ 3).

FIG. 9 shows MIP-MWCNT-Ti prepared in example 1 of the present invention₃C₂T_x-a representation of the reproducibility, selectivity and interference resistance of PDDA/GCE; (A) MIP-MWCNT-Ti₃C₂T_x-DPV curves of PDDA/GCE for 5 different electrodes; (B) MIP-MWCNT-Ti₃C₂T_x-PDDA/GCE vs 1mg mL^-1Response currents of ginsenoside Rb1(a), ginsenoside Re (b), ginsenoside Rg1(c), ginsenoside Rd (d) and ginsenoside Rg3 (e); (C) MIP-MWCNT-Ti₃C₂T_xInterference immunity of PDDA/GCE.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the defects of complex equipment, high instrument maintenance cost, complex sample pretreatment process, time consumption and the like of the conventional method for detecting the ginsenoside Rg3, the invention provides an electrochemical sensor, a preparation method and application thereof in detecting the ginsenoside Rg 3.

The invention provides an electrochemical sensor which comprises an electrode, wherein a composite material and a molecularly imprinted polymer are attached to the surface of the electrode, the composite material is a composite of a multi-walled carbon nanotube and titanium carbide, and a template of the molecularly imprinted polymer is ginsenoside Rg 3.

The invention adopts the multi-walled carbon nano-tube and the titanium carbide for compounding, generates the advantages of cooperative and rapid electron transfer capability, large electrochemical area, good catalytic activity and the like, and is beneficial to amplifying electrochemical signals, thereby improving the sensitivity of the sensor.

The specificity of the electrochemical sensor to the ginsenoside Rg3 is increased by adopting the molecularly imprinted polymer with the ginsenoside Rg3 as the template.

According to the invention, through the coordination and cooperation of the multi-walled carbon nanotube and the titanium carbide composite material and the molecularly imprinted polymer with the ginsenoside Rg3 as the template, the formed electrochemical sensor has high regeneration capacity, the electrochemical sensor is favorable for recycling, and the detection cost is further reduced.

The polymeric monomer of the molecularly imprinted polymer can be o-phenylenediamine, pyrrole, phenols and the like, wherein the effect of adopting the o-phenylenediamine is better. In some examples of this embodiment, the composite material is a composite of multi-walled carbon nanotubes, titanium carbide, and diethylene glycol diacrylate phthalate. Researches show that when the diethylene glycol diacrylate phthalate is added into the composite material, the dispersion stability effect on the composite material can be achieved, and therefore the detection performance of the electrochemical sensor on the ginsenoside Rg3 can be further improved.

In some embodiments of this embodiment, the molecularly imprinted polymer is poly-o-phenylenediamine.

In some examples of this embodiment, the mass ratio of the multi-walled carbon nanotubes to the titanium carbide is 2.5 to 3.5: 1.

In some examples of this embodiment, the electrode surface is sequentially covered with a composite layer and a molecularly imprinted polymer.

The invention also provides a preparation method of the electrochemical sensor, which comprises the steps of adding the multi-walled carbon nanotube and titanium carbide into a solvent containing diethylene glycol diacrylate phthalate for compounding to obtain a composite material of the multi-walled carbon nanotube, the titanium carbide and the diethylene glycol diacrylate phthalate, and modifying the surface of an electrode by adopting the composite material to form a modified electrode; and performing electropolymerization on the surface of the modified electrode by using o-phenylenediamine as a functional monomer and ginsenoside Rg3 as a template molecule to form a molecularly imprinted polymer, thus obtaining the electrochemical sensor.

Research shows that compared with single-walled carbon nanotubes, the multi-walled carbon nanotubes have unique surface effect, good mechanical strength, good conductivity and strong catalytic performance, and are easy to functionalize, so that the obtained electrochemical sensor has better detection effect.

The invention adopts electropolymerization (electrochemical polymerization), and has the advantages of good controllability, excellent repeatability, difficult deformation of imprinting cavities and the like. The molecularly imprinted polymer prepared by the method has better selectivity and adsorption capacity.

In some examples of this embodiment, the compounding process is sonication. The method is beneficial to the compounding of the multi-wall carbon nano-tube and the titanium carbide, and simultaneously can insert the phthalic acid diethylene glycol diacrylate into the multi-wall carbon nano-tube and between layers of the titanium carbide, thereby increasing the activity of the material.

In some examples of this embodiment, the compounding is followed by a centrifugal wash and then water is added to make the composite solution. The method can remove the uncombined material and increase the convenience of the electrode modification by the composite material.

In one or more embodiments, the centrifugal washing is performed using a mixed solution of ethanol and water. The rotation speed of centrifugal washing is 13000-15000 rpm.

In some examples of this embodiment, the electrode is polished and cleaned prior to modifying the electrode. Is beneficial to the modification effect of the electrode.

In some embodiments of this embodiment, the buffer solution for electropolymerization is an acetic acid solution.

In some examples of this embodiment, electropolymerization is carried out at a potential in the range of 0 to 1.2V and at a scan rate of 50 to 150 mV.s^-1. The number of polymerization cycles also influences the detection performance of the electrochemical sensor, and when the number of polymerization cycles is 10, the film thickness of the molecularly imprinted polymer is more suitable, and the detection performance is better.

In some examples of this embodiment, electropolymerization is performed to elute the template with a mixed solution of methanol and acetic acid. Research shows that the elution time can also influence the detection performance of the electrochemical sensor, and when the elution time is 11.5-12.5 min, the response current is higher and the detection performance is better.

In some examples of this embodiment, the mass ratio of o-phenylenediamine to ginsenoside Rg3 is 1: 0.9-1.1. Research shows that the mass ratio of o-phenylenediamine to ginsenoside Rg3 influences the detection effect of the electrochemical sensor, and when the mass ratio is 1: 0.9-1.1, the recognition site is more appropriate and the detection effect is better.

The third embodiment of the invention provides an application of the electrochemical sensor in detection of ginsenoside Rg 3.

In a fourth embodiment of the invention, the method for detecting the ginsenoside Rg3 is provided, wherein the electrochemical sensor is used as a working electrode, potassium ferricyanide is used as an electrochemical active probe, and a solution to be detected containing the ginsenoside Rg3 is subjected to electrochemical detection.

In some examples of this embodiment, three-electrode electrochemical detection is employed.

In some examples of this embodiment, the electrochemical detection is performed using Cyclic Voltammetry (CV)/or Differential Pulse Voltammetry (DPV).

The voltage range of CV detection is-0.4V-0.8V, and the scanning rate is 40-60 mV s^-1. The voltage range of DPV is from-0.2 to 0.7V, and the scanning speed is 40 to 60 mV.s^-1。

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

The preparation method of the electrochemical sensor is shown in figure 1 and comprises the following steps:

1. 1.5mg of MWCNT (purchased from Nanjing Xiancheng nanomaterial science and technology Co., Ltd.) and 0.5mg of Ti were weighed₃C₂T_x(purchased from Nanjing Xiapong nanomaterial science and technology Co., Ltd.) was dispersed in 1mL of a 0.5 wt% PDDA solution. Sonication for 2h, stirring for 120min, followed by 4 washes with water and ethanol (v/v,1:1) by centrifugation at 14000rpm for 5min each. Discarding the supernatant, adding water in equal amount to 1mL to obtain uniform nanocomposite suspension, and marking the nanocomposite as MWCNT-Ti₃C₂T_xPDDA, stored at 4 ℃ until use.

2. The naked GCE is pre-polished by alumina powder with the particle size of 0.3 mu m and 0.05 mu m to obtain a smooth mirror surface, and then the mixed solution of ultrapure water and ethanol is used for ultrasonic treatment for 3min to remove physical adsorption substances. Before modifying the electrode, the cleaned electrode is placed in N₂And (5) drying.

3.7 μ L of MWCNT-Ti₃C₂T_x-PDDA suspension (2 mg. mL)^-1) Carefully dropping the solution on the surface of GCE, and drying the modified electrode in the air for about 40min to obtain MWCNT-Ti₃C₂T_x-PDDA/GCE. Then MWCNT-Ti₃C₂T_x-PDDA/GCE was immersed in 5mL of a solution containing 1 mg/mL^-1o-PD、1mg·mL^-10.1 mol.L of ginsenoside Rg3^-1Acetic acid buffer (ABS, pH 5.2). The electropolymerization process is carried out in a potential range of 0-1.2V, and the scanning rate is 100mV s^-1And the circulation is performed for 10 times.

4. And (3) eluting the electropolymerized electrode in a methanol-acetic acid solution (v/v,9:1) for 12min to remove the ginsenoside Rg3 template. Washing the obtained electrode with deionized water, and drying at 37 ℃ to obtain MIP-MWCNT-Ti₃C₂T_xPDDA sensor (denoted MIP-MWCNT-Ti)₃C₂T_x-PDDA/GCE)。

Example 2

This example is the same as example 1, except that: in the step 3, ginsenoside Rg3 is not added in the electropolymerization process, and NIP-MWCNT-Ti is obtained₃C₂T_xPDDA sensor (note as NIP-MWCNT-Ti)₃C₂T_x-PDDA/GCE)。

Example 3

This example is the same as example 1, except that: omitting step 1, and using no MWCNT-Ti in step 3₃C₂T_x-PDDA modifies GCE. A MIP sensor (denoted as MIP-GCE) is obtained.

Example 4

This example is the same as example 1, except that: in step 1, MWCNT is added, and in step 3, Ti is directly adopted₃C₂T_x-PDDA modifies GCE. Obtaining MIP-Ti₃C₂T_x-PDDA sensor (denoted MIP-Ti)₃C₂T_x-PDDA/GCE)。

Example 5

This example is the same as example 1, except that: in step 1, Ti is added₃C₂T_xAnd in the step 3, directly adopting MWCNT-PDDA to modify GCE. Obtaining MIP-MWCNT-PDDA sensor (marked as MIP-MWCNT-PDDA/GCE).

The performance of the prepared electrochemical sensor was measured as follows.

Electrochemical analysis:

the electrochemical detection adopts a standard three-electrode electrochemical testing device, a glassy carbon electrode modified by a nano material and a molecularly imprinted polymer is used as a working electrode, and silver/silver chloride (Ag/AgCl) and a platinum wire are respectively used as a reference electrode and a counter electrode. Selecting potassium ferricyanide K₃[Fe(CN)₆]As an electrochemical active probe, cyclic voltammetry and differential pulse voltammetry are adopted to evaluate the electrochemical performance of the sensor. It was observed that the electrochemical probe could reach the electrode surface through the etching chamber. When the ginsenoside Rg3 molecules are re-attached to these cavities, they can block the arrival of probes and the transfer of electrons. Therefore, the reduction of electrochemical signal is proportional to the amount of re-adsorption of ginsenoside Rg3 molecules on the MIP. The change in redox probe current response (Δ I) was calculated as the difference in oxidation peak current measured before and after reabsorption of ginsenoside Rg 3. All electrochemical experiments were carried out at a concentration of 0.5 mol.L^-110mM K of KCl₃[Fe(CN)₆]In solution. The voltage range of CV detection is-0.4V-0.8V, and the scanning rate is 50mV s^-1. The voltage range of the DPV is from-0.2 to 0.7V, and the scanning speed is 50mV s^-1. All electrochemical experiments were performed at room temperature.

And (3) analyzing an actual sample:

3 real samples of the ginseng Rg3 tablets, the ginseng extract powder and the ginseng tabletted candies are selected for determination. The detection process comprises the following steps: after grinding, the sample was weighed and diluted to 1000. mu.g.mL with deionized water^-1A sample solution. Adding ginsenoside Rg3 with specified concentration into the sample to perform recovery rate test; 600. mu.L of the sample solution and 400. mu.L of 1000. mu.g.mL^-1After the ginsenoside Rg3 standard solution is mixed to 1000 μ L, 8 μ L of the mixed solution is added into the molecular imprinting electrode for standard recovery rate determination.

Analysis of results

1、MWCNT-Ti₃C₂T_x-characterization of PDDA.

As shown in fig. 2A, multi-walled carbon nanotubes were observed to be hollow tubular structures. As shown in FIG. 2B, Ti can be clearly seen₃C₂T_xThin-layer structures with a PDDA of the order of hundreds of nanometers. FIG. 2C is Ti₃C₂T_xSchematic representation of the attachment to the surface of the MWCNT tubular structure. As can be seen, the synthesized product had MWCNT-Ti of 200nm in size₃C₂T_xStructural features of PDDA. As can be seen from fig. 1D, the sem image shows that the MWCNT has a tubular structure. A typical accordion-like Ti can be seen in FIG. 1E₃C₂T_xA multilayer structure. As shown in FIG. 1F, Ti₃C₂T_xThe bent MWCNTs are wrapped and gradually cross-linked to form a nano porous network, so that the transfer of electrons and protons is promoted, and the titanium carbide is effectively wrapped in the carbon nanotubes and uniformly distributed. From the above characterization, MWCNT-Ti was determined₃C₂T_xPDDA nanocomposites have been successfully constructed.

2. And (4) modifying the working electrode.

MWCNT-PDDA and Ti are added to improve the electrochemical performance of the working electrode₃C₂T_x-PDDA and MWCNT-Ti₃C₂T_xPDDA modifies the working electrode. At 10mM K₃[Fe(CN)₆]CV experiments were performed in solution to study the electrochemical performance of bare and modified electrodes. As shown in fig. 2G, it can be observed that both the bare electrode and the modified electrode have a distinct redox peak attributed to ferricyanide ion. The results show that the peak current of the redox increases gradually with the modification of the electrode. MWCNT-PDDA/GCE (curve c) and MWCNT-Ti₃C₂T_xThe redox peak of PDDA/GCE (curve d) is significantly higher than that of GCE (curve a) and Ti₃C₂T_xPDDA/GCE (curve b), in which MWCNT is-Ti₃C₂T_xThe redox peak of-PDDA/GCE is maximal. This is probably because MWCNT-Ti₃C₂T_xthe-PDDA significantly increases the conductivity of the electrode, thereby facilitating electron transfer. In addition, DPVs of sensors based on different modified electrodes were recorded (FIG. 2H), and MWCNT-Ti was observed₃C₂T_xThe peak current of the PDDA/GCE is larger than that of other modified electrodes. The results show that MWCNT-Ti₃C₂T_xPDDA-modified GCE significantly improved the electricityElectrochemical performance of the electrode. Thus, in subsequent experiments, the composite was used to modify the working electrode.

3、MIP-MWCNT-Ti₃C₂T_xPreparation and morphological characterization of PDDA electrodes.

MWCNT-Ti₃C₂T_xElectropolymerization of-PDDA/GCE in polymer solution of ginsenoside Rg3 and o-PD (m/m,1:1) to form MIP film on the surface of the electrode. The thickness of the MIP film is controlled by the number of polymerization cycles. The resulting CV curve is shown in FIG. 3. The curves show that the peak current drops sharply with increasing number of scans, remaining unchanged after 10 scans, because the synthesized MIP film prevents the redox label from entering the electrode surface. These results indicate MIP-MWCNT-Ti₃C₂T_x-PDDA/GCE was successfully prepared.

The appearance of the electrode is characterized by adopting a scanning electron microscope. As shown in FIG. 4A, in MWCNT-Ti₃C₂T_xAfter electropolymerization on PDDA/GCE, a uniform and smooth molecularly imprinted film was observed. Subsequently, the elution of the ginsenoside Rg3 molecules resulted in an increase in the pores on the surface of the modified electrode (fig. 4B), indicating that the template molecules were successfully eluted from the polymer. For ginsenoside Rg3 in FIG. 4C in MIP-MWCNT-Ti₃C₂T_xReabsorption on PDDA/GCE, it can be observed that the imprinted cavities on the rough surface are effectively filled, indicating MIP-MWCNT-Ti₃C₂T_xPDDA/GCE has specific imprinting sites. With MIP-MWCNT-Ti₃C₂T_xSEM image of FIG. 4D shows NIP-MWCNT-Ti in comparison to PDDA/GCE₃C₂T_xThe PDDA/GCE surface is much more hazy and dense. The results show that MWCNT-Ti₃C₂T_xThe surface of the-PDDA nano composite material successfully synthesizes the molecularly imprinted membrane.

4. The electrochemical performance of the electrode is modified.

The electrochemical behavior and characteristics of the different modified layers were analyzed using cyclic voltammetry. So as to contain 0.5 mol.L^-110mM K of KCl₃[Fe(CN)₆]The solution was used as a supporting electrolyte, and the electron transfer capacity of the modified layer was analyzed by CV. Results of the experimentAs shown in FIG. 5A, MWCNT-Ti can be seen₃C₂T_xThe redox peak current of the-PDDA nano composite material after modification on GCE reaches about 120 muA (curve a), and the conductivity of the sensor can be greatly improved. And MWCNT-Ti₃C₂T_xMIP-MWCNT-Ti compared to PDDA/GCE₃C₂T_xThe peak current of the PDDA (curve b) is significantly reduced, probably because the formation of MIP films hinders the conduction of electrons. After the template is removed, a plurality of three-dimensional imprinting cavities can be generated, the electron diffusion is enhanced, and the oxidation-reduction reaction is accelerated. Therefore, the signal enhancement is due to effective template removal (curve c). In addition, when ginsenoside Rg3 was added to the surface of the MIP electrode, the peak current was greatly reduced (curve d), indicating that ginsenoside Rg3 was present in MIP-MWCNT-Ti₃C₂T_xThe surface of PDDA/GCE has specific adsorption. Curve e shows, NIP-MWCNT-Ti₃C₂T_xThe PDDA/GCE is polymerized without template molecules, and the electric signal is lowest because a dense film is formed on the surface of the electrode.

In order to improve the sensitivity of the sensor, 4 different sensing interfaces (example 1 and examples 3-5) are prepared and compared with the current response conditions of the ginsenoside Rg3 with different concentrations. MIP-Ti as shown in FIG. 6₃C₂T_xThe current response Δ I of the-PDDA/GCE electrode (FIG. 6A) is significantly higher than that of the MIP-GCE electrode (FIG. 6B), due to Ti₃C₂T_x-increase of specific surface area of GCE after PDDA modification. Surprisingly, the Δ I value of MIP-MWCNT-PDDA/GCE (FIG. 6C) was 40 times that of GCE. The carbon tubes are supposed to accelerate the electron transfer of the electrochemical reaction on the surface of the GCE, and effectively amplify the current signals. With MIP-MWCNT-Ti₃C₂T_xcomparing-PDDA/GCE (FIG. 6D), it can be found that the response current of MIP-MWCNT-PDDA/GCE to ginsenoside Rg3 is 1 mg/mL from the concentration of ginsenoside Rg3^-1To 1.5 mg. mL^-1、1.5mg·mL^-1To 2 mg. mL^-1The decrease was about 40 μ A. In addition, the sensing interface is 2 mg-mL^-1The response current of the ginsenoside Rg3 is very small, which indicates that the ginsenoside Rg3 is not suitable for detection in a large concentration range. Therefore, after comprehensive considerationSelecting MIP-MWCNT-Ti₃C₂T_xPDDA/GCE is the optimal sensing interface.

The kinetics of the molecularly imprinted electrochemical sensor was studied by analyzing the effect of the scanning rate on the redox current. In 10mM potassium ferricyanide solution, the scanning rate was varied from 10 mV. multidot.s^-1Increase to 150mV · s^-1MIP-MWCNT-Ti was detected₃C₂T_xElectrochemical Properties of PDDA/GCE. It can be seen from fig. 5B that there is a good linear relationship between the square root of the scan rate and the peak current density. The maximum current of the redox reaction increases linearly with increasing scan rate. In addition, the distance between the redox peaks is increasingly greater. Based on these results, the peak current to the oxidation peak (Ipa) and reduction peak (Ipc) is related to the square root of the scan rate (v @)^1/2) A linear fit was performed. The final linear regression equation is Ipa-21.69 v^{1 /2}－40.29，Ipc＝-23.24v^1/2+28.29, correlation coefficients 0.9991 and 0.9986, respectively (fig. 5C). The calculations indicate that the electrochemical signal is the result of a diffusion-controlled surface-catalyzed reaction.

5. And (4) optimizing experimental conditions.

In order to improve the selectivity and sensitivity of the MIP electrochemical sensor, the influence of factors such as the number of polymerization cycles, the ratio of functional monomers to template molecules, elution time and the like on the performance of the MIP electrochemical sensor is examined. The DPV technology is adopted to research the molecular imprinting electrochemical sensor pair of 1 mg.mL^-1The electrochemical response of the ginsenoside Rg3 is realized, and the electrochemical performance of the ginsenoside Rg3 is evaluated. The thickness of the molecularly imprinted membrane often affects the number of imprinting cavities and the diffusion rate of the ginsenoside Rg3, which can be controlled by adjusting the number of polymerization cycles of electropolymerization. Fig. 7A shows the effect of the number of turns of polymerization on the current response. The research shows that the response current of the oxidation peak of the ginsenoside Rg3 is correspondingly increased along with the increase of the number of polymerization cycles, which indicates that more imprinting sites are formed. At 10 turns, the response current is maximum. This is because the film is too thin to form a stable film, while the polymer film is too thick to inhibit the diffusion rate of ginsenoside Rg 3. Therefore, in the next measurement, 10 rounds were selected as the bestNumber of polymerization cycles.

The ratio of template molecules to functional monomers has a significant effect on the number of imprinted sites in the polymer matrix, which in turn affects the performance of the MIP sensor. Preparing MIP-MWCNT-Ti with different proportions (3:1, 2:1, 1:2 and 1:3) of ginsenoside Rg3 and o-PD₃C₂T_x-a PDDA sensor. As shown in fig. 7B, the current response Δ I increases with the increase of the functional monomer. This may be because fewer monomers are unable to bind enough template molecules, resulting in fewer recognition sites on the MIP membrane. When the ratio is greater than 1:1, Δ I begins to decrease, probably due to the decrease in recognition sites or binding cavities caused by excess monomer. The result shows that the optimal ratio of the ginsenoside Rg3 to the o-PD is 1: 1.

The effect of elution time on the removal of template molecules was examined, as shown in FIG. 7C. The current response increased with longer elution times, indicating that the template molecule was gradually removed. Extracting ginsenoside Rg3 from the blot part in methanol-acetic acid mixed solution (v/v,9:1) to regenerate MIP sensor. The maximum response current of the MIP sensor was reached at 12min, so 12min was chosen as the optimal elution time.

6. The sensor performance characteristics are analyzed electrically.

To evaluate the performance of the fabricated sensors, MIP-MWCNT-Ti was used under optimal conditions₃C₂T_xPDDA DPV tests were performed on ginsenoside Rg3 at different concentrations. As shown in FIG. 8A, the peak current of MIP sensor decreases with the increase of the concentration of ginsenoside Rg3, and is 10-2000 μ g/mL^-1Within the range, the current response (Δ I) is linear with the concentration of ginsenoside Rg 3. The regression equation is that the Delta I is 0.042c +2.499, and the detection limit (S/N is 3) is 0.34 mu g/mL^-1(FIG. 8B). In the formula, delta I is the difference value of the oxidation peak current intensity before and after the incubation of the ginsenoside Rg3, and c is the concentration of the ginsenoside Rg 3. From the curve fit, we calculated the sensitivity of the sensor to be 0.5945 μ A μ g mL^-1·cm^-2Thus, the MIP sensor prepared newly has higher sensitivity. Because the existing detection method for detecting the content of the ginsenoside Rg3 is less, the sensor is expected to become an electrochemical-based sensorA new detection method of the optical signal response.

7. Repeatability, selectivity and interference immunity studies.

The reproducibility of electrochemical sensors is a major parameter affecting the applicability of molecularly imprinted electrochemical sensors. Fig. 9A records the peak currents of five modified electrodes when MIP electrodes elute template molecules under the same conditions. The Relative Standard Deviation (RSD) of 5 electrodes was 2.67%, and the results were satisfactory. The structural analogs of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re and the like are researched by adopting DPV, and the selectivity of the sensor is discussed. As shown in fig. 9B, the constructed sensor has a strong signal response to ginsenoside Rg3 with a response current 60 times (a) that of ginsenoside Rb1 and 4.5 times (c) that of ginsenoside Rg 1. Interference immunity is an important indicator for evaluating the performance of MIP sensors. The research shows that the content of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re and other substances is 1mg mL^-1Potential interference in the presence of ginsenoside Rg 3. The results in FIG. 9C show that there is no significant effect after 5 fold addition of interfering substances. The above results demonstrate that MIP sensors have good selectivity and specificity, which can be attributed to the abundant cavities in the polymer matrix being complementary in size, shape and function to the template.

8. And (3) detecting the ginsenoside Rg3 in the real sample.

To further demonstrate the feasibility of this approach, MIP-MWCNT-Ti was used₃C₂T_x3 samples of ginsenoside Rg3 tablets (each tablet contains 23.7mg Rg3), ginseng extract powder (each bag contains 25mg Rg3) and ginseng tablet candy (each tablet contains 20mg Rg3) are detected by PDDA/GCE. After grinding the sample, 1000. mu.g/mL of deionized water was added^-1A sample solution. 600. mu.L of the sample solution and 400. mu.L of 1000. mu.g.mL^-1After the ginsenoside Rg3 standard solution is mixed to 1000 μ L, 8 μ L of the mixed solution is added into the molecular imprinting electrode for standard recovery rate determination. Table 1 shows the measurable results.

TABLE 1 ginsenoside Rg3 actual sample analysis results (n ═ 3)

Table 1 shows that the recovery rate of the method is 96.93-105.35%, and the molecular imprinting electrochemical sensor has good quantitative analysis capability on ginsenoside Rg3 in a complex sample.

Discussion of the related Art

Based on surface molecular imprinting technology and MWCNT-Ti₃C₂T_xThe PDDA nano material amplification technology establishes a molecular imprinting electrochemical sensor for detecting the ginsenoside Rg3 with high specificity and high sensitivity. Porous MWCNT-Ti₃C₂T_xthe-PDDA nanocomposite effectively provides a large electrochemically active area and excellent electrical conductivity. The specific cavity of the MIP layer of the ginsenoside Rg3 molecule can simultaneously identify and quantify Rg3, so that the ginsenoside Rg3 molecule has high selectivity and anti-interference performance. Further, MWCNT-Ti₃C₂T_xThe combination of-PDDA and MIP has good synergistic effect on the electrocatalytic oxidation of the ginsenoside Rg 3. Prepared MIP-MWCNT-Ti₃C₂T_xThe PDDA/GCE has a high regeneration capacity. The ginsenoside Rg3 is 10-2000 microgram/mL^-1The concentration range and the response current show good linear relation, and the detection limit is 0.34 mu g/mL^-1. The established electrochemical detection method is successfully applied to the detection of the ginsenoside Rg3 in an actual food sample, and the recovery rate is good.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.