CN114609204B - CMK-8 and GNs combined modified electrode, electrochemical sensor and application - Google Patents

Abstract

The invention relates to a working electrode of an electrochemical sensor with CMK-8 and GNs combined modification, wherein the surface of the working electrode is subjected to combined modification by adopting a mixture of CMK-8 and GNs; the modification method of the working electrode comprises the following steps: s1, polishing and grinding the glassy carbon electrode until the working surface is smooth, and airing after washing clean with purified water; s2, preparing CMK-8 dispersion liquid and GNs dispersion liquid; s3, mixing the GNs dispersion liquid and the CMK-8 dispersion liquid, and carrying out ultrasonic treatment to obtain a mixed dispersion liquid; s4, coating a certain amount of mixed dispersion liquid on the polished working surface of the glassy carbon electrode, and drying to obtain the CMK-8-GNs@GCE glassy carbon working electrode. The electrochemical sensor of the invention enables the baicalin detection to be simpler and faster, has higher sensitivity, extremely low detection limit and good repeatability and stability, and solves the problems of low sensitivity, long time consumption or high cost, complex pretreatment process, large using amount of organic solvent and the like of the existing baicalin detection method.

Description

CMK-8 and GNs combined modified electrode, electrochemical sensor and application

Technical Field

The invention relates to the technical field of modification of electrochemical sensors, in particular to an electrode modified by combination of CMK-8 and GNs, an electrochemical sensor comprising the electrode and application of the electrode in detection of baicalin.

Background

The radix scutellariae is the dry root of the Chinese medicinal plant radix scutellariae, which is a famous medicinal material. It is one of natural medicines and has been widely used in traditional Chinese medicine for thousands of years. Baicalin is one of the most important biological activities in baikal skullcap root. In the medical field, the baicalin has the functions of antiallergic, anxiolytic, antioxidant, anti-inflammatory and free radical scavenging, besides, the baicalin also has the capability of helping to treat hypertension, cervical cancer, traumatic brain injury and cell injury, and can play a role in treating the avian influenza of mice in animal treatment and help the growth of garrupa. In addition, baicalin has the effects of diminishing inflammation and removing acnes in the field of cosmetics. In China, the public health department requires that the content of baicalin in baikal skullcap root is not less than 8.0%. The content of baicalin in the baicalin clinical application specified in Japanese pharmacopoeia is more than 10%. Therefore, it is important to establish or develop a method or apparatus for accurately, rapidly, sensitively, economically and effectively detecting baicalin content in baikal skullcap root.

At present, the detection method of baicalin mainly comprises thin layer chromatography, ultraviolet spectrophotometry, high performance liquid chromatography-electrochemical detection method, liquid chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-mass spectrometry combined method, capillary electrophoresis method and the like. However, the sensitivity of uv and thin layer chromatography is relatively low. The high performance liquid chromatography method has high sensitivity and stability, but has the advantages of long time consumption, expensive instrument, complex pretreatment process and large amount of used organic solvent. Compared with the traditional medicine detection research, the electrochemical method has the advantages of simplicity, high sensitivity, good stability, low cost and the like, and has been applied to traditional medicine detection research. For example, electrochemical method is used to determine the components of the traditional Chinese medicine such as quercetin, protocatechuic aldehyde and baicalin. In recent years, various functional materials, particularly carbon nanomaterials such as grapheme carbon nanoplatelets GNs and carbon nanotube CNTs have been used to modify electrodes to improve the sensitivity and selectivity of electrochemical sensors. The mesoporous carbon CMK-8 with the cubic la3d structure is a novel non-silicon-based mesoporous material and is commonly used in the field of batteries due to the excellent conductivity. Graphene is a novel two-dimensional carbon nanomaterial GNs, which has a unique single-layer carbon atom structure, and thus has excellent optical, electrical, thermal and mechanical energy. The two carbon nano materials are often applied to detection of various traditional Chinese medicine components in electrochemical experiments, but the combined use of GNs and CMK-8 for detection of the medicine components has not been reported yet.

Disclosure of Invention

First, the technical problem to be solved

In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides an electrode modified by combination of CMK-8 (cubic la3d structure mesoporous carbon) and GNs (graphene carbon nanoplatelets), an electrochemical sensor comprising the electrode, and an application of the electrode in detecting baicalin. The electrochemical sensor of the invention enables the baicalin detection to be simpler and faster, has higher sensitivity, extremely low detection limit and good repeatability and stability, and solves the problems of low sensitivity, long time consumption or high cost, complex pretreatment process, large using amount of organic solvent and the like of the existing baicalin detection method.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

in a first aspect, the present invention provides a working electrode of an electrochemical sensor that is jointly modified by CMK-8 and GNs, wherein the working electrode surface is jointly modified by a mixture of CMK-8 and GNs; the modification method of the working electrode comprises the following steps:

s1, polishing and grinding the glassy carbon electrode until the working surface is smooth, and airing after washing clean with purified water;

s2, preparing CMK-8 dispersion liquid and GNs dispersion liquid;

s3, mixing the GNs dispersion liquid and the CMK-8 dispersion liquid, and carrying out ultrasonic treatment to obtain a mixed dispersion liquid;

s4, coating a certain amount of mixed dispersion liquid on the polished working surface of the glassy carbon electrode, and drying to obtain the CMK-8-GNs@GCE glassy carbon working electrode.

According to the preferred embodiment of the invention, in S1, alumina polishing powder with the particle size of 0.05 mu m is used for polishing and grinding the glassy carbon electrode, the 8-shaped method is used for polishing and grinding until the working surface is smooth, and an infrared lamp is placed for airing after purified water is washed clean; in S2, the concentration of the CMK-8 dispersion liquid and the GNs dispersion liquid is 2mg/L; in S3, the GNs dispersion liquid and the CMK-8 dispersion liquid are mixed according to the volume ratio of 1: 2-4.

According to a preferred embodiment of the invention, in S3, the GNs dispersion and the CMK-8 dispersion are mixed according to a volume ratio of 1:3, mixing.

According to a preferred embodiment of the invention, in S4, the coating amount of the mixed dispersion is 8-10. Mu.L.

According to a preferred embodiment of the invention, in S4, the coating amount of the mixed dispersion is 10. Mu.L.

In a second aspect, the present invention also provides an electrochemical sensor for detecting baicalin content in baikal skullcap root, which comprises the working electrode of any one of the embodiments, a counter electrode and a reference electrode. Preferably, the reference electrode is a calomel electrode and the counter electrode is a titanium rod.

In a third aspect, the present invention provides the use of an electrochemical sensor according to any one of the embodiments described above for detecting baicalin.

According to the handover embodiment of the invention, differential pulse voltammetry or cyclic voltammetry is adopted when the baicalin content is detected; when differential pulse voltammetry is adopted, PBS buffer solution with pH=7 is used as electrolyte; the potential for enriching baicalin is 0.4V-0.6V, the enrichment time is 860-960s, and the stirring speed is 1500-2000r/min. More preferably, a PBS buffer solution with ph=7 is used as the electrolyte; the potential for enriching baicalin is 0.4V, the enrichment time is 860s, and the stirring speed is 1500r/min.

When the baicalin content is detected, the baicalin peak current of the electrolyte increases along with the increase of the pH value within the range of 4-7, and the peak current reaches the maximum when the pH value is 7. Since baicalin is easily hydrolyzed under alkaline conditions, peak current decreases at pH > 7. Thus, the optimal pH of the buffer was determined to be 7. Wherein, the peak current of baicalin changes along with the change of the enrichment potential, the highest current peak appears when the potential is 0.4V, and the peak current of baicalin obviously decreases when the potential is more than 0.4V and less than 0.4V.

When the baicalin content is detected, the current increases with the increase of the enrichment time and is lower than 860s Shi Feng, the current reaches the maximum at 860s and starts to decrease after 860s, so that the optimal enrichment time is 860s.

When the baicalin content is detected, the peak current gradually increases along with the increase of the stirring speed, the current is maximum at the rotating speed of 1500r/min Shi Feng, and the current gradually decreases at 1500-2000r/min. According to analysis, the stirring speed is too high, the vibration intensity of the stirrer is also increased, the testing environment is unstable, and the other reason is that too fast stirring is caused by too many bubbles, so that a part of enrichment sites are occupied, and the baicalin enrichment is reduced. Therefore, the optimal stirring speed was 1500r/min.

According to the handover example of the present invention, when differential pulse voltammetry is used, the measurement conditions are:

the pH=7 of 0.2MPBS buffer solution is taken as electrolyte, the constant potential is used for enriching baicalin, the enrichment potential is E=0.4V, the enrichment time t=860 s, the stirring speed is 1500r/min, and the measurement parameters are as follows: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

According to the handover embodiment of the invention, a standard solution of baicalin is prepared and a sample to be detected is prepared before detection;

the standard solution is a standard solution of baicalin, wherein the baicalin powder is subjected to DMF volume fixing and ultrasonic dispersion to obtain 0.002M;

the preparation method of the sample to be tested comprises the following steps: grinding Scutellariae radix decoction pieces into fine powder, sieving with 50 mesh sieve, weighing 1.0g Scutellariae radix powder, extracting baicalin with 60% ethanol as solvent, mixing at a feed-liquid ratio of 1:16, ultrasonically treating for 30-40min, centrifuging for 4-6min, collecting supernatant, and filtering with 0.22 μm filter membrane; repeatedly extracting the residue with 60% ethanol for 2 times according to the above method, mixing the three filtrates, rotary evaporating with rotary evaporator until crystals are separated out, and removing all ethanol; and (3) re-dissolving the mixture by using 10mLDMF after spin drying to prepare baicalin sample liquid, and diluting the baicalin sample liquid by 100 times by using DMF (dimethyl formamide), thus obtaining a sample to be detected.

(III) beneficial effects

The invention adopts CMK-8 and GNs to combine to coat and modify the working surface of the glassy carbon electrode, adopts Differential Pulse Voltammetry (DPV) to compare the electrochemical performances of baicalin on different carbon material modified electrodes, and experimental results show that under the same experimental conditions, the working surface of the glassy carbon electrode is 1.0X10 ^-6 The oxidation peak currents of mol/L baicalin at CMK-8-GNs@GCE, CMK-8@GCE, GNs-GCE and GCE are 81 mu A, 33 mu A, 2 mu A and 0.4 mu A respectively, and the result shows that the response currents of the baicalin at CMK-8-GNs@GCE are 2.5 times, 42 times and 202.5 times that of the electrodes of the CMK-8@GCE, GNs-GCE and GCE. Based on the response current of the GCE electrode, the enhancement multiple of the response current of baicalin on the CMK-8-GNs@GCE electrode is 2 times of the product of the CMK-8@GCE and the GNs-GCE. Therefore, after the CMK-8 and the GNs are adopted to jointly modify the glassy carbon electrode, the CMK-8 and the GNs have synergistic effect on the electrode detection sensitivity, and the synergistic effect is beyond expectations.

Based on the good electrochemical catalytic performance of the CMK-8-GNs@GCE electrode to baicalin, the invention also constructs a baicalin electrochemical sensor with high sensitivity. Under the optimal condition, the oxidation peak current of baicalin on the CMK-8-GNs@GCE electrode and the concentration of baicalin are in the range of

1.0×10 ^-9 ～3.2×10 ^-7 In linear relation within M range, I _pa (μA)＝0.4129C(nmol/L)

+1.5404 (r=0.9989), the detection limit of which is 1.0×10 ^-9 M (S/n=3). The electrochemical sensor is successfully applied to the detection of baicalin in a traditional Chinese medicine baical skullcap root sample, and the labeling recovery rate is 113.5-116.9%. The detection device is simple, easy to operate, low in detection limit, wide in linear range, convenient and quick to detect baicalin in the baical skullcap root, good in reproducibility and beneficial to realizing automatic detection of baicalin an actual sample.

The invention further optimizes the mixing proportion and the total coating amount of the CMK-8 and GNs combined modified glassy carbon electrode, optimizes the detection conditions including parameters such as pH of electrolyte, enrichment potential for enriching baicalin, enrichment time, stirring speed and the like, so as to provide the optimal detection conditions and obtain the maximum response current in the actual detection process of baicalin, improve the sensitivity of an electrochemical sensor to baicalin detection, further reduce the detection limit, improve the detection repeatability, stability and reliability, and enhance the anti-interference capability. In conclusion, the invention provides a means for accurately, rapidly, sensitively, economically and effectively detecting the baicalin content in the baical skullcap root.

Drawings

FIG. 1 shows that the CMK-8-GNs@GCE, CMK-8@GCE, GNs@GCE and GCE electrodes are arranged in a matrix with the concentration of 1.0X10 ^-6 Electrochemical impedance spectrum in mol/L baicalin PBS solution.

FIG. 2 is a cyclic voltammogram of CMK-8-GNs@GCE in an electrolyte containing baicalin and without baicalin.

FIG. 3 shows that CMK-8-GNs@GCE (a), CMK-8@GCE (b), GNs@GCE (c), GCE (d) are contained in a composition of 1.0X10 ^-6 CV curve in mol/L baicalin electrolyte.

FIG. 4 shows that CMK-8-GNs@GCE (a), CMK-8@GCE (b), GNs@GCE (c), GCE (d) are contained in a composition of 1.0X10 ^-6 mol/L baicalin electrolyte DA PV curve.

FIG. 5 shows different proportions of GNs: CMK-8 (1:1, 1:2, 1:3, 1:4) modified CMK-8-GNs@GCE electrodes in the presence of 1.0X10 ^-6 DPV current response measured in mol/L baicalin electrolyte.

FIG. 6 is a graph of CMK-8-GNs@GCE electrodes modified with mixed dispersions (total concentration 2 mg/mL) at 1.0X10 ^-6 DPV current response measured in mol/L baicalin electrolyte.

FIG. 7 is a graph showing the measurement of baicalin (1.0X10) in PBS electrolytes at different pH values for CMK-8-GNs@GCE electrode ^-6 mol/L) of the DPV peak current response at the time of the test.

FIG. 8 is 1X 10 ^-6 DPV peak current response of mol/L baicalin at different enrichment potentials (A), enrichment time (B) and stirring speed (C).

FIG. 9 (A) is 1X 10 ^-6 A relation diagram of mol/L baicalin and redox peak current and potential under different sweeping speeds; (B) A linear relation graph of oxidation-reduction peak current and potential at different scanning speeds; (C) DPV curves for CMK-8-GNs@GCE electrodes in PBS buffer at different pH values (g.fwdarw.l is pH from 4.fwdarw.9); (D) For pH and peak potential (E _p ) Is a relationship of (2).

FIG. 10 is a schematic illustration of the mechanism of the electrode reaction of baicalin on CMK-8-GNs@GCE.

FIG. 11 (A) shows DPV curves of baicalin at various concentrations; and (B) is a standard curve of baicalin with different concentrations.

FIG. 12 shows the effect of the heat at 1X 10 ^-6 Adding 1×10 mol/L baicalin solution ^-4 mol/L Ca ²⁺ Glucose, xanthine, vitamin C, al ³⁺ In the case of CMK-8-GNs@GCE, a baicalin selective histogram.

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings. In each of the following examples and comparative examples, the DPV test conditions were as follows unless otherwise specified (the specification is followed by a specific description): coating amount 10 μl, ph=7 with 0.2M PBS buffer solution as electrolyte, enrichment potential e=0.4V, enrichment time t=860 s, stirring speed 1500r/min, and measurement parameters are: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

Example 1

The embodiment is a preparation method of a glassy carbon electrode jointly modified by CMK-8 and GNs, which comprises the following steps:

(1) Glassy carbon electrode treatment

Polishing and grinding the glassy carbon electrode (with the diameter of 3 mm) by using 0.05 mu m alumina polishing powder, grinding and polishing by a 8-shaped method until the working surface is smooth, washing clean by purified water, and then placing an infrared lamp for airing. And (3) using a three-electrode working system (a working electrode is a glassy carbon electrode, a reference electrode is a calomel electrode, a counter electrode is a titanium rod), carrying out electrochemical characterization on the polished glassy carbon electrode by using a cyclic voltammetry in potassium ferricyanide electrolyte, wherein the peak potential difference is smaller than 80mv, and proving that the glassy carbon electrode used in the experiment has good conductivity.

(2) CMK-8 and GNs combined modified glassy carbon electrode

Weighing 20mg of prepared CMK-8 and 20mg of graphene in a 10mL volumetric flask, adding DMF (dimethyl formamide) to a volume to scale marks, and respectively preparing 2mg/mL of CMK-8 dispersion liquid and 2mg/mL of GNs dispersion liquid. After the volume is fixed, the mixture is put into an ultrasonic instrument for ultrasonic treatment for 20min, and a CMK-8 dispersion liquid and a graphene dispersion liquid which are uniformly dispersed are respectively obtained. And mixing the graphene dispersion liquid and the CMK-8 dispersion liquid in a volume ratio of 1:3 by using a pipette, and performing ultrasonic treatment for 20min to prepare the CMK-8-GNs composite material. And transferring 10 mu L of CMK-8-GNs dispersion liquid by using a liquid transferring gun, dripping the dispersion liquid on the surface of the polished and dried GCE electrode for 3 times, and drying by using an infrared lamp to obtain the CMK-8-GNs@GCE modified electrode.

Comparative examples 1 to 2

Comparative example 1 was prepared by dropping 10. Mu.L of 2mg/mL of a CMK-8 dispersion alone onto the polished and dried surface of the GCE electrode 3 times, and drying with an infrared lamp to obtain a CMK-8@GCE modified electrode. Comparative example 2 was prepared by dropping 10. Mu.L of a 2mg/mL GNs dispersion alone onto the polished and dried surface of the GCE electrode 3 times, and drying with an infrared lamp to obtain a GNs@GCE modified electrode.

The impedance of the four electrodes of the bare Glassy Carbon Electrode (GCE), the Graphene (GNs), the CMK-8 and the GNs-CMK-8 modified glassy carbon electrode is tested and compared, and the electrochemical behaviors of baicalin on the bare Glassy Carbon Electrode (GCE), the Graphene (GNs), the CMK-8 and the GNs-CMK-8 modified glassy carbon electrode are compared by adopting a Differential Pulse Voltammetry (DPV) method and a Cyclic Voltammetry (CV) method.

The test comparison method and comparison result are as follows:

(1) Preparation of standard solution

Weighing 0.002g baicalin powder in a 10mL volumetric flask, and performing ultrasonic dispersion for 20min by using DMF to fix the volume to the scale mark to obtain 0.002M baicalin standard solution.

(2) Electrochemical impedance testing

In the presence of 1.0X10 ^-6 Electrochemical impedance tests were performed on GCE, GNs@GCE (comparative example 2), CMK-8@GCE (comparative example 1) and CMK-8-GNs@GCE (example 1) in mol/L baicalin PBS solution (pH=7), respectively.

As a result, as shown in FIG. 1, the Nyquist plot of the CMK-8@GCE electrode consisted of a semicircle in the high frequency region and a straight line in the low frequency region, with smaller semicircle indicating smaller electron transfer resistance and closer straight line to 90℃indicating faster diffusion rate. The semicircle in the high frequency region can be seen as the electron transfer rate resistance order: CMK-8-GNs@GCE < CMK-8@GCE < GNs@GCE < GCE, which indicates that the CMK-8-GNs recombination effectively reduces the electrode and enhances the electrode conductivity. Compared with other electrodes, the CMK-8-GNs@GCE modified electrode is in a straight line which is more similar to 90 degrees in a low-frequency region, so that the modified CMK-8-GNs@GCE has reduced resistance, is more beneficial to the diffusion of electrolyte on the surface of the electrode, and is more sensitive and effective for detecting baicalin.

(3) The cyclic voltammogram of CMK-8-GNs@GCE (example 1) in baicalin-containing and baicalin-free electrolytes was tested.

In the presence of 1.0X10 ^-6 Cyclic voltammograms of CMK-8-gns@gce (example 1) were tested in a PBS solution of mol/L baicalin (ph=7) and in a PBS solution without baicalin (ph=7). As shown in fig. 2, wherein curve a corresponds to an electrolyte containing baicalin and curve b corresponds to an electrolyte containing no baicalin. As is clear from FIG. 2, the current response of CMK-8-GNs@GCE to baicalin was very remarkable in the potential range of-0.15V to 0.6V.

(4) Electrochemical behaviors of baicalin on different carbon material modified electrodes are studied by adopting Cyclic Voltammetry (CV).

In the presence of 1.0X10 ^-6 In a PBS solution of mol/L baicalin (pH=7), electrochemical sensing performance of the CMK-8-GNs@GCE electrode (example 1), the CMK-8@GCE electrode (comparative example 1), the GNs@GCE electrode (comparative example 2) and the GCE electrode on baicalin was compared.

As a result, as shown in FIG. 3, almost no redox peak of baicalin was observed on bare glassy carbon electrode GCE. Baicalin showed a very weak redox peak on the gns@gce electrode (comparative example 2), indicating that graphene has a weak electrocatalytic effect on baicalin. While the background current of the gns@gce electrode (comparative example 2) is also quite weak. This is mainly because: although graphene has a very large specific surface area, in the process of modifying an electrode, two-dimensional graphene may be re-stacked on the surface of a glassy carbon electrode, so that the electrochemical active area of the electrode is severely reduced, and the electrochemical sensing performance of the electrode is poor. And the oxidation-reduction peak current of the baicalin on the CMK-8@GCE electrode (comparative example 1) is obviously increased, which proves that the CMK-8 has obvious enhancement effect on the electrochemical oxidation reduction of the baicalin. The three-dimensional cubic mesoporous structure of CMK-8 is mainly beneficial, so that the three-dimensional cubic mesoporous structure still maintains a higher electrochemical active area in the electrode modification process.

The oxidation-reduction peak of baicalin on CMK-8-GNs@GCE prepared in example 1 is further increased, which shows that the CMK-8 and graphene composite material has a synergistic enhancement effect on the electrochemical oxidation-reduction reaction of baicalin. This is because, as the two are combined, the graphene establishes a bridge connecting between the mesoporous carbon and the mesoporous carbon, so that not only is the electrochemical activity of CMK-8-GNs@GCE remarkably increased, but also the attachment sites are increased, the electron transfer rate is improved, the resistance is reduced, and the electron transfer of the baicalin electrochemical reaction is promoted, so that the CMK-8-GNs@GCE shows an enhanced current response signal.

(5) The electrochemical performance of baicalin on different carbon material modified electrodes is compared by adopting Differential Pulse Voltammetry (DPV)

In the presence of 1.0X10 ^-6 In a PBS solution of mol/L baicalin (pH=7), CMK-8-GNs@GCE electrode (example 1) was compared,Electrochemical sensing properties of baicalin by CMK-8@GCE electrode (comparative example 1), GNs@GCE electrode (comparative example 2) and GCE electrode. As shown in FIG. 4, the composition contains 1.0X10 ^-6 In PBS solution of mol/L baicalin, the oxidation peak currents of the baicalin in CMK-8-GNs@GCE, CMK-8@GCE, GNs-GCE and GCE are 81 mu A, 33 mu A, 2 mu A and 0.4 mu A respectively. The results show that: baicalin shows response current at CMK-8-GNs@GCE which is 2.5 times, 42 times and 202.5 times that of CMK-8@GCE, GNs-GCE and GCE electrode.

The comparison experiments of the (2) - (5) show that the baicalin has the best electrochemical catalytic performance on the CMK-8-GNs@GCE electrode.

Example 2

This example was based on the preparation of CMK-8-GNs@GCE electrode in example 1, with only the mixing volume ratio of GNs dispersion to CMK-8 dispersion being changed to 1:1, 1:2, 1:3, 1:4, but the total concentration of GNs and CMK-8 still remained 2mg/mL, and the mixed dispersion drop coating amount still remained 10. Mu.L. The response currents of the modified CMK-8-GNs@GCE electrodes with different volume ratios were tested by Differential Pulse Voltammetry (DPV). As shown in FIG. 5, the peak current response of baicalin increased with the increase in the volume ratio of CMK-8 in the mixed dispersion. However, the increase was to a maximum at GNs: CMK-8=1:3, a minimum at GNs: CMK-8 ratio of 1:1, and a response current lower at 1:2 and 1:4 than at GNs: CMK-8=1:3. It can be seen that the optimal compounding ratio of GNs to CMK is 1:3 when modifying the GCE electrode.

Example 3

In this example, on the basis of the preparation of CMK-8-GNs@GCE electrode in example 1, the dripping amount of the mixed dispersion was changed to 0. Mu.L, 2. Mu.L, 4. Mu.L, 6. Mu.L, 8. Mu.L, 10. Mu.L, 12. Mu.L, and the average was obtained in three replicates. The total concentration of GNs and CMK-8 in the mixed dispersion was still kept at 2mg/mL. The response current of the modified CMK-8-GNs@GCE electrodes was measured using Differential Pulse Voltammetry (DPV) using different volumes of mixed dispersions.

As a result of the test, as shown in FIG. 6, the corresponding current gradually increased as the coating amount increased, and at a coating amount of 8 to 10. Mu.L, the response current reached a larger value, more preferably 10. Mu.L, at which time the response current reached a maximum value. When the coating amount was further increased to 12. Mu.L, the electrode resistance was increased and the peak current of oxidation was decreased. In addition, the response current is also small when the coating amount is less than 8. Mu.L.

Example 4

In the embodiment, the influence of PBS electrolytes with different pH values on baicalin oxidation peak current is tested by adopting a Differential Pulse Voltammetry (DPV). The test conditions were: preparing PBS buffer solution with pH=4, 5, 6, 7, 8, 9, dissolving baicalin standard substance in the PBS solution to obtain 1.0X10 ^-6 mol/L baicalin, 3 in parallel are arranged each. The DPV test conditions are as follows: coating amount 10 μl, using 0.2M PBS buffer solution as electrolyte, enrichment potential E=0.4V, enrichment time t=860 s, stirring speed 1500r/min, and measuring parameters: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

As a result, as shown in FIG. 7, in the range of pH4-7 of the PBS buffer solution, the baicalin peak current increased with increasing pH, and the peak current reached the maximum at pH 7. Since baicalin is easily hydrolyzed under alkaline conditions, peak current decreases at pH > 7. Thus, the buffer has an optimal pH of 7.

Example 5

In this example, peak currents obtained from different enrichment potentials were used when the baicalin content was tested using Differential Pulse Voltammetry (DPV). The DPV test conditions are as follows: PBS, 1×10 at ph=7 ^-6 The mol/L baicalin and the coating amount are 10 mu L, 0.2M PBS buffer solution is used as electrolyte, the enrichment potential is E=0V, 0.2V, 0.4V, 0.6V and 0.8V potentials are used for carrying out parallel enrichment on the baicalin, the enrichment time t=860 s, the stirring speed is 1500r/min, and the measurement parameters are: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

As a result, as shown in FIG. 8A, the peak current of baicalin was also changed with the change of the enrichment potential, but the change did not have a linear relationship. Wherein, the highest current peak occurs at the potential of 0.4V, and the current is reduced both above 0.4V and below 0.4V Shi Feng.

Example 6

In the embodiment, when the baicalin content is tested by adopting a Differential Pulse Voltammetry (DPV), constant potential of 0.4V is used for enriching different durations, and then a DPV curve is tested. The DPV test conditions are as follows: PBS, 1×10 at ph=7 ^-6 The mol/L baicalin, the coating amount of 10 mu L, 0.2M PBS buffer solution as electrolyte, the enrichment potential of E=0.4V potentiostatic enrichment baicalin, the enrichment time t=0 s, 260s, 460s, 660s, 860s, 960s and 1060s are subjected to parallel experiments, the stirring speed is 1500r/min, and the measurement parameters are: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

As a result, as shown in FIG. 8B, the peak current before the enrichment of 860s increases with the increase of the enrichment time, the peak current is at a larger value at 860-960s, and starts to decrease after 860s reaches the maximum peak current. The optimal enrichment time was thus determined to be 860s.

Example 7

In the embodiment, when the baicalin content is tested by adopting a Differential Pulse Voltammetry (DPV), constant potential of 0.4V is adopted to enrich the baicalin, the baicalin electrolyte is stirred at different stirring speeds in the enrichment process, the enrichment time is 860s, and then a DPV curve is tested. The DPV test conditions are as follows: PBS, 1×10 at ph=7 ^-6 Performing parallel experiments with mol/L baicalin, coating amount of 10 mu L, 0.2M PBS buffer solution as electrolyte, enrichment potential of E=0.4V potentiostatic enrichment of baicalin, enrichment time t=860 s, stirring speed of 0r/min, 500r/min, 1000r/min, 1500r/min, 1800r/min and 2000r/min, and measuring parameters as follows: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

As a result, as shown in FIG. 8C, the peak current gradually increased with increasing stirring speed, and the current was larger at a rotational speed of 1500-2000r/min Shi Feng, and the current was maximized at 1500r/min Shi Feng, and gradually decreased beyond 1500-2000r/min. The reason is mainly that the stirring speed is too high, the vibration intensity of the stirrer is also high, the testing environment is unstable, and the reason is that too fast stirring bubbles are excessively generated, occupy a part of enrichment sites and lead to the reduction of baicalin enrichment. Therefore, the optimal stirring speed was 1500r/min.

Example 8

In the embodiment, CV test is adopted to study the influence of different scanning rates (V) on the detection of baicalin peak current (I). The test conditions were: baicalin concentration of 1.0X10 ^-6 In mol/L, the electrolyte was PBS buffer solution with pH=7, and cyclic voltammetry test was performed using a scanning rate of 0.05V/s, 0.1V/s, 0.15V/s, 0.2V/s, 0.25V/s, 0.3V/s, 0.35V/s, 0.45V/s, 0.55V/s, 0.65V/s, 0.75V/s, 0.85V/s.

The experimental results are shown in fig. 9A, in which the detected redox peak current increases with increasing scanning rate. As can be seen from FIG. 9B, the current response of baicalin shows a good linear relationship with the scanning rate, and the linear equation of the oxidation peak current and the scanning rate is I _pa =31.844+264.48 v (r= 0.9978); the linear equation of the reduction peak current and the sweep is I _pc = -33.229-202.99 v (r=0.9967). The experimental results show a good linear relationship between current response and scan rate, which indicates that the electrode reaction of baicalin on CMK-8-GNs@GCE is mainly controlled by adsorption.

Example 9

In this example, the electrochemical behavior of baicalin at different pH values was studied using the DPV test, and the results are shown in fig. 9C. As the pH increases, the oxidation peak position of baicalin moves negatively, the current gradually increases and decreases, and as shown in fig. 9D, the linear equation is ep= -0.043ph+0.4188 (r=0.9918), and the linear slope is close to the theoretical value of-0.059V/pH of the reversible system at 25 ℃. This indicates that the reaction process of baicalin on the electrode is an oxidation-reduction reaction process with equal numbers of electrons and protons. The redox reaction of baicalin on CMK-8-GNs@GCE is considered to be a reaction involving 2 proton 2 electrons, and the reaction mechanism is shown in FIG. 10.

Example 10

The DPV method is adopted to test DPV curves of baicalin with different concentrations on CMK-8-GNs@GCE under the optimal test condition, and the graph shows that as the concentration of baicalin increases, the oxidation peak current response value of baicalin also increasesLarge. The optimal test conditions are as follows: PBS, 1×10 at ph=7 ^-6 mol/L baicalin, coating amount of 10 mu L, 0.2M PBS buffer solution as electrolyte, enrichment potential of E=0.4V potentiostatic enrichment baicalin, enrichment time t=860 s, stirring speed of 1500r/min, and measuring parameters: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

As can be seen from fig. 11A, as the baicalin concentration increases, the oxidation peak current response value of baicalin also increases. As can be seen from FIG. 11B, the oxidation peak current of baicalin is 1.0X10 ^-9 ～3.2×10 ^-7 Shows good linear relation in the mol/L range, and the linear regression equation is I _pa (μa) = 0.4129c (nmol/L) +1.5404, r=0.9989, detection limit 1.0×10 ^-9 mol/L (S/n=3). Further, the detection method of the invention has high sensitivity and low detection limit for detecting baicalin.

Example 11

The present embodiment compares the advantages of using different electrochemical detection methods for detecting baicalin. Wherein the DM-CD-GNs@GCE electrode is from document 1; au-scnt@gce electrode from document 2; DDMIMPF ₆ The @ MWNTsE electrode is from document 3; moS (MoS) ₂ The @ GCE electrode is from document 4. The comparison results are shown in the following table:

note that: DM-CD-GNs@GCE is a glassy carbon electrode modified by 2, 6-dimethyl-cyclodextrin functionalized modified graphene hybrid nano sheets; DDMIMPF ₆ The @ MWNTsE is a 1-dodecyl-3-methylimidazole hexafluorophosphate modified multi-wall carbon nano tube composite electrode; moS (MoS) ₂ The @ GCE is a molybdenum disulfide modified glassy carbon electrode; au-SCNT@GCE is a glassy carbon electrode modified by gold and a chopped multiwall carbon nanotube.

As can be seen from the comparison, the CMK-8-GNs@GCE electrode has a significantly lower detection limit when detecting baicalin, and the linear range is mainly concentrated in a low concentration range, so that the CMK-8-GNs@GCE electrode is very suitable for quantitative detection of low-concentration baicalin.

Example 12

This example was used to test the repeatability and stability of electrochemical sensors using CMK-8-GNs@GCE electrodes in detecting baicalin. The testing method comprises the following steps: the same CMK-8-GNs@GCE electrode pair was used at a concentration of 1X 10 ^-6 The DPV test was performed under optimal conditions (see example 10) on a mol/L baicalin solution, and the measurement was performed in parallel for 7 times. The experimental results showed an RSD of 2.57%, indicating that the CMK-8-GNs@GCE electrode had good reproducibility. CMK-8-GNs@GCE was placed in a refrigerator at 4℃for 48 hours and then assayed, and it was found that there was little effect on the detection of baicalin. Thus, the CMK-8-GNs@GCE electrode has good stability.

Example 13

The embodiment is used for testing the anti-interference performance of an electrochemical sensor using a CMK-8-GNs@GCE electrode when detecting baicalin. The testing method comprises the following steps: selecting some common substances (including CaCl ₂ Glucose, xanthine, vitamin C and AlSO ₄ ) Baicalin of known concentration was disturbed to test the immunity of the electrochemical detection. During the test, the test method comprises the steps of 1X 10 ^{- 6} CaCl with 100 times concentration is respectively added into a mol/L baicalin working system ₂ Glucose, xanthine, vitamin C and AlSO ₄ DPV detection is performed. As shown in FIG. 12, the current response of baicalin after the addition of the interferents is not greatly different from that of baicalin under the optimal conditions, and the baicalin has good anti-interference capability on the interferents.

Example 14

In the embodiment, an electrochemical sensor is composed of a CMK-8-GNs@GCE electrode, a counter electrode (titanium rod) and a reference electrode (calomel electrode), and the content of baicalin in the baical skullcap root decoction pieces is actually detected under the optimal detection condition.

(1) Preparing a sample to be tested:

grinding Scutellariae radix decoction pieces into fine powder, and sieving with 50 mesh sieve. Weighing 1.0g of baicalin powder, and extracting baicalin with 60% ethanol as solvent. Mixing the materials at a feed liquid ratio of 1:16, performing ultrasonic treatment for 40min, centrifuging for 4min, and collecting supernatant and passing through a 0.22 μm nylon filter membrane. Ultrasonic extracting the residue with 60% ethanol for 40min, and repeating the extraction for 2 times. The three filtrates were combined and rotary evaporated to crystal precipitation with a rotary evaporator to remove all ethanol. And (5) re-dissolving the mixture by using 10mL of DMF after spin drying to prepare baicalin sample liquid. And diluting the sample liquid by 100 times to obtain a sample to be tested. The sample to be detected is dissolved in the electrolyte and then detected by the sensor.

(2) Preparation of standard solution

Weighing 0.002g baicalin powder in a 10mL volumetric flask, and performing ultrasonic dispersion for 20min by using DMF to fix the volume to the scale mark to obtain 0.002M baicalin standard solution.

(3) Determining test conditions: coating amount 10 μl (CMK-8:gns=3, total concentration 2 mg/mL), ph=7 as electrolyte with 0.2M PBS buffer solution, enrichment potential e=0.4V, enrichment time t=860 s, stirring speed 1500r/min, measurement parameters were: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

(4) 4 mu L of sample solution is added into 20mL of PBS buffer solution for DPV test, the Huang Qingan concentration in the sample solution is detected to be 102nM, and the average value of the baicalin content in the baicalin powder is calculated to be 227.62mg/g. And 3 baicalin standard solutions with different concentrations are respectively added into the sample liquid, recovery rate experiments are carried out, and the result is shown in the following table, and the measured standard recovery rate is 113.5-116.9%, which shows that the method is effective for detecting baicalin extracted from the baicalin.

In conclusion, the CMK-8 and graphene composite modified glassy carbon electrode can obviously reduce the resistance of the electrode, enhance the current response signal intensity of baicalin on the electrode and improve the response sensitivity of the electrode to baicalin. In addition, the invention optimizes the actual detection conditions, and obtains the optimal conditions that the proportion of GNs to CMK-8 is 1:3, the total concentration is 2mg/L, the coating amount is 10 mu L, the pH value of buffer solution is=7, the enrichment potential is E=0.4V, the enrichment time t=860 s and the stirring speed is 1500r/min. The electrochemical sensor provided by the invention has the advantages of proper baicalin content in the detection of the baicalin, strong detection repeatability and stability, extremely low detection limit, and reliability and development prospect.

The CMK-8 provides more attachment sites for adsorbing the drug components due to good mesopores; graphene has a large specific surface area and high conductivity and electrocatalytic activity. Graphene is a carbon nano sheet, and the mesoporous of CMK-8 is not easy to reduce the specific surface area due to impurities or other reasons by modifying the graphene in the three-dimensional structure of CMK-8, and also serves as a bridge for connecting mesoporous carbon with mesoporous carbon, so that the resistance is reduced. The specific surface area of the mixed CMK-8-GNs is increased, so that the attachment sites of the medicine are increased, and the reaction is more sensitive. Experiments prove that under the same experimental conditions, baicalin shows response current at CMK-8-GNs@GCE which is 2.5 times, 42 times and 202.5 times that of CMK-8@GCE, GNs-GCE and GCE electrodes. Based on the response current of the GCE electrode, the enhancement multiple of the response current of baicalin on the CMK-8-GNs@GCE electrode is 2 times of the product of the CMK-8@GCE and the GNs-GCE. Therefore, after the CMK-8 and the GNs are adopted to jointly modify the glassy carbon electrode, the CMK-8 and the GNs have synergistic effect on the electrode detection sensitivity, and the synergistic effect is beyond expectations.

Description of the different electrodes in example 11:

[1]Wang F，Lv M X，Lu K，et al.Electrochemical behaviors of baicalin at an electrochemically activated glassy carbon electrode and its determination in human blood serum[J].Journal of the Chinese Chemical Society，2012，59(7)：829-835.

[2]Ji Y L，Wang G F，Fang B.Electrocatalysis and determination of baicalin at a nano-Au single wall carbonnanotube modified electrode[J].Chinese Journal of Analysis Laboratory,2010，29(6)：11-14.

[3] liu Dongju research on a high-sensitivity electrochemical detection method of flavonoids [ D ]. Shandong university of Specification.2015.

[4]Chen G，Zhang H W，Ye J N.Determination of baicalein,baicalin and quercetin in Scutellariae radix and its preparations by capillary electropHoresis with electrochemical detection[J].Talanta，2000，53(2)：471-479.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (8)

1. A method for detecting baicalin using an electrochemical sensor, the electrochemical sensor comprising: a working electrode, a counter electrode and a reference electrode; the surface of the working electrode is subjected to joint modification by adopting a mixture of CMK-8 and GNs; the modification method of the working electrode comprises the following steps:

s1, polishing and grinding the glassy carbon electrode until the working surface is smooth, and airing after washing clean with purified water;

s2, preparing CMK-8 dispersion liquid and GNs dispersion liquid;

s3, mixing the GNs dispersion liquid and the CMK-8 dispersion liquid, and carrying out ultrasonic treatment to obtain a mixed dispersion liquid;

s4, coating a certain amount of mixed dispersion liquid on the polished working surface of the glassy carbon electrode, and drying to obtain the CMK-8-GNs@GCE glassy carbon working electrode.

2. The method according to claim 1, wherein in S1, alumina polishing powder with the particle size of 0.05 μm is used for polishing and grinding the glassy carbon electrode, the 8-shaped technique is used for polishing and grinding until the working surface is smooth, and the glassy carbon electrode is placed in an infrared lamp for airing after purified water is washed clean; in S2, the concentration of the CMK-8 dispersion liquid and the GNs dispersion liquid is 2mg/L; in S3, the GNs dispersion liquid and the CMK-8 dispersion liquid are mixed according to the volume ratio of 1: 2-4.

3. The method according to claim 1, wherein in S3, GNs dispersion and CMK-8 dispersion are mixed in a volume ratio of 1:3, mixing.

4. The method according to claim 1, wherein in S4 the coating amount of the mixed dispersion is 8-10 μl.

5. The method according to claim 1, wherein in S4 the coating amount of the mixed dispersion is 10 μl.

6. The method according to claim 1, wherein differential pulse voltammetry or cyclic voltammetry is used in detecting baicalin content; when differential pulse voltammetry is adopted, PBS buffer solution with pH=7 is used as electrolyte; the potential for enriching baicalin is 0.4V-0.6V, the enrichment time is 860-960s, and the stirring speed is 1500-2000r/min.

7. The method according to claim 1, wherein when differential pulse voltammetry is used, the measurement conditions are:

the pH=7 of 0.2MPBS buffer solution is taken as electrolyte, the constant potential is used for enriching baicalin, the enrichment potential is E=0.4V, the enrichment time t=860 s, the stirring speed is 1500r/min, and the measurement parameters are as follows: the potential interval is-1-0.4V, the amplification is 10mV, the pulse width is 0.06s, the sampling interval is 0.02s, the pulse period is 0.5s, the amplitude is 50mV, and the sensitivity is 100 mu A.

8. The method of claim 1, wherein a standard solution of baicalin is prepared and a sample to be tested is prepared prior to detection;

the standard solution is a standard solution of baicalin, wherein the baicalin powder is subjected to DMF volume fixing and ultrasonic dispersion to obtain 0.002M;

the preparation method of the sample to be tested comprises the following steps: grinding Scutellariae radix decoction pieces into fine powder, sieving with 50 mesh sieve, weighing 1.0g Scutellariae radix powder, extracting baicalin with 60% ethanol as solvent, mixing at a feed-liquid ratio of 1:16, ultrasonically treating for 30-40min, centrifuging for 4-6min, collecting supernatant, and filtering with 0.22 μm filter membrane; repeatedly extracting the residue with 60% ethanol for 2 times according to the above method, mixing the three filtrates, rotary evaporating with rotary evaporator until crystals are separated out, and removing all ethanol; and (3) re-dissolving the mixture by using 10mLDMF after spin drying to prepare baicalin sample liquid, and diluting the baicalin sample liquid by 100 times by using DMF (dimethyl formamide), thus obtaining a sample to be detected.