Preparation and application of polyamide thin-layer two-way chromatography two-channel electrochemical sensor

Abstract

The invention belongs to the technical field of preparation of electrochemical sensors, and discloses preparation of a polyamide thin-layer two-way chromatography two-channel electrochemical sensor, and the preparation method comprises the following steps: S1, preparing an electrochemical test strip through silk-screen printing for detecting oxidation peak current values of two substances ECH and VB; s2, according to the pattern of the electrochemical test strip, an unfolding cylinder is designed, and the unfolding cylinder is composed of a cover plate on the upper portion and an unfolding groove on the lower portion; and S3, improving the electrochemical test strip by using beta-CD-coated MWCNTs (multi-walled carbon nanotubes) by adopting a dispensing method. The electrochemical sensing test strip capable of rapidly detecting two active components (ECH and VB) of Ma is prepared through a silk-screen printing technology. Beta-CD (at) MWCNTs are modified on a working electrode, and specific adsorption to target molecules is enhanced through inclusion of beta-CD, so that the detection sensitivity is improved. Meanwhile, the polyamide thin layer is immobilized on the electrode, so that the sample pretreatment step is simplified, and the electrode can detect the Ma active component more accurately and quickly.

Description

Technical Field

[0001] This application belongs to the field of electrochemical sensor fabrication technology, specifically relating to the fabrication and application of a polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor. Background Technology

[0002] Cistanche deserticola (Ma) is a plant of the genus Cistanche in the family Orobanchaceae of the order Lamiaceae[1]. It has the effects of tonifying kidney yang, nourishing essence and blood, and moistening the intestines and relieving constipation. Ma contains rich bioactive components, mainly including phenylethanol glycosides, iridoids and their glycosides. The active components include echinacoside, verbascoside, iridoids and lignans, among which echinacoside and verbascoside have a variety of biological activities and important pharmacological effects. Echinacoside (ECH) has a wide range of pharmacological activities such as neuroprotection, anti-inflammation, antioxidation, antiviral, and cardioactivity; Verbascoside (VB) has pharmacological effects such as antitumor, anti-inflammation, anti-skin damage, neuroprotection, and improvement of learning and memory[6]. Both have a wide range of clinical application value.

[0003] Thin-layer chromatography (TLC) is a simple, rapid, and economical chromatographic analysis technique widely used in chemistry, biology, medicine, food, and other fields for the separation, identification, and quantitative analysis of components in mixtures. Polyamide thin-layer chromatography (PAM-TLC) is a technique that uses polyamide as the stationary phase. Polyamide is a high-molecular-weight polymer with abundant amide groups, capable of forming hydrogen bonds and other interactions with various compounds. Therefore, PAM-TLC has a unique advantage in separating polar compounds. The separation principle of PAM-TLC is similar to other thin-layer chromatography techniques, based on the differences in the distribution of different substances between the stationary phase (polyamide) and the mobile phase. The amide groups of polyamide can form hydrogen bonds with polar groups such as hydroxyl, amino, and carboxyl groups in compounds, thus affecting the distribution behavior of compounds between the stationary and mobile phases. The more polar the compound, the stronger the interaction with the polyamide and the slower its migration rate, and vice versa.

[0004] ECH is composed of two glucose units linked by a phenylethanol glycoside group, while VB is composed of one glucose and one rhamnose unit linked by a phenylethanol glycoside group. ECH and VB have similar structures, so traditional three-electrode sensors cannot separate them. Therefore, a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor was designed to solve the problem that traditional electrodes cannot measure ECH and VB quickly due to their similar structures. Summary of the Invention

[0005] This application provides the preparation and application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor, aiming to solve the problem that existing technologies cannot easily and rapidly measure ECH and VB, thus reducing detection sensitivity.

[0006] In the first aspect, the preparation of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor includes the following steps:

[0007] S1: Electrochemical test strips are prepared by screen printing and used to detect the oxidation peak current values ​​of two substances, ECH and VB.

[0008] S2: Based on the pattern of the electrochemical test strip, design the development cylinder, which consists of an upper cover plate and a lower development groove;

[0009] S3: Improve the electrochemical test strip with β-CD@MWCNTs by drop coating method: transfer the aqueous MWCNTs slurry to a centrifuge tube, add an appropriate amount of β-CD solution, sonicate, and finally obtain β-CD@MWCNTs material, store at room temperature, drop coat it on the surface of the working electrode, dry and store.

[0010] S4: Preparation of polyamide thin film: Cut out the positions where polyamide film needs to be coated using a silicone pad of thickness according to the electrode design diagram; weigh polyamide powder in a beaker, add formic acid solution, stir to dissolve it into a transparent state; then add ethanol solution and stir until homogeneous;

[0011] S5: Spread a small amount of the liquid obtained in S3 evenly on the prepared silicone pad, then spray water mist onto the polyamide pad one by one with a spray gun to precipitate a white solid. Finally, let it air dry naturally to obtain the PAM / β-CD@MWCNTs / SPEs sensor.

[0012] Furthermore, the specific steps of S1 are as follows:

[0013] S1.1: Make the corresponding screen template according to the required electrode pattern and size;

[0014] S1.2: Silver paste, carbon paste and insulating layer are sequentially transferred onto the substrate through a screen template using a doctor blade to prepare an electrochemical test strip;

[0015] S1.3: Specify the x and y axes of the electrochemical test strip for insertion into the developing tank for experiments.

[0016] Furthermore, the electrode includes two circular electrodes and one square electrode. The circular electrodes are the working electrodes, used to detect ECH and VB, respectively. The square electrode is the counter electrode, and a silver wire is connected externally as a reference electrode during the experiment.

[0017] Furthermore, the experiment includes the following two steps:

[0018] (1) Using the x-axis as the base, insert the electrode into the developing cylinder and spot the sample at the spotting point. The sample is developed along the y-axis, and the front of the developing agent reaches the top of the polyamide film. The electrode is then removed and dried.

[0019] (2) With the y-axis as the base, insert the electrode into the expansion cylinder, connect the contact to the adapter cable, spot the sample at the spotting point, expand the sample along the x-axis, and continuously perform LSV scanning. The two standards arrive at the working electrode one after another for subsequent testing.

[0020] Furthermore, the developing cylinder is 104mm long, 30mm wide, and 70mm high. A sample dispensing hole is designed at the rear of the developing cylinder to match the position of the sensor sample dispensing point. The width of the test strip contact point is reserved in the cover of the developing cylinder for fixing the position of the test strip and connecting the adapter cable.

[0021] Furthermore, the PAM / β-CD@MWCNTs / SPEs sensor can be inserted into the developing cylinder with the x-axis as the base. A mixed standard solution of 10 μL LECH and VB is added to the spotting point through the reserved sample application hole. Under the action of the developing solvent, the mixed solution slowly develops along the y-axis.

[0022] Furthermore, the developing solvent is composed of a combination of alcohol, acetic acid, and phosphate buffer.

[0023] Secondly, the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor is used to detect two active components, ECH and VB, in Cistanche deserticola. The linear relationship between the ECH oxidation peak current value and its concentration is as follows:

[0024] Ipa=0.0933C+6.0353R2=0.9975;

[0025] The linear relationship between the VB oxidation peak current value and its concentration is as follows:

[0026] Ipa=0.0918C+9.8667R2=0.9980.

[0027] Compared with the prior art, this application has at least the following beneficial effects:

[0028] Based on further analysis and research into existing technical problems, this application presents an electrochemical sensing test strip capable of rapidly detecting two active components (ECH and VB) of Ma using screen printing technology. Modifying the working electrode with β-CD@MWCNTs enhances the specific adsorption of target molecules through the inclusion effect of β-CD, thereby improving detection sensitivity. Simultaneously, immobilizing a polyamide thin layer on the electrode simplifies sample pretreatment steps, enabling the electrode to detect the active components of Ma more accurately and rapidly.

[0029] Meanwhile, the electrochemical sensing test strip prepared in this application exhibits good linearity in the concentration range of ECH and VB from 50 to 500 μmol / L, with a LOD of 1 μmol / L. This electrochemical sensing test strip utilizes the characteristics of ECH and VB, and therefore a dual working electrode design with an external reference electrode enables rapid detection of the concentrations of ECH and VB. Multiple electrochemical scans using different electrodes showed that the RSDs of ECH and VB were both less than 5%, indicating good reproducibility of the method. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the fabrication of a polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to one embodiment of this application.

[0031] Figure 2 A schematic diagram of the design of the development tank in the fabrication of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0032] Figure 3 SEM characterization images of MWCNTs (A, C) and β-CD@MWCNTs (B, D) during the fabrication of a polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0033] Figure 4 Electrochemical response of SPE in probe solution at different stages and (A) cyclic voltammetry and (B) electrochemical impedance spectroscopy at one stage in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0034] Figure 5 The UV-Vis spectrum of inclusion complexes formed from mixed solutions in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0035] Figure 6 The UV-Vis spectra of the electrochemical response of SPEs at 500 μMECH at different stages of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application.

[0036] Figure 7 Electrochemical response of SPEs at 500 μMVB at different stages in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0037] Figure 8 A comparison chart showing the optimization of β-CD@MWCNTs nanomaterial dosage in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0038] Figure 9The result of LSV continuous scanning of ECH(A) and VB(B) for 30 cycles in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0039] Figure 10 Electrochemical response diagrams of different concentrations of ECH(AC) and VB(DF) in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0040] Figure 11 A comparison of the anti-interference test results of PAM-TLC / β-CD@MWCNTs / SPEs against ECH(A) and VB(B) and interfering substances in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0041] Figure 12 ECH and VB and interfering substances thin-layer chromatograms are provided in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application;

[0042] Figure 13 The graph shows the reproducibility (A, B) and stability (C, D) test results of PAM-TLC / β-CD@MWCNTs / SPEs in the application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in one embodiment of this application. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0044] Example 1

[0045] The main symbol abbreviations designed in the following embodiments are as follows:

[0046] Main Symbol Abbreviation Reference Table

[0047] Abbreviation English name Chinese name Ma Cistanche deserticola Cistanche deserticola ECH Echinacosi de Echinacoside VB Verbascosi de Verbascoside TLC Thin Layer Chromatography Thin-layer chromatography PAM-TLC Polyami de Thin Layer Chromatography Polyamide thin-layer chromatography SPES Screen-Printed Electrochemi cal Strips Screen-printed electrochemical test strips MWCNTs Multi-Walled Carbon Nanotubes Multi-walled carbon nanotubes β-CD β-Cyclodextrin β-Cyclodextrin MB Methylene Blue Methylene blue CV Cyclic voltammetry Cyclic voltammetry

[0048] like Figure 1-2 As shown, the preparation of the polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in this application includes the following steps:

[0049] S1: Electrochemical test strips are prepared by screen printing to facilitate subsequent detection of the oxidation peak current values ​​of ECH (echinacea glycoside) and VB (verbasaloside). The specific steps are as follows:

[0050] like Figure 2As shown: S1.1: According to the required electrode pattern and size, make the corresponding screen template. The electrode includes two circular electrodes and one square electrode. The circular electrodes are working electrodes (WE1, WE2), which are used to detect ECH and VB, respectively. The square electrode is the counter electrode (CE). During the experiment, a silver wire (0.3 mm in diameter) is connected externally as a reference electrode (RE). The two working electrodes share the counter electrode and the reference electrode, forming two independent electrochemical systems.

[0051] S1.2: Silver paste, carbon paste, and insulating layer are sequentially transferred onto the substrate using a doctor blade through a screen template to produce an electrochemical test strip. During the operation, it is necessary to control the angle, pressure, and speed of the doctor blade to ensure that the paste fills the mesh evenly and forms a wet film layer of uniform thickness on the substrate.

[0052] Figure 1 In the diagram, the light gray area represents the silver paste-printed wires and contacts, the dark gray area is the carbon paste-printed working area, the green area is the insulating layer (dielectric oil), the scale lines facilitate recording the subsequent polyamide thin-layer sample placement, and the vertical lines above the electrodes are used to fix the test strip orientation during testing; both are printed using carbon paste. The red box area represents the polyamide plating area, and the red dots mark the sampling holes.

[0053] S1.3: Specify the x and y axes of the electrochemical test strip to facilitate subsequent insertion into the developing tank for experiments.

[0054] The experiment includes the following two steps:

[0055] (1) Using the x-axis as the base, insert the electrode into the developing cylinder and spot the sample at the spotting point. The sample is developed along the y-axis, and the front of the developing agent reaches the top of the polyamide film. Remove the electrode and let it dry.

[0056] (2) With the y-axis as the base, insert the electrode into the expansion cylinder, connect the contact to the adapter cable, spot the sample at the spotting point, expand the sample along the x-axis, and continuously perform LSV scanning. The two standards arrive at the working electrode one after another for subsequent testing.

[0057] S2: Based on the pattern of the electrochemical test strip, a developing cylinder was designed. The developing cylinder consists of an upper cover plate and a lower developing groove, measuring 104mm in length, 30mm in width, and 70mm in height. A sample application hole was designed at the rear of the developing cylinder to match the sensor's sample application point. The cover of the developing cylinder was pre-drilled to accommodate the test strip's contact points, for fixing the test strip's position and facilitating the connection of the adapter cable. Then, 3D printing technology was used to print the test strip using transparent resin material. Finally, a hole (1mm in diameter) was drilled at the edge of the cover plate for connecting an external silver wire as a reference electrode.

[0058] S3: An improved electrochemical test strip using a drop-coating method was developed with β-CD@MWCNTs: Aqueous MWCNTs (multi-walled carbon nanotubes) slurry was transferred to a centrifuge tube, and an appropriate amount of 1000 μM β-CD solution was added. The mixture was sonicated for 20 min to obtain the final β-CD@MWCNTs material, which was then stored at room temperature. The slurry was then drop-coated onto the working electrode surface, dried at 50°C, and stored for subsequent characterization and testing.

[0059] S4: Preparation of polyamide thin film: Cut out the positions where polyamide film needs to be coated using a 0.8 mm thick silicone pad according to the electrode design diagram; weigh 1 g of polyamide powder (200-400 mesh) in a beaker, add 6 mL of 80% formic acid solution, stir until it dissolves and becomes transparent, and let it stand for 10 min; then add 3 mL of 70% ethanol solution, stir until homogeneous, and let it stand for 20 min;

[0060] S5: Spread a small amount of the liquid obtained in S3 evenly on the prepared silicone pad, then spray water mist onto the polyamide pad one by one with a spray gun to precipitate a white solid. Finally, let it air dry for 12 hours to obtain the PAM / β-CD@MWCNTs / SPEs sensor.

[0061] The prepared PAM / β-CD@MWCNTs / SPEs sensor was inserted into the developing tank with the x-axis as the base. A 10 μL ECH and VB mixed standard solution was added to the spotting point through the pre-reserved sample loading port. Under the action of the developing solvent, the mixed solution slowly developed along the y-axis until the solvent front reached the upper end of the polyamide (ECH and VB simultaneously reached the corresponding working electrode parallel position). The sensor was removed and dried, then inserted back into the developing tank with the y-axis as the base. The electrochemical workstation was connected, and LSV continuous scanning was started under optimal experimental conditions (voltage 0.2V-0.8V, scan rate 10mV / s). Once the standard reached the working electrode, its oxidation peak current value was recorded.

[0062] Electrochemical detection: Naked SPEs, MWCNTs / SPEs, β-CD@MWCNTs / SPEs and PAM / β-CD@MWCNTs / SPEs were tested by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in a 5.0 mM [Fe(CN)6]3- / 4- solution containing 0.1 M KCl.

[0063] According to the 2020 edition of the Chinese Pharmacopoeia, 2 μL of the mixed standard of LECH and VB was spotted onto a polyamide thin-layer plate. Methanol, acetic acid, and water were used as developing solvents. The plate was then removed, dried, and examined under ultraviolet light. The mass ratio of methanol, acetic acid, and water was 2:1:7.

[0064] In this application, the developing solvent is used as the electrolyte. The water in the developing solvent is replaced with phosphate buffer, and the final developing solvent is methanol:acetic acid:0.01M pH 7.2 PBS. The mixed standard solution is then separated by thin-layer chromatography.

[0065] Example 2

[0066] The polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor provided in this application is used to detect two active components, ECH and VB, in Cistanche deserticola, specifically including the following:

[0067] Take approximately 1 g of the powdered product, pass it through a No. 4 sieve, and accurately weigh it. Place it in a 100 ml brown volumetric flask, accurately add 50 ml of 50% methanol, seal tightly, shake well, weigh, soak for 30 min, and sonicate for 40 min (power 250 W, frequency 35 kHz). Cool, weigh again, add 50% methanol to make up the lost weight, shake well, let stand, take the supernatant, filter, and take the filtrate. Spot 10 μL of the prepared sample and perform LSV testing according to step S6. Then, add low, medium, and high concentration standard solutions for spiking to verify the sensor's practicality. Finally, select Cistanche deserticola medicinal materials from three different regions—Gansu, Inner Mongolia, and Xinjiang—for testing.

[0068] Example 3

[0069] like Figure 3As shown, the surface morphology and structure of the material sample and electrode surface were analyzed using a scanning electron microscope (SEM) with a high-energy electron beam. The MWCNTs exhibited a helical shape. After the addition of β-CD, a thin film formed on the surface of the MWCNTs. Simultaneously, at the same magnification, the image after β-CD modification was more blurred, which is due to the poor conductivity of β-CD, indirectly indicating that β-CD was assembled on the surface of the MWCNTs. To further confirm the modification process, electrochemical and ultraviolet spectrophotometric methods were used to verify it.

[0070] Example 4

[0071] like Figure 4 As shown, electrochemical test strips modified with different stages of nanomaterials were subjected to CV and EIS scans in a 5.0 mM [Fe(CN)6]3- / 4-(KCl) solution. Bare SPEs, MWCNTs / SPEs, and β-CD@MWCNTs / SPEs were tested in solution. Bare SPEs showed the lowest peak current. With different modifications to MWCNTs and β-CD@MWCNTs, the electrochemical signal of the test strips increased, indicating that MWCNTs enhance the sensor's sensitivity and increase the effective working area of ​​the working electrode. β-CD forms a host-guest inclusion complex with the probe solution, loading it onto the electrode surface for redox reactions, thus significantly increasing the peak current. PAM / β-CD@MWCNTs / SPEs were tested in a solid-liquid chromatographic environment. The smaller sample loading, slower electron transfer rate, and higher resistance resulted in a lower oxidation peak current, a wider redox peak distance, and a rightward shift of the oxidation peak voltage.

[0072] From electrochemical impedance spectroscopy ( Figure 4 As shown in -B), under the same electrochemical conditions, a semi-circular arc appears in the high-frequency region (the semi-circular arc is the diameter of the capacitive arc, representing the charge transfer resistance; its decrease indicates a reduction in the electron transfer resistance of the ferricyanide pair on the electrode surface). The bare SPE shows a clear semi-circle in the Nyquist fit, with an impedance value of 263.3 Ω. The MWCNT / SPEs interface resistance is 1 μΩ, almost a straight line, while the β-CD@MWCNT / SPEs interface resistance is 120.2 Ω. The semi-circular arc is significantly smaller than that of the bare SPEs but larger than that of the MWCNT / SPEs, because β-CD itself is not conductive, thus increasing the resistance. The PAM / β-CD@MWCNTs / SPEs interface resistance is 393.4 Ω, because the polyamide thin layer on the electrode surface is solid, affecting the electron transfer rate and is not conductive itself.

[0073] Example 5

[0074] like Figure 5-7 As shown, the feasibility of PAM / β-CD@MWCNTs / SPEs is verified. The specific details are as follows:

[0075] (1) Ultraviolet-visible spectrophotometry was used to demonstrate that guest molecules (ECH and VB) can form inclusion complexes with host molecules (β-CD). The ultraviolet-visible spectra of methanol solutions of ECH (50 μM) and VB (50 μM) were recorded with and without β-CD (1000 μM). After the addition of β-CD, the ultraviolet absorption intensities of both ECH and VB increased compared to solutions of ECH and VB alone, but their absorption bands remained unchanged. This may be because the addition of β-CD increases the solubility of ECH and VB in water, and also implies the formation of host-guest inclusion complexes between the host and guest molecules.

[0076] (2) Electrochemical methods were used to demonstrate the effect of the modified material on the electrochemical response of ECH and VB. The results are as follows: Figure 6 and Figure 7 As shown, bare SPEs showed the lowest response to ECH and VB. After modification with MWCNTs, the peak current increased, indicating that MWCNTs increased the effective working area of ​​the electrochemical sensor and increased the electron transfer rate. After modification with β-CD@MWCNTs, the peak current was significantly enhanced again, indicating that a host-guest inclusion complex was formed between the host molecule and the guest molecule, further proving the experimental conclusions of UV-Vis spectrophotometry.

[0077] Among them, after a polyamide thin layer is deposited on β-CD@MWCNTs / SPEs, since the detection environment of the first three is in solution, while the detection environment of PAM / β-CD@MWCNTs / SPEs is in chromatographic solid-liquid state, the sample loading is smaller, the electron transfer rate is slower, and the resistance is larger. Therefore, the oxidation peak current is reduced, the peak shape is wider, and the peak position is shifted to the right.

[0078] Example 6

[0079] like Figure 8 and Figure 9 As shown, the concentration of β-CD@MWCNTs and the number of scan cycles were optimized. The specific steps for optimizing the concentration of β-CD@MWCNTs are as follows:

[0080] MWCNT slurry was mixed with 1000 μM β-CD solution to prepare MWCNT concentrations of 2 wt%, 4 wt%, 6 wt%, 8 wt%, 10 wt%, and 12 wt%. CV scans were performed in the probe solutions under conditions of -0.2–0.6 V and a scan rate of 10 mV / s. As the MWCNT concentration increased, the oxidation peak current gradually increased, reaching its highest value at a MWCNT concentration of 8 wt%, after which the oxidation peak current began to decrease. Therefore, a MWCNT concentration of 8 wt% was selected as the optimal experimental condition.

[0081] The specific details of optimizing the number of scan cycles are as follows:

[0082] Each individual analyte underwent 30 LSV scans. ECH, under the influence of the developing solvent, first reached the working electrode (WE1) region, peaking at the 7th scan, and then slowly decreased. VB followed, reaching the working electrode (WE2) region, peaking at the 13th scan, and then slowly decreasing thereafter. Ultimately, 10 LSV scans were performed on ECH followed immediately by 5 LSV scans on VB. Scans 5-9 of ECH were selected for linearity data representation, and scans 11-15 of VB were selected for linearity data representation.

[0083] Example 7

[0084] like Figure 10 As shown, different concentrations (10-500 μM) were set to allow the PAM / β-CD@MWCNTs / SPEs sensor to detect the linearity and range of ECH and VB under optimal experimental conditions. 10 μL of sample was spotted into the developing solvent and run until both analytes reached the electrode region. LSV scanning was performed at a scan rate of 1 mV / s and a scan interval of 0.2-0.8 V. Data for ECH were selected from 270-720 s, and data for VB were selected from 720-1170 s. The final experimental results are shown below. Figure 10 As shown in Figures A and D, the oxidation peak current increases as the analyte approaches the electrode region with the developing solvent, until the highest ECH value is 450-540 s (data). Figure 10 As shown in Figure B), the highest value of VB is 900-990s (data graph as shown). Figure 10 After E (as shown in Figure E), the analyte leaves the electrode region along with the developing solvent, thus the oxidation peak current gradually decreases. There is a good linear relationship between concentration and oxidation peak current. The calculated detection limit is 1 × 10⁻⁶ M (S / N = 3). The linear relationships between the concentrations of ECH and VB and their oxidation peak currents are as follows:

[0085] ECH: Ipa(μA)=0.0933C(μM)+6.0353R2=0.9975

[0086] VB: Ipa(μA)=0.0918C(μM)+9.8667R2=0.9980

[0087] Therefore, the PAM / β-CD@MWCNTs / SPEs prepared in this application exhibit a lower detection limit and a wider linear range.

[0088] Example 8

[0089] like Figure 11 and Figure 12 As shown, four substances—quercetin, caffeic acid, rhein, and isorhamnetin—were added to a 250 μM mixed standard solution of ECH and VB using a PAM-TLC / β-CD@MWCNTs / SPEs sensor. The concentrations of these four interfering substances were 10 times the concentrations of ECH and VB. ECH and verbascose, as phenylethyl glycosides, are mainly affected by environmental factors such as pH, temperature, light, oxidation, and enzymatic hydrolysis, undergoing glycosidic bond hydrolysis and ester bond cleavage to generate caffeic acid, glucose, and other substances. The final experimental results are as follows. Figure 12 As shown in the figure, the individual ECH and VB responses are almost indistinguishable from the current responses of the analytes and various ion mixtures. Calculations show that the differences in current responses between the two are 98.98% (ECH) and 94.00% (VB). Figure 2-13 C represents four interfering substances that were run in a developing solvent of methanol: glacial acetic acid: 0.01M PBS (pH 7.2) = 2:1:7. The experimental results show that the polyamide thin film has a good separation effect. Therefore, the sensor can still accurately detect ECH and VB even in the presence of high concentrations of interfering substances, and has excellent anti-interference ability.

[0090] Example 9

[0091] like Figure 13 As shown, using optimal experimental conditions and the same method, 15 PAM / β-CD@MWCNTs / SPEs sensors were prepared and tested with a mixed standard solution of ECH and VB (500 μM). The relative standard deviations (RSDs) obtained were 2.08% (ECH) and 2.40% (VB), indicating that the sensors have good reproducibility and can meet the requirements of practical applications. Furthermore, the stability of the modified electrode was tested by storing the same batch of sensors at room temperature for 15 days. The current response values ​​showed little difference compared to the initial values, with RSDs of 2.48% (ECH) and 4.79% (VB), indicating that the prepared sensors have good stability.

[0092] Test case

[0093] Under optimal experimental conditions, spiked solutions from three different origins were analyzed to determine the content of ECH and VB in Ma. To further confirm the accuracy and reliability of the sensor, high-performance liquid chromatography (HPLC) was used as the standard detection method for Echinacea oleifera dried fruit and VB to detect the above samples. The results obtained from the two detection methods were compared and analyzed.

[0094]

[0095]

[0096] The detection results of the two methods showed no significant difference, confirming that the sensor constructed in this application has accurate and reliable detection results, and can accurately detect the content of Viola tianshanense, ultimately realizing the application of detection of its active ingredients.

[0097] In the fabrication and application of the aforementioned polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor, an electrochemical sensing test strip capable of rapidly detecting two active components (ECH and VB) of Ma was prepared using screen printing technology. Modifying the working electrode with β-CD@MWCNTs enhances the specific adsorption of target molecules through the inclusion effect of β-CD, thereby improving detection sensitivity. Simultaneously, immobilizing a polyamide thin layer on the electrode simplifies sample pretreatment steps, enabling the electrode to detect the active components of Ma more accurately and rapidly.

[0098] Meanwhile, the electrochemical sensing test strip prepared in this application exhibits good linearity in the concentration range of ECH and VB from 50 to 500 μmol / L, with a LOD of 1 μmol / L. This electrochemical sensing test strip utilizes the characteristics of ECH and VB, and therefore a dual working electrode design with an external reference electrode enables rapid detection of ECH and VB concentrations. Multiple electrochemical scans using different electrodes showed that the RSDs of ECH and VB were both less than 5%, indicating good reproducibility of the method. Over time, after being stored at room temperature for 15 days, the sensor maintained stable signal strength, making it suitable for long-term storage and use. Strong resistance to common interfering substances such as quercetin, caffeic acid, isorhamnetin, and rhein demonstrates the excellent selectivity of the method.

[0099] In summary, the electrochemical sensing test strip constructed in this paper is characterized by its ease of operation and low cost, making it suitable for rapid on-site detection of traditional Chinese medicinal materials for quality control. It overcomes the shortcomings of traditional methods (such as HPLC) which are expensive and time-consuming. It can achieve rapid and sensitive detection of the active ingredient Ma at room temperature, showing broad prospects in fields such as medical diagnosis and environmental monitoring.

[0100] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. Preparation of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor, characterized in that, The preparation method includes the following steps: S1: Electrochemical test strips are prepared by screen printing and used to detect the oxidation peak current values ​​of two substances, ECH and VB. S2: Based on the pattern of the electrochemical test strip, design the development cylinder, which consists of an upper cover plate and a lower development groove; S3: Improve the electrochemical test strip with β-CD@MWCNTs by drop coating method: transfer the aqueous MWCNTs slurry to a centrifuge tube, add an appropriate amount of β-CD solution, sonicate, and finally obtain β-CD@MWCNTs material, store at room temperature, drop coat it on the surface of the working electrode, dry and store. S4: Preparation of polyamide thin film: Cut out the positions where polyamide film needs to be coated using a silicone pad of thickness according to the electrode design diagram; weigh polyamide powder in a beaker, add formic acid solution, stir to dissolve it into a transparent state; then add ethanol solution and stir until homogeneous; S5: Spread a small amount of the liquid obtained in S3 evenly on the prepared silicone pad, then spray water mist onto the polyamide pad one by one with a spray gun to precipitate a white solid. Finally, let it air dry naturally to obtain the PAM / β-CD@MWCNTs / SPEs sensor.

2. The fabrication of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 1, characterized in that, The specific steps of S1 are as follows: S1.1: Make the corresponding screen template according to the required electrode pattern and size; S1.2: Silver paste, carbon paste and insulating layer are sequentially transferred onto the substrate through a screen template using a doctor blade to prepare an electrochemical test strip; S1.3: Specify the x and y axes of the electrochemical test strip for insertion into the developing tank for experiments.

3. The preparation of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 2, characterized in that, The electrode consists of two circular electrodes and one square electrode. The circular electrodes are the working electrodes, used to detect ECH and VB, respectively. The square electrode is the counter electrode, and a silver wire is connected externally as a reference electrode during the experiment.

4. The preparation of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 2, characterized in that, The experiment includes the following two steps: (1) Using the x-axis as the base, insert the electrode into the developing cylinder and spot the sample at the spotting point. The sample is developed along the y-axis, and the front of the developing agent reaches the top of the polyamide film. The electrode is then removed and dried. (2) With the y-axis as the base, insert the electrode into the expansion cylinder, connect the contact to the adapter cable, spot the sample at the spotting point, expand the sample along the x-axis, and continuously perform LSV scanning. The two standards arrive at the working electrode one after another for subsequent testing.

5. The fabrication of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 1, characterized in that, The developing cylinder is 104mm long, 30mm wide, and 70mm high. The sample application hole at the rear of the developing cylinder is designed to match the position of the sensor sample application point. The width of the test strip contact point is reserved in the cover of the developing cylinder for fixing the position of the test strip and connecting the adapter cable.

6. The fabrication of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 1, characterized in that, The PAM / β-CD@MWCNTs / SPEs sensor can be inserted into the developing cylinder with the x-axis as the base. A mixed standard solution of 10 μL LECH and VB is added to the spotting point through the reserved sample application hole. Under the action of the developing solvent, the mixed solution slowly develops along the y-axis.

7. The preparation of the polyamide thin-layer bidirectional chromatography dual-channel electrochemical sensor according to claim 6, characterized in that, The developing solvent is composed of a combination of alcohol, acetic acid, and phosphate buffer.

8. The application of a polyamide thin-layer two-dimensional chromatography dual-channel electrochemical sensor, characterized in that, This method is used to detect two active components, ECH and VB, in Cistanche deserticola. The linear relationship between the ECH oxidation peak current value and its concentration is as follows: Ipa=0.0933C+6.0353R2=0.9975; The linear relationship between the VB oxidation peak current value and its concentration is as follows: Ipa=0.0918C+9.8667R2=0.9980。

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