Preparation method and application of flexible electrochemical sensor
By fabricating a flexible electrochemical sensor with gold nanostructures on a polyurethane sponge scaffold, the instability problem of hydrogen peroxide detection in three-dimensional cell culture was solved, and sensitive, rapid, and stable real-time monitoring of hydrogen peroxide was achieved.
Patent Information
- Application Number
- CN202310896210.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-20
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2043-07-20
AI Technical Summary
Existing technologies make it difficult to monitor the release of biomolecules, especially hydrogen peroxide, from cells in real time during three-dimensional cell culture, and traditional planar electrodes lead to unstable and unreliable signal detection.
Using polyurethane foam as a scaffold, a flexible electrochemical sensor was fabricated by electrochemically depositing gold nanostructures and combining them with a PDMS base. This separated the working area and the clamping area, enabling real-time monitoring of hydrogen peroxide during cell culture.
It achieves sensitive and rapid detection of hydrogen peroxide, possesses good biocompatibility and mechanical stability, can work stably under tensile and bending conditions, and is suitable for real-time monitoring of three-dimensional cell culture.
Smart Images
Figure CN116973420B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of electrochemical detection technology, specifically relating to a method for preparing and applying a flexible electrochemical sensor. Background Technology
[0002] In vitro cell culture has made significant contributions to cell physiology research, but it is typically conducted in a two-dimensional plane. In a two-dimensional plane, the surface area of cells in contact with the culture medium is always larger than the surface area in contact with other cells, which hinders the accurate description of in vivo physiological conditions and distorts normal cell function. In recent years, three-dimensional cell culture technology has developed rapidly, greatly promoting the development of fields such as tissue regeneration, physiological research, and drug screening. Three-dimensional cell culture bridges the gap between two-dimensional models and in vivo models. This method not only simulates the in vivo microenvironment better but also has the advantages of low cost, time-saving, and easy control.
[0003] Electrochemical technology, characterized by high sensitivity, rapid response, experimental simplicity, and low cost, is a powerful tool for real-time monitoring of biological information. Combining electrochemical biosensors with three-dimensional cell culture is crucial for assessing biochemical responses in cell biology. Hydrogen peroxide (H2O2), one of the major reactive oxygen species in cellular metabolism, plays a vital biological role as a signaling molecule in signal transduction, inflammation, tumorigenesis, and neurodegenerative damage. Real-time monitoring of its release in three-dimensional cultured cells helps to gain a deeper understanding of pathological processes and molecular mechanisms. Several attempts have been made to sense biomolecules released from cells in three-dimensional culture in real time. However, these studies used planar electrodes, meaning that only biosignaling molecules diffused to the electrode surface could be detected. Furthermore, in three-dimensional culture models, the long diffusion distance from cells to planar electrodes leads to significant recombination or degradation of unstable biomolecules, resulting in unreliable results.
[0004] To address the above issues, three-dimensional conductive scaffolds will become an excellent and effective candidate material for realizing real-time sensing and three-dimensional culture. Polyurethane foam has a large number of pores on its surface and inside, and has good elasticity, so it can be used as a three-dimensional scaffold. However, due to the excellent tensile properties of polyurethane foam, it is difficult to control its working area when used as the substrate of the working electrode in actual detection work. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing a flexible electrochemical sensor. This flexible electrochemical sensor is used for hydrogen peroxide detection and exhibits good biocompatibility, excellent tensile properties, and stable electrochemical sensing performance.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a flexible electrochemical sensor, comprising the following steps:
[0007] (1) The pretreated polyurethane sponge was immersed in conductive carbon slurry dispersion to obtain a conductive support.
[0008] (2) The conductive scaffold obtained in step (1) is immersed in chloroauric acid aqueous solution for electrochemical deposition to obtain a gold nanostructure layer modified conductive scaffold.
[0009] (3) Place the gold nanostructure layer modified conductive scaffold obtained in step (2) into the groove of the PDMS base, and use PDMS to separate the gold nanostructure layer modified conductive scaffold into a working area and a clamping area to obtain the working electrode.
[0010] (4) Assemble the working electrode obtained in step (3) with the electrochemical workstation, counter electrode, reference electrode, electrolytic cell and electrolyte. Immerse the working area in the electrolyte and connect the clamping area to the electrochemical workstation to obtain a flexible electrochemical sensor.
[0011] Furthermore, in step (1), the density of the polyurethane foam is 60 ppi.
[0012] Further, in step (1), the pretreatment method is to cut the polyurethane sponge, wash it alternately with ultrapure water and ethanol 2 to 3 times, and dry it at 60°C in a vacuum drying oven.
[0013] Further, in step (1), the soaking is performed 4 to 5 times. After each soaking, the soaked polyurethane sponge is taken out and centrifuged at 1000 rpm for 2 minutes, and then cured at 50°C for 30 minutes.
[0014] Further, in step (1), the ratio of conductive carbon paste to diluent in the conductive carbon paste dispersion is 1:4.
[0015] Further, in step (2), the concentration of the chloroauric acid aqueous solution is 10 mM, and the electrochemical deposition is performed by cyclic voltammetry in a potential range of -0.6 to 0.7 V.
[0016] Furthermore, in step (3), the amount of PDMS applied is 0.2 mL.
[0017] Furthermore, in step (4), the reference electrode is an Ag / AgCl wire, and the counter electrode is a Pt wire.
[0018] The flexible electrochemical sensor was prepared by the above method.
[0019] The above-mentioned flexible electrochemical sensor is used in the detection of hydrogen peroxide.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] (1) The working electrode is prepared by placing polyurethane sponge as a support in the groove of the PDMS base. The PDMS base can effectively fix the working area. At the same time, the PDMS base can provide a good stretchable conductive substrate for the working electrode, which can meet the mechanical load research in the subsequent cell culture process. In addition, the special porous structure of polyurethane sponge can provide abundant active sites for carbon paste and nano gold modified materials, which solves the insulation problem of polyurethane sponge.
[0022] (2) The working electrode is designed as a working area and a clamping area. The clamping area is used to connect the electrochemical workstation to assemble a flexible electrochemical sensor. On the one hand, the working area can be used for long-term three-dimensional cell culture by the flexible electrochemical sensor, which can realize the rapid transport of nutrients and metabolites during cell culture. On the other hand, the working area uses electrodeposited gold nanoparticles, which have good electrocatalytic ability for intracellular reactive oxygen species. The polyurethane sponge used as a scaffold has excellent elasticity and also provides good mechanical stability, enabling the flexible electrochemical sensor to monitor the release of H2O2 from cells under chemical and mechanical loads in real time. Attached Figure Description
[0023] Figure 1 This is a diagram illustrating the fabrication process of the flexible electrochemical sensor in Example 1;
[0024] Figure 2 These are scanning electron microscope images of the working electrode of Example 1: (A) bare polyurethane sponge, (B) conductive scaffold, (C) conductive scaffold modified with gold nanostructure layer at 10 μm magnification, and (D) conductive scaffold modified with gold nanostructure layer at 1 μm magnification.
[0025] Figure 3 This is a cyclic voltammogram of the flexible electrochemical sensor in 0.5 mM hydroxymethyl ferrocene according to Example 1;
[0026] Figure 4 The images show cyclic voltammetry diagrams of the flexible electrochemical sensor from Example 1 after being bent in 0.5 mM hydroxymethyl ferrocene with different degrees of stretching (A) and different radii (B).
[0027] Figure 5 The diagram shows the current response (A) and the linear relationship (B) between the concentration of hydrogen peroxide and the corresponding current signal value when the flexible electrochemical sensor of Example 1 is applied to the analysis of hydrogen peroxide standard solution after the continuous addition of different concentrations of hydrogen peroxide.
[0028] Figure 6 This is a scanning electron microscope image of HeLa cells cultured for 8 hours using the working electrode in Example 1;
[0029] Figure 7This is a graph showing the measurement results of the flexible electrochemical sensor from Example 1 applied to the release of hydrogen peroxide from cells.
[0030] Among them, 1 is the reference electrode, 2 is the counter electrode, 3 is the working electrode, 31 is the PDMS base, 32 is the gold nanostructure layer modified conductive scaffold, 33 is the PDMS separation region, 4 is the electrolytic cell, 5 is the electrolyte, and 6 is the electrochemical workstation. Detailed Implementation
[0031] The technical solutions of the embodiments of the present invention will be described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. There are no particular restrictions on the source of all raw materials used in the present invention; they can be purchased from the market or prepared according to conventional methods known to those skilled in the art.
[0032] Example 1
[0033] The flexible electrochemical sensor in this embodiment is prepared by the following method:
[0034] (1) The pretreated polyurethane sponge was immersed in conductive carbon paste dispersion 5 times. After each immersion, the immersed polyurethane sponge was taken out and centrifuged at 1000 rpm for 2 min, and then cured at 50℃ for 30 min to obtain a conductive support. The density of the polyurethane sponge was 60 ppi. The pretreatment method was to cut the polyurethane sponge into 15 mm × 4 mm × 3 mm, wash it twice with ultrapure water and ethanol alternately, and dry it completely in a vacuum drying oven at 60℃. The ratio of conductive carbon paste to diluent in the conductive carbon paste dispersion was 1:4.
[0035] (2) The conductive scaffold obtained in step (1) is immersed in an aqueous chloroauric acid solution for electrochemical deposition to obtain a gold nanostructure layer modified conductive scaffold; wherein the concentration of the aqueous chloroauric acid solution is 10 mM, and the electrochemical deposition is performed by cyclic voltammetry in a potential range of -0.6 to 0.7 V.
[0036] (3) After mixing the PDMS prepolymer and curing agent, place it in a vacuum drying oven for 30 minutes to remove air bubbles, place it in a 3D printed model, and cure it at 85°C for 30 minutes to obtain a PDMS base with a groove. The groove size is 15mm×4mm×3mm. Place the gold nanostructure layer modified conductive scaffold obtained in step (2) in the groove of the PDMS base, and pour PDMS into the PDMS separation area of the gold nanostructure layer modified conductive scaffold to separate the working area and the clamping area of the gold nanostructure layer modified conductive scaffold to obtain the working electrode. The PDMS pouring amount is 0.2mL; the size of the working area is 4mm×4mm×3mm.
[0037] (4) Assemble the working electrode obtained in step (3) with the electrochemical workstation, counter electrode, reference electrode, electrolytic cell and electrolyte. Immerse the working area in the electrolyte and connect the clamping area to the electrochemical workstation to obtain a flexible electrochemical sensor.
[0038] The fabrication process of the flexible electrochemical sensor in this embodiment is as follows: Figure 1 As shown, (A) is a flowchart of the preparation of the working electrode, (B) is a schematic diagram of the microstructure of the working electrode, and (C) is a schematic diagram of the structure of the flexible electrochemical sensor. The gold nanostructure layer modified conductive scaffold 32 is placed in the groove of the PDMS base 31. The PDMS separating region 33 separates the gold nanostructure layer modified conductive scaffold 32 into a working region and a clamping region. The working region is the gold nanostructure layer modified conductive scaffold part with fine circles, and the clamping region is the gold nanostructure layer modified conductive scaffold part with coarse circles. The flexible electrochemical sensor is assembled from the working electrode 3, the electrochemical workstation 6, the counter electrode 2, the reference electrode 1, the electrolytic cell 4, and the electrolyte 5. The working region is immersed in the electrolyte, and the clamping region is connected to the electrochemical workstation 6.
[0039] The morphology of the working electrode in Example 1 was characterized using scanning electron microscopy (SEM) images, and the results are as follows: Figure 2 As shown, (A) bare polyurethane sponge; (B) conductive scaffold; (C) conductive scaffold modified with gold nanostructure layer at 10 μm magnification; (D) conductive scaffold modified with gold nanostructure layer at 1 μm magnification. It can be seen that the bare polyurethane sponge possesses a good three-dimensional porous framework structure, which is beneficial for the growth of catalysts and support materials, and its surface is relatively smooth and flat. After modification with conductive carbon paste and gold, the surface of the polyurethane sponge framework becomes rough. Further magnification of the gold nanostructure layer-modified conductive scaffold clearly shows that the gold nanoparticles are uniformly and tightly loaded on the conductive scaffold. This indicates that the porous structure of the sponge and the large rough surface area of the carbon paste provide favorable conditions for the electrodeposition of gold on the electrode surface, playing a good supporting role in the growth of gold.
[0040] Cyclic voltammetry of a flexible electrochemical sensor in 0.5 mM hydroxymethyl ferrocene was measured using an electrochemical workstation, as shown below. Figure 3 As shown, the working electrode of this embodiment is compared with the carbon paste electrode. The carbon paste electrode does not have a modified gold nanostructure layer. Due to the good conductivity of gold clusters, the peak current of the prepared flexible electrochemical sensor is significantly increased, which proves that the gold nanostructure effectively expands the electroactive surface area of the sensor, thereby improving the conductivity of the material.
[0041] The mechanical performance test results of the flexible electrochemical sensor in Example 1 are as follows: Figure 4As shown, (A) different degrees of stretching, and (B) bending with different radii. Electrochemical measurements were performed after stretching the working electrode to different set lengths on the test platform. It can be seen that under different degrees of stretching, even under high strain conditions, the CV curves recorded in the redox probe are only slightly affected by the stretching. The working electrode was wrapped around cylinders with different radii of curvature and bent. Cyclic voltammetric responses in 0.5 mM hydroxymethylferrocene were measured under different degrees of bending. The peak current and potential did not change significantly, demonstrating the excellent electrochemical stability of the electrode.
[0042] Hydrogen peroxide standard solutions of different concentrations were prepared using 0.1M phosphate buffer solution at pH 7.4, and the results are as follows. Figure 5 As shown in Figure 1, the current response graph (A) and the linear relationship between the hydrogen peroxide concentration and the corresponding current signal value are obtained after continuous addition of different concentrations of hydrogen peroxide (B). With the continuous addition of hydrogen peroxide, the response current increases accordingly. Even at a low concentration of 20 nM, a clear response signal can be observed, reflecting good electrochemical sensing capability. The response current shows a linear relationship with the hydrogen peroxide concentration between 20 nM and 43 μM, with a correlation coefficient of 0.999 and a detection limit of 1.5 nM. This indicates that the prepared flexible electrochemical sensor is sensitive and rapid for the electrooxidation reaction of H2O2, and has broad application prospects in real-time monitoring of H2O2 release.
[0043] Example 2
[0044] Detection of hydrogen peroxide in cells
[0045] HeLa cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, and stored in a 37°C, 5% CO2 humidified incubator. After electrode sterilization, HeLa cells (20 μL, 5 × 10⁶ cells / mL) were transferred to a culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. 6 cellsmL -1 Inoculate onto the sterilized working electrode described above, such as... Figure 6 As shown, after 8 hours of culture, the cells adhered tightly to the scaffold surface and had formed a typical spindle shape.
[0046] The release of H2O2 from HeLa cells cultured on the working electrode surface was monitored using an amperometric method. HeLa cells were stimulated with 200 ng / ml PMA, and the amperometric response of the flexible electrochemical sensor was recorded. Results are as follows: Figure 7As shown, upon PMA stimulation, HeLa cells immediately generated an electrical signal within a short period of time. As a control, the cell-free sensor did not generate any measurable signal upon stimulation, indicating that the increase in current is based on hydrogen peroxide produced by the cells. Conversely, no obvious amperometric signal was observed when catalase was added. This is because catalase selectively decomposes H2O2, resulting in a decrease in the amount of H2O2 released by the cells.
[0047] The above description is merely an exemplary embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.
Claims
1. The application of a flexible electrochemical sensor in the detection of hydrogen peroxide, characterized in that, The fabrication method of the flexible electrochemical sensor includes the following steps: (1) The pretreated polyurethane sponge was immersed in a conductive carbon slurry dispersion to obtain a conductive support. (2) The conductive scaffold obtained in step (1) is immersed in an aqueous solution of chloroauric acid for electrochemical deposition to obtain a gold nanostructure layer modified conductive scaffold; (3) Place the gold nanostructure layer modified conductive scaffold obtained in step (2) into the groove of the PDMS base, and use PDMS to separate the working area and the clamping area of the gold nanostructure layer modified conductive scaffold to obtain the working electrode; (4) Assemble the working electrode obtained in step (3) with the electrochemical workstation, counter electrode, reference electrode, electrolytic cell and electrolyte, immerse the working area in the electrolyte, and connect the clamping area to the electrochemical workstation to form a flexible electrochemical sensor. HeLa cells were seeded onto a sterile working electrode and cultured. The cells adhered tightly to the surface of a gold nanostructure layer-modified conductive scaffold. HeLa cells were stimulated with PMA, and the amperometric response of the flexible electrochemical sensor was recorded. The release of H2O2 from HeLa cells cultured on the working electrode surface was monitored by amperometry.
2. The application according to claim 1, characterized in that, In step (1), the density of the polyurethane foam is 60 ppi.
3. The application according to claim 1, characterized in that, In step (1), the pretreatment method is to cut the polyurethane sponge, wash it alternately with ultrapure water and ethanol 2-3 times, and dry it at 60°C in a vacuum drying oven.
4. The application according to claim 1, characterized in that, In step (1), the soaking is performed 4 to 5 times. After each soaking, the soaked polyurethane sponge is taken out and centrifuged at 1000 rpm for 2 minutes, and then cured at 50°C for 30 minutes.
5. The application according to claim 1, characterized in that, In step (1), the ratio of conductive carbon paste to diluent in the conductive carbon paste dispersion is 1:
4.
6. The application according to claim 1, characterized in that, In step (2), the concentration of the chloroauric acid aqueous solution is 10 mM, and the electrochemical deposition is performed by cyclic voltammetry in a potential range of -0.6 ~ 0.7 V.
7. The application according to claim 1, characterized in that, In step (3), the amount of PDMS applied is 0.2 mL.
8. The application according to claim 1, characterized in that, In step (4), the reference electrode is an Ag / AgCl wire, and the counter electrode is a Pt wire.